Cost Centres, Profit Centres, Investment Centres Essay Example for Free

Cost Centres, Profit Centres, Investment Centres Essay The increasing complexity of today’s business environment makes it virtually impossible for most firms to be controlled centrally. Decentralisation is a necessary response to this increasing complexity and involves the delegation of decision-making responsibility by senior management to sub-ordinates. The structure is such that decision making is dispersed to various units within the organisation, with managers at various levels making key decisions relating to their centre of responsibility. These centres of organisational activity are known as responsibility centres and may be defined ‘as a unit of a firm where an individual manager is held responsible for the unit’s performance. ’1 The performance of each centre and its manager is measured and controlled through a system of responsibility accounting which is based on the principles of locating responsibility and tracing costs/revenue/investments etc. to the individual managers who are primarily responsible. The division of the firm into separately identifiable units of responsibility allows for more accurate measurement of managerial performance because local information is more thorough. Overall, in order to obtain an accurate measurement of managerial performance, measures should be based on elements which the manager can control or significantly influence. There are three main types of responsibility centre. A cost centre is the lowest level of responsibility, and performance is measured in terms of the costs incurred by it. Cost centres do not generate revenue and therefore have no profit objectives, which differentiates it from profit and investment centres. Managers of cost centres are accountable only for controllable costs and are not responsible for level of activity or long-term investment decisions. Managerial performance is measured by efficiency of operations in terms of the quantity of inputs used in producing a given output. The basis of this type of measurement lies in comparing actual inputs to budgeted controllable costs or some predetermined level that represents efficient utilisation. Cost control and efficiency of operations are the main elements of this type of unit. However, costs in general can be difficult to measure, trace and allocate and it can be difficult to differentiate between controllable and uncontrollable costs. This poses a major drawback for the evaluation of cost centres and their management, since cost is its main element of measurement. The focus being mainly on costs, makes this centre some-what weak in terms of evaluation and measurement of managerial performance. Cost centres can be split into two different types; standard cost centres and discretionary cost centres. In the former, measurement is exercised by comparing standard cost with actual cost. Variances would be indicative of the efficiency of the centre and therefore its managers’ performance. Discretionary cost centres are centres where output cannot be measured in financial terms, for example advertising and publicity, RD etc. ‘Control normally takes the form of ensuring that actual expenditure adheres to budgeted expenditure for each expense category.’2 However, a major problem with this type of responsibility centre is the measurement of the effectiveness of expenditure and the determination of the efficiency of the centre itself and its management. A profit centre offers an additional element to the measurement process in that both inputs and outputs are measured in monetary terms. The manager of a profit centre has increased autonomy as s/he is responsible for revenue as well as costs; hence it is easier to measure the effectiveness and efficiency of managerial performance in financial terms. ‘In this situation, managers are normally free to set selling prices, choose which markets to sell in, make product-mix and output decisions and select suppliers.’3 A profit centre differs form a cost centre in that its main objective is to maximise profit and the performance of the manager is measured in terms of profit made. Top executives allocate assets to a profit centre, and the manager is responsible for using these assets to make a profit. Each profit centre has a profit target and has the authority to adopt such policies that are necessary to achieve these targets. Profit centre managers are evaluated by comparing actual profit to targeted profit. Profit analysis using profitability ratios or segmented income statements are used as a basis for evaluating managerial performance. The major issue with profit statements is the difficulty in deciding what is controllable or traceable, and in order to assess the managers’ performance rather than the economic performance of the unit, measures must be based on controllable profit only. Another difficulty arises in allocating revenue and costs to profit centres, as it is unlikely that the profit centre is completely independent. This has prompted many firms to use multiple performance measures such as a balanced scorecard, which measures non-financial as well as financial elements of the unit. The measurement of profit is also compounded by the use of transfer prices and agreeing on its ‘fairness’. Transfer prices are allocated to goods transferred from one unit to another within a firm. The implication of transfer prices is that for the selling unit it will be a source of revenue and for the receiving unit it is an element of cost, and as a result each division may act in its own interests. Transfer pricing therefore has a significant bearing when calculating revenues, costs and profits of responsibility centres. The choice of transfer pricing method is important because it affects goal congruence as well as performance measurement. However, it is difficult to determine the correct transfer price, as there are a wide variety of methods available, varying from negotiation to approaches based on the market or based on cost. The investment centre manager has increased responsibility in comparison to the cost and profit centre managers and as a result there are further options for managerial performance measurement by top management. The investment centre manager has responsibility for revenue and costs, and also has the authority to make capital investment decisions. This type of unit represents the highest level of managerial autonomy. An investment centre differs from a profit centre in that investment centre management is evaluated on the basis of the rate of return earned on the assets employed or the residual income earned, while profit centre management is evaluated on the basis of excess revenue over expenses for the period. The manager in charge has the objective of profitability, depending not only on sales but also on profitability of the capital used. Overall, investment centres offer the broadest basis for measurement in the sense that managerial performance is measured not only in terms of profits, but also in terms of assets employed to generate those profits. Performance can be measured using a variety of tools, and this ensures that the drawbacks of one method are overcome by the merits of another. This in turn leads to more accurate results and is one of the main reasons why investment centres are so popular as a means of managerial performance measurement in large companies. Both the effectiveness and the efficiency of the manager can be assessed by reference to the accounting data available. Investment centres offer many qualities required for good managerial performance measurement. For example, they provide incentives to the unit manager, they can recognise long-term objectives as well as short-term objectives and the increased responsibility means there are more controllable factors for use in performance measurement calculations. Return on investment is a measurement approach in common use in investment centres. This method has the advantage of being simple and easy to calculate. ‘ROI expresses divisional profit as a percentage of the assets employed in the division.’4 It has the further advantage of motivating managers to achieve the best return on investments in order to achieve the associated rewards. ROI provides a return measure that controls the size and is comparable to other measures. It can be used as a common denominator for comparing the returns of similar businesses, such as other divisions within the group or outside competition. It is widely used and most managers understand what the measure reflects. However, some complications arise in the calculation of this method. For example, difficulties regarding the calculation of profit, some of which are described above. Profit can be defined in a number of ways and this enables the figure to be manipulated. In the case of the figure for investments, the question arises whether this should be total assets (gross or depreciated), total operating assets or net total assets. The result would differ in each case, but if consistency is maintained throughout the organisation, decisions would remain unaffected. Another difficulty that may arise in relation to this method is that managers may focus on self-interests rather than the overall goal of the organisation and some profitable opportunities may be ignored because s/he fears potential dilution of existing successful endeavours. Furthermore, ROI does not adequately recognise risk. A manager who generates a large ROI result may be investing in riskier assets which may not be consistent with organisational goals. Use of ROI as a managerial performance measure can lead to under or over investment in assets or incorrect asset disposal decisions, in order to achieve the result the manager requires to accomplish his reward. To overcome some of the above difficulties, many firms use residual income to evaluate managerial performance. This method seeks to motivate managers to invest where the expected returns exceed the cost of capital. For the purpose of managerial performance measurement, ‘it compares the controllable contribution of an investment with the targeted rate of return.’5 There is a greater possibility that managers will be encouraged to act in the best interests of the company. Another advantage of this method is that it is more flexible because different cost of capital rates can be applied for different levels or risk. Though ROI and RI operate on a similar basis, RI proves better in certain circumstances. For example, if ROI is chosen as the measuring technique, managers may be reluctant to make additional investments in fixed assets as it may bring down the ROI for their centre. RI calculation results would be more accurate in these situations. However, residual income does not overcome the problem of determining the value of assets or the figure to be used for profit. If RI is used in a short-term perspective, it can over-emphasise short-term performance at the expense of long-term performance. Investment projects with positive net present values can show poor ROI and RI results in early years, leading to rejection of projects by managers. Residual income also experiences problems in comparing managerial performance in divisions of different sizes. The manager of the larger division will generally show a higher RI because of the size of the division rather then superior managerial performance. Another drawback for this method is that it requires an estimate of the cost of capital, a figure which can be difficult to calculate. Economic value added is an extension of the residual income measurement. It measures surplus value created by total investments which include funds provided by banks, shareholders etc. Its key element is the emphasis on after-tax operating profit and the actual annual cost of capital. The latter aspect differentiates it from the RI measure, which uses the minimum expected rate of return. EVA is a further step towards encouraging centre managers to concentrate on the overall goal of the organisation rather than their own self interests, hence reducing dysfunctional behaviour. The above measures are financial measures. As stated previously, it is important also to study non-financial aspects, such as customer satisfaction, quality, internal processes, growth etc. in order to get a more complete picture when measuring managerial performance. The above measures also focus on performance within the investment centre and do not consider the performance relative to overall company objectives. In conclusion, it can be stated that in order to assess managerial performance as opposed to the economic performance of the division, it is vital to make a distinction between the controllable and uncontrollable elements used in the chosen calculations. Each measurement technique is not without limitations, but these difficulties can be overcome by using a wide variety of measurement tools and striking the right balance between them. Of the three types of responsibility centre, an investment centre can be considered to yield better results, as it allows for the broadest basis for measurement, making it widely popular as a means of managerial performance measurement. 1 C. Drury, Management and Cost Accounting, 6th Ed. P. 653 2 C. Drury, Management and Cost Accounting, 6th Ed. P. 654 3 C. Drury, Management and Cost Accounting, 6th Ed. P. 654/655 4 C. Drury, Management and Cost Accounting, 6th Ed. P. 845 5 IPA Manual, Management Accounting, P 239

Porters Five Forces Analysis Marketing Essay

Porters Five Forces Analysis Marketing Essay Introduction The main purpose of applying the five forces analysis is to identify the key factors in the industrial environment that influence the organizations capabilities to position itself in order to merit competitive advantage. It is a framework for industry analysis and business strategy development formed by Michael Porter. An industry is a group of firms that market products which are close substitutes for each other (e.g. the car industry, the hotel industry).Some industries are more profitable than others, the answer lies in understanding the dynamics of competitive structure in an industry. Porters Five Forces Model is one of the most influential analytical models for assessing the nature of competition in an industry. Porter explains that there are five forces that determine industry attractiveness and long-run industry profitability. These five competitive forces are the threat of new competitors entry, the threat of substitutes, the bargaining power of buyers, the bargaining power of suppliers, and the degree of rivalry between existing competitors. Porters five forces diagram. http://www.b2binternational.com/china/images/stories/sections/porters_five_forces.gif Source: www.valuebasedmanagement.net Introduction to hotel industry A hotel is an institution that provides a short-term paid residence. In the past, hotels were just a small room with a bed, cupboard, and a table, but now it has totally changed to something else. Nowadays hotels are luxurious residences that include different types of facilities. Most of the hotels now include spas, swimming pools, fitness centers, conferences rooms and international restaurants. Even the rooms are now bigger and include many comfort facilities. Malaysian Association of Hotels (MAH)  was established in 1974. It is now being officially recognized as a National Hotel Association. Now it sets the regulations and minimum acceptable levels for being a legal verified hotel in Malaysia. It has 2,184 registered members and 17 more hotels in the next 3 years. Table: Hotels and rooms supply 2010/2011 Bargaining power of suppliers The term suppliers comprises all sources for inputs that are needed in order to provide goods or services. The two key suppliers to the Hotel industry are labors and real estate Over all the suppliers in this market are defined as property owners, developers and real estate companies, interior design and furnishings companies, architects, management and training service providers, marketing companies, industry consultants and ICT manufacturers. Category Rating 1-10 Remarks Number of suppliers 6 (medium) Considerable no. of local and Chinese contractors Small number of quality training providers and skilled employees. Availability of substitute 6(medium) Substitutes for property (real estate agents), designers, and employees are available. Switching cost category 2 (low) -substitute for hotel are few.. Suppliers threat of forward integration 2 (low) Suppliers are highly unlikely to forward integrate into the hotel business Industrys treat backward integration 5 (high) -hotels could backward integrate to own real estate company. They could have their own training wing. Contribution to quality 5 (high) -Property development and real estate companies add to the quality so does skilled labor and quality training Contribution to cost 2 (low) -Most suppliers are much smaller companies compared to hotel companies. -Hence hotel companies have a much higher bargaining power. suppliers contribution to cost is low Overall, the number of suppliers for the Hotel industry is quite large and each supplier is very small in size compared to the leading players in the industry. These few powerful players are indispensable to the suppliers. Substitutability of the suppliers is also quite feasible and inexpensive. Switching between real estate agents is not goingto affect a particular Hotel company significantly. However in terms of quality, training centers for employees and ICT manufacturers who provide IT systems thatfor property management are relatively more difficult to replace. Therefore in terms of substitute suppliers industry attractiveness is moderately high. Unlike the supplier is threat of  forward integration, Industry is threat of backwardintegration is pretty high since large hotel chains like ITC or IHCL  would have no qualms expanding into the real estate  business or developing employee training facilities in-house. Similarlythe industry is contribution to both cost and quality isrelatively high. Overall bargaining power of suppliers is low and industry is attractiveness in terms of supplier bargaining power is high (4). BARGAINING POWER OF BUYERS The bargaining power of buyers determines how much customers can impose pressure on margins and volumes. The end-users of the high-end hotel industry are:- Leisure traveler Business traveler Customers who require space for conferences or other events Category Rating 1-10 Remarks -Number of Buyers 7(high) -Buyers are numerous and small in size.- Losing one customer cannot going to make a difference. Their bargaining power is low -Availability of substitutes: (medium) -Multiple substitutes for a given hotel or brand is available -Informal accommodation for friends and family is available alternative -Corporate guest houses for the business traveler -Switching cost: 2(low) -Switching costs arenegligible Buyers are price sensitiveexcept in the -Buyers threat of backward integration: 5(high) Customers are will notconstruct a hotel or buy a place of residence for each place they visit. -Contribution to quality 2(low) Additional facilities suchas spas, gyms etc. are usedmy hotels to improve thequality of customers stay -contribution to cost 5(high) Brand image is veryimportant in this industry and leads to extra cost, Additional amenities,training of staff, locationrent (like close to airport)etc. -Buyers profitability 2(low) Low buyers profitability- In the mid -segment, there are numerous buyers, of very small profitability In the premium segment, buyers are very affluent, and they have greater bargaining power comparedto the mid-segment Industrys threat of forward integration. 4(medium) -low chances or forward integration This industry has many customers who are relatively very small in size. Loss of a single customer has little impact on a hotel company and this drives down the buyers bargaining power. Similarly buyers threat of backward integration is almost impossible and so the industry is under threat of forward integration. However the industry does have several substitutes such as camping and recreational vehicles for tourists, corporate guesthouses for business travelers and other informal means of accommodation with friends and family. Switching cost for all these options is very low, except for the RV. Apart from the provision of accommodation, hotels also provide additional facilities and services such as restaurants, gyms, spas, conference halls, ball rooms, lounges etc. Therefore their contribution to quality as well as cost for the buyer is very high. Barriers of entry Category Rating 1-10 Remarks Economies of scale 5(high) High economies of scale- Very important to operatea chain of hotels in multiplelocations, especially for the premium segment. This reduces thedependence on tourismtrends at any given location Product differentiation 4(medium) Highly differentiated- Brand names and valuesare very important in attracting and retaining customers -brand identity 4(medium) Brand is very important. -switching cost 2(low) -low switching cost -capital requirement 4(medium) -capital intensive. -staff, dà ©cor, infrastructure e.t.c is very expensive. -Access to technology 3(moderate) -ICT is very important for property management. -Access to raw material 4(medium) -Labor, land and other essentials are easy to obtain. -government protection 3(moderate) -The tourism industry receives government. -exit barriers 2(low) -High exit barriers. Specialized assets for the industry. Brand names are very important in the hotel industry. Companies use their strong brand names to attract new customers and retain old ones. Besides, economies of scale are also a huge factor in this industry. Profitability of hotel chains is drastically higher than individual operations. A new entrant cannot compete with established players in terms of quality, price and even services. If they cannot establish significant economies of scale.Being a capital intensive industry with a large amount of it tied down in fixed costs, makes entry more difficult. Similarly high exit barriers due to specializedassets make the industry less attractive.The hospitality industry is strongly influenced by travel and tourism trends. Government protection for the tourism industry is very high and this in turn rubs off on the hotel industry making it thereby making the industry attractive in general. Competitive power of rivalry players This aspect describes the intensity of competition between existing players (companies) in an industry. High competitive pressure results or leads to pressure on price margins and on profitability for every single company in the industry. The following table shows the analysis of the rivalry between hotels. factors Ratings (5) Remarks. No. of competitions 4 (high) Small number of large operators Industry growth 3 (medium) Annual growth rate of 15% Fixed cost 1 (low) Highly capital intensive differentiations 4 (high) Strong brand name commands a very high price premium. Switching cost 2 (moderate) Low cost switching to similar brands Openness to terms of sale 4 (high) Price, taxes etc. are known Excess capacity 2 (moderate) Only 70% rooms occupied Strategic stakes 2 (moderate) Although large hotel companies have diversified they still have a majority stake in the hotel industry. Summary Porter Five Forces Factor Current Future Rating(5) Key Rationale Rating Key Rationale Threat of New Entrants 4   reasonable     5 sensible   Competitive Rivalry 4   reasonable   5 sensible   Threat of Substitute Products 3 average   4 reasonable   Supplier Relative Buying Power 5   sensible   4 reasonable   Buyer Relative Buying Power 4        reasonable 4   reasonable Conclusion Porter five forces analysis was used effectively to determine the hotel industries in Malaysia based on treat of new entrants, competitive rivalry, and treat of substitute products, suppliers-relative buying power, and buyer-relative buying power. Hotel is a very flourishing industry in Malaysia with not so many substitutes so the treat of substitute products is very low. Rivalry between hotels is not very high because rivalry is based on classification (5-star hotels compete against other 5-star hotels). Finally in the future relative buying powers will decrease because there will be many new entrants.

Ernie Barnes: Research of the Football Artist Essay -- history

Ernie Barnes: Research of the Football Artist Ernie Barnes was and still is one of the most popular and well-respected black artists today. Born and raised in Durham, North Carolina, in 1938, during the time the south as segregated, Ernie Barnes was not expected to become a famous artist. However, as a young boy, Barnes would, â€Å"often [accompany] his mother to the home of the prominent attorney, Frank Fuller, Jr., where she worked as a [housekeeper]† (Artist Vitae, The Company of Art, 1999). Fuller was able to spark Barnes’ interest in art when he was only seven years old. Fuller told him about the various schools of art, his favorite painters, and the museums he visited (Barnes, 1995, p. 7). Fuller further introduced Barnes to the works of such artists as, Raphael, Michelangelo, and Correggio, which later influenced Barnes’ mannerist style of painting. As a young boy Barnes was â€Å"introverted and shy† (p. 8). He wasn’t able to fight like the other young boys his age, and quickly became a punching bag for bullies. The after school brawls became so severe that Barnes’ mother asked his principal to allow him to leave school fifteen minutes early everyday. After viewing the extent of Barnes’ bruises, the principal had no choice but to comply. On the other hand, once the other children learned that Barnes could draw they no longer laughed and made fun of him, â€Å"They just watched [him draw] in silent awe† (p. 8). When Barnes entered junior high school, he became interested in dating and knew that the only way he could get attention from the girls was to play junior varsity football. Therefore, he joined the team, and was dubbed too sensitive for the game, and later quit the team. However, when Barnes entered high school, he was put on a bodybuilding program, by the high schools weight lifting coach, Mr. Tucker, who showed a genuine interest in Barnes’ drawings. Through Mr. Tucker’s constant encouragement, Barnes was able to reinvent himself, graduating from high school with twenty-six football scholarships, as well as the respect of the community (Artist Vitae, 1999). Before Barnes went to college, at North Carolina College (now North Carolina Central University), he impregnated a young girl and was forced to marry her in order to save face, and his first child was born in 1957. Although Barnes’ marriage was not a successful one, he adored his newbor... ...ers football team. Completes â€Å"A Dream Unfolds†, commission for National Basketball Association commemorating their 50th anniversary. Private commissions (5). Receives Treasure of Los Angeles award, Central City Associatio  · 1998: â€Å"The Advocate† donated by Donna Arnold to North Carolina Central University School of Law. Begins paintings for traveling exhibition, Visual Poem of Human Experience. Private commissions (6).  · 1999: Private commissions (2). Continues to work on paintings for traveling exhibition, Visual Poems of Human Experience (The Company of Art, Chronology 1999). Bibliography Barnes, Ernie (1995). From Pads to Palette. Waco, Texas: WRS Publishing. Huyett, Pat. (2000). Mbembe: High Aspirations [Online]. Available: http://cctr.umkc.edu/~phuyett/mbembe.html [2001, March 19]. The Company of Art. (1999). Artist Vitae [Online]. Available: http://www.erniebarnes.com/bio.html [2001, March 19]. The Company of Art. (1999). Chronology [Online]. Available: http://www.erniebarnes.com/chronology.html [2001, March 19]. The October Gallery. (2000, May 19). About the Artist [Online]. Available: http://www.octobergallery.com/sbarnes.htm [2001, March 19].

Friendship in The Pact :: The Pact Relationships Essays

Friendship in The Pact Friendship is a huge part of everyone's life, whether they know it or not. In some way shape or form everyone needs relationships. In the book The Pact, friendship is huge. Three boys George, Sam, and Rameck become best friends and you could say that they save each others lives. Not physically but in a sense that without the pact they made there lives might not be where they are today. I can relate this book to a very good friend of mine that got caught up in a bad situation. I’ll use the name â€Å"Bob†. We live in the small town of Cape May, NJ and everyone knows everyone. Not always the best situation for people like Bob. He and I became friends before any of these awful things started to happen. Bob got caught up in the wrong crowd one summer and begandealing cocaine. At the time I was un- aware of this. I began to notice a change in him around the middle of the summer, I asked him if there was anything wrong or if I could do anything for him. Bob wouldn’t tell me what was going on, he said, â€Å"I don’t want to hurt you.† With this statement, â€Å"I don’t want to hurt you.† I immediately knew that something was seriously wrong. At the time I didn’t know what, but I was going to find out. So I started snooping around, and I did my own investigation. Some may say that it was me being nosy, but I know that I’m sure glad I was. It’s a good thing I did too. In my snooping around I found out that Bob was dealing cocaine. It was at this time that I realized Bob’s situation was real, and I would have to do something about it. I thought Bob simply needed to get away from where he knew everyone, and drugs were so easily accessible. Bob and I then sat down to talk. This was not easy for me to do and I’m sure it wasn’t easy for him to hear. I mean, picture someone you think of as a little sister sitting you down and saying, â€Å"Bob, I know all about you dealing cocaine.† It takes a lot to stand up to your friends, but it takes a lot more to just sit and watch their lives go down the drain.

Comparing Families of Fifty Years Ago with Families of Today :: Compare Contrast Comparison

The definitions of a family today and a family in the past are far from similar. The definitions may have some similarities but they have changed dramatically in many more ways. 50 years ago, families had rules that were stricter and families were closer in the sense of a relationship. Although some families today are more distant from each other and have fewer rules to maintain order, there are still some that maintain the same styles of the families 50 years ago. Families have changed a lot but still have some similarities depending on their home-life. Families today just do not seem to spend time with each other. The mother and father both have jobs and tend to not be home for the children after school therefore causing more independence among each member of the family. Children that have more independence make mistakes on their own without being warned about them. Independent children have no guidance and get out of hand because there is not a strong boss type figure around most of the time to help distinguish the difference between right and wrong. Eating dinner as a family is a major tradition that has been forgotten as the years have gone by and caused more separation among the family. A family 50 years ago that did not eat dinner together would be a strange one but today it seems to be of the norm. Once again, the job affects this aspect of the family as well. People are just too busy at work or too tired to even participate in dinner at home anymore. Either families just do not have time for each other anymore or they j ust are not together due to divorce. Divorce is another thing that was like â€Å"illegal† 50 years ago. Divorce is tragic for the children in the family today and causes the children to hate both or one of the parents. Divorce causes total chaos. This may result in the children to be neglected because single parents need to work to support themselves and their children. Thus, causing no guidance for a growing child. Losing touch with family ties have caused a lot of trouble and will continue to cause more. Families now and 50 years have similarities among the pile of differences. Comparing Families of Fifty Years Ago with Families of Today :: Compare Contrast Comparison The definitions of a family today and a family in the past are far from similar. The definitions may have some similarities but they have changed dramatically in many more ways. 50 years ago, families had rules that were stricter and families were closer in the sense of a relationship. Although some families today are more distant from each other and have fewer rules to maintain order, there are still some that maintain the same styles of the families 50 years ago. Families have changed a lot but still have some similarities depending on their home-life. Families today just do not seem to spend time with each other. The mother and father both have jobs and tend to not be home for the children after school therefore causing more independence among each member of the family. Children that have more independence make mistakes on their own without being warned about them. Independent children have no guidance and get out of hand because there is not a strong boss type figure around most of the time to help distinguish the difference between right and wrong. Eating dinner as a family is a major tradition that has been forgotten as the years have gone by and caused more separation among the family. A family 50 years ago that did not eat dinner together would be a strange one but today it seems to be of the norm. Once again, the job affects this aspect of the family as well. People are just too busy at work or too tired to even participate in dinner at home anymore. Either families just do not have time for each other anymore or they j ust are not together due to divorce. Divorce is another thing that was like â€Å"illegal† 50 years ago. Divorce is tragic for the children in the family today and causes the children to hate both or one of the parents. Divorce causes total chaos. This may result in the children to be neglected because single parents need to work to support themselves and their children. Thus, causing no guidance for a growing child. Losing touch with family ties have caused a lot of trouble and will continue to cause more. Families now and 50 years have similarities among the pile of differences.

Critical Review for a Research Article Based

INTRODUCTION English language is the main international English. It is also said that English is the language of progress and development. In this age of internet and globalization, the use of English has increase tremendously. Most of the non-speaking English countries take great importance in English education. China and Malaysia being one of them. Ministry of Education (MOE) in Malaysia has been trying for years to improve the standard of English language especially in communication and writing by implementing efforts such as research on methods that could be applicable in Malaysian schools.English language has been accorded for second language in Malaysia as stated on Article 152 and been given due attention for years. Teaching English can be declared as challenge in Malaysia because the subject has always been argued, doubted, changed for many times that the issues has never been completely resolved. Meanwhile, MOE keep trying to improvise the teaching and learning process in sc hools from early education to the highest level by employing suitable means and aids such as bringing in modern teaching methods from Western countries.Communicative teaching methods and grammar-translation has been discussed, implemented on English language teaching in China based on the article by Jin, Singh, and Li (2005). This paper will provide the critical review of it, and the relevance of the methods in Malaysian Schools. 2. 0 COMPARISON BETWEEN COMMUNICATIVE LANGUAGE TEACHING AND GRAMMAR-TRANSLATION Based on the article by Jin, Singh and Li (2005) the CLT and Grammar-Translation method might not be applicable to all teaching situation.While the final result of the study was in favour of CLT methods, the gaps between the results on test paper for two groups are low. Futhermore, the research done by Rao (2002) concluded that students claim that using Grammar-translation method will be more suitable for class session in China. Since teaching is deeply rooted in the local philo sophy, culture, and basic concepts of education, the students’ learning styles and habits in language acquisition must be considered. Although the grammar-translation method is out of favor, students accustomed to this method may still derive benefit from it. Feature Article  Country School  Allen CurnowFor example, Chinese students generally show great interest in language structures and linguistic details when they are learning a language. â€Å"We would like to know what happens, because if we understand the system, we can use English more effectively† (Harvey 1985). Therefore, in teaching English to Chinese students, appropriate grammar analysis is essential, especially for beginners. Limited utilization of translation from or to the target language is an indispensable part of teaching. Vocabulary work and pattern drills are also ways of familiarizing the student with sentence structures.This information helps learners acquire linguistic competence. The main features of GT are: 1. It is teacher centred and does not cater for the learner’s individual needs 2. The emphasis is on grammar learning through verb drills, the translation of written texts and the memorization of wordlists 3. The focus is on the product rather than the process of learning; 4. Language is viewed as a body of knowledge rather than an instrument for communicating and functioning effectively in the real world 5.Linguistic practice is confined to the memorization of words and rules 6. Instruction aims at the mastery of the written medium rather than oral communication 7. Accuracy rules over fluency 8. Correction is all-out and punitive 9. The L2-model adopted is elitist and so is the educational philosophy 10. Feedback on learner performance is not likely to be helpful as it is solely accuracy-based But instead of teaching grammar traditionally and drilling grammar patterns, teachers need to relate teaching grammar and pattern drills to meaning and use.In other words, language structure practice should be used in contexts that involve some basic principles of appropriateness. This is the exact area that the traditional ESL teaching has long overlooked—teaching English for a communicative purpose. Thus, English teaching should be partly communicatively oriented, so students can acquaint themselves with appropriate language usage. the main pedagogical principles advocated by CLT are: 1. It is pupil-centred rather than teacher-centred 2 The emphasis is on communication and effective interactional skills 3.The focus is on the process rather than the product of learning; 4. Language is viewed as a skill to learn rather than a body of language  to pass on to the pupil 5. Linguistic practice occurs through communicative activities 6. Instruction aims at the mastery of all of the four core language skills 7. Fluency rules over accuracy 8. Correction is selective and non-judgmental 9. The L2-model adopted is flexible and can deviate from the L2-standard Form 3. 0 RELEVANCE TO MALAYSIAN SCHOOLS. In Malaysia, the teaching of English language starts early, as early as in kindergarten between the ages of 4 to 5.Students were exposed to many kinds of teaching methods from the traditional approach such as gra mmar-translation and towards more modern one like CLT. It is common for teachers to support the one that is more effective and theoretically sound basis for teaching. After independence, the changes was made for education in Malaysia where instead of using English language in school, the medium of teaching and learning process changes to Malay. Thus, affecting the teaching methods in Malaysia where teachers will provide materials and lecturers to students and teacher-centred classroom were practiced.Malaysia introduced the communicative syllabus in 1970. Back then, the study of grammar was considered not ‘fashionable’ and out of date. Students were not taught how to build correct sentences in English. Too much emphasis was placed on spoken English. Role-play and how to respond to given situations took centre stage. CLT method is one of the famous methods used by teachers and claimed to be more effective rather than GT. Krashen and other SLA theorist stress that language learning comes about through learning language communication rather than through practicing.Johnson (1984) and Littlewood (1984) consider that the acquisition of communication competence in a language is an example of skill development. On the other hand, the draw backs from this method are the need of an authentic materials and interaction between learners with them using only the target language as means of communication. Comparing to schools in urban area, most of the schools in rural area lacks the means of these authentic materials and ready interaction from outside. Malaysian people used Malay, Chinese, and Indian to communicate especially in rural area where English has not been used at all.This in turn will affect the communication grasp of students in rural schools where the proficiency in English are low. Thus, in turn, students will refuse to join in the interaction by being silent and it will hinder the teaching and learning process as they cannot catch up to the class lesson. The students in rural school still depend on teacher translation as their lack of exposition to English language will lower their proficiency. They could not speak the language fluently and need to learn the language structures and understand it from basic and practice by their own.It is different for students enrol in urban area where they were pre-disposed to the languages already. Most of them start to learn English from child where their cultural environment and socio-economy were contrasting from students from rural schools. They might have help with their parents and family and the more modern school system in the city. Technologies were blooming in this area where the students’ proficiency is slightly higher. Because they might be aware of the language and have practice them, communicative approach can be adapted to their teaching and learning process.But even so, teachers claimed that practising CLT method in class will take too much time and works as it requi re complete involvement from all students because of its learner-centred style. Because of this, the lack of practice will somehow affect the learning of the grammar structure and glaring mistakes from students for writing will form as CLT method is divulging more into developing students’ communicative approach. Yes, their speaking skill will be better as will their listening skill but that will not promise them 100% correctness in spelling and grammatical uses.The solutions to this are that both students should try to adapt both methods in learning a second language in Malaysia. The students need to be taught on how to learn not only of the language but to practice the language as frequently as possible. The CLT method can help the students develop an insight into the language and prepare them into an environment where the language are the only one use to communicate. On the other hand, grammar-translation will help the students practice the language on their own where ther e is no exposition of the language around them.Teachers can provide materials for these students to facilitate or encourage them to learn it independently without being too dependent on teacher. CONCLUSION Various efforts, on both national and individual levels, have been poured into the strategies to improve students’ ability or command of English language. There are of course many methods in second language teaching which include the Grammar Translation Approach. This approach was historically used in teaching Greek and Latin and later modern languages.Experienced teachers said that if they did not engage the help of the mother tongue, the lesson would involve a lot more time and resulting in students that were indifferent and psychologically were not there. ESL teaching in Malaysia, with its traditional setting, is markedly different from that in the United States and Great Britain in that it is conducted in different social and cultural contexts. Yet this does not mean th at the communicative approach is not applicable in such a context. By practicing CLT alone, it might be hard for teachers to attract students to participate and thus wasting time in trying to facilitate them.So, as to make this approach work well in here, we must reconcile it with the traditional grammar-translation method that is still popularly used in Malaysia. REFERENCES 1- Lingjie Jin, Michael Singh, Liqun Li; Communicative Language Teaching In China: Misconceptions, Applications And Perceptions. (2005) Australian Association For Research In Education. 2- Nor Hashimah Jalaluddin, Norsimah Mat Awal, Kesumawati Abu Bakar; The Mastery Of English Language Among Lower Secondary School Students In Malaysia: A Linguistic Analysis, European Journal Of Social Sciences – volume 7, number 2, 2008. – Mohd. Faisal Hanapiah (1993); English Language And The Language Of Development: A Malaysian Perspective. Department Of Modern Language, Jurnal Kemanusian. Page 106-120. 4- Rao Zh enhui; Modern Vs. Traditional, Bureau Of Educational And Cultural Affairs, Office Of English Language Programs. Taken on Oct 2012 from http://eca. state. gov. 5- Hyacinth Gaudart; English Language Teaching In Malaysia: A Historical Account, The English Teacher  Vol Xvi December 1987. 6- Kesumawati Abu Bakar, Nor Zakiah Abdul Hamid, Dr. Norsimah Mat Awal, Assoc.Prof. Dr. Nor Hashimah Jalaluddin; First Language Influence On Second Language Performance: A Study Of Common English Grammatical Errors Among Rural Secondary School Students. Taken on oct 2012 from http://repo. uum. edu. my. 7- Prof Puan Sri Dr Rohaty; (June 28, 2009) Teaching English by Using Bahasa Malaysia, Taken On Oct 2012 from http://rohaty-education. blogspot. com. 8- Dr Gianfranco Conti,(2011) Grammar Translation And Communicative Language Teaching Compared, taken on oct 2012 from http://languageteachingbyconti. blogspot. com

Titration Journal

E r J. Biochem. 40,177-185 (1973) u. Intracellular Titration of Cyclic AMP Bound to Receptor Proteins and Correlation with Cyclic-AMP Levels in the Surviving Rat Diaphragm Lien DO KHAC,Simone HARBON Hubert J. CLAUSER and lnstitut de Biochimie, Universit6 de Paris-Sud, Orsay (Received April 9/July 17, 1973) Extracts prepared from rat diaphragms incubated with or without theophylline and/or epinephrine have been tested for their total cyclic AMP content and for their ability to bind exogenously added cyclic [â€Å"]AMP.Less cyclic [3H]AMP can be bound inthe extracts after theophylline and/or epinephrine treatment indicating that the rise in cyclic AMP level was accompanied by a n increase in the quantity of cyclic AMP bound intracellularly to the cyclic AMP-dependent protein kinases. Maximum cyclic AMP binding capacities, as measured by total cyclic AMP exchanges, were however identical in all cases. Accurate estimations of intracellular binding of cyclic AMP have been correlated with the level of cyclic AMP in the tissue : the reaction seems to obey simple saturation kinetics, a n apparent intracellular K d for cyclic AMP has been evaluated as 330 nM.The findings are consistent either with a real difference in the intracellular binding constant as compared to that measured in vitro (28 nM) or with the fact that the cyclic nucleotide in the cell may not all be available for the kinase protein receptors. They also suggest that the method described may prove useful for studying any possible intracellular control beyond the step of cyclic AMP synthesis.Regulation of cellular metabolism by adenosine 3†² :5†²-monophosphate (cyclic AMP) [I], its mediation through complex protein kinases [2,3] and the mechanism of the activation of these enzymes [4–61 have been well documented within the past years in the eukaryotic cell. Activation has been demonstrated to occur according to Equation (1) through a n interaction of cyclic AMP with the regulatory subuni t (R) of the enzyme, leading to a dissociation of this subunit from the catalytic subunit (C) which is thus activated. RC cyclic AMP + R cyclic AMP C . (1) + +However completely satisfactory correlations between the levels of intracellular cyclic AMP and its ultimate metabolic effects have been in many cases difficult to obtain. Striking examples for this situation are to be found in the results of Craig et al. [7] in rat diaphragm, of Stull and Mayer [8] in rabbit skeletal muscle concerning the regulation of phosphorylase activation, of Schaeffer et al. [9] and Miller et al. [lo] concerning regulation of glycogen metabolism in adrenalectomized rats, and of Harbon and Clauser [Ill This work is dedicated to Professor E. Lederer for his 65 th anniversary. Abbreviations.Cyclic AMP; adenosine 3†²: 5†²-monophosphate. in the rat uterus stimulated by prostaglandin El or E,. I n all these cases, cyclic AMP levels may be elevated without eliciting the expected metabolic responses. Two hypotheses have been formulated to explain these obvious discrepancies, either a decrease in the activation of the enzymes mediating cyclic AMP action within the cell, or a compartmentalization of the intracellular nucleotide. Hence it seems necessary to measure directly the degree to which the first step of the activation sequence (Equation 1)reflects the apparent intracellular cyclic AMP concentrations.This might be achieved by establishing in intact cells or tissues, correlations between the levels of intracellular cyclic AMP under welldefined physiological conditions, the extent to which it is bound to the specific receptor protein and the extent to which the complex protein kinases are in the active state. Satisfactory correlations between cyclic AMP levels and protein kinase activation have been recently established in various tissues by Corbin et al. [I21 and Soderling et al. [13].The present work was to investigate if correlations could also be obtained between intracell ular cyclic AMP levels and the amounts of intracellular cyclic AMP bound to receptor protein (R cyclic AMP) in the surviving rat diaphragm incubated with or without theophylline and epinephrine. The results reported demonstrate that – E r J. Biochem. 40 (1973) u. 178 Intracellular Titration of Cyclic AMP-Receptor Protein Binding precise titrations of endogenous cyclic AMP bound versus cyclic AMP present in the intact tissue may be obtained.An apparent Kd value for the intracehlar cyclic AMP binding is observed which differs widely from the K d of the same binding established in vitro [14-161. This method may prove to be useful for studying the modification of cyclic AMP binding under conditions where the formation and breakdown of cyclic AMP does not seem to be affected. A preliminary report of these results has been presented [17]. MATERIALS AND METHODS Cylic AMP was obtained from P L Biochemicals Inc. , theophylline and Tris from Merck (Darmstadt), Na,ATP 4 H,O, L-epinephri ne bitartrate from Calbiochem.Cellulose ester membrane filters (HA 0. 45 pm, 24 mm) were purchased from Millipore Corp. All reagents used were products of Prolabo (reagent grade). Cyclic [3H]AMP was a product of New England Nuclear Inc. , specific activity 24 Ci/ mmol. Animals were Wistar rats weighing about 200 to 300 g and fasted 24 h before the experiments. Tissue homogenizations were performed with an Ultra Turrax homogenizer. – The reaction mixture for the binding assay contained in a final volume of 250 p1, 20 mM TrisHC1 buffer pH 7. 5, 10 mM MgCI,, 6. 7 mM theophylline and cyclic [3H]AMP a t various concentrations as indicated.The reaction was initiated by the addition of a n aliquot of diaphragm extracts equivalent to 70- 150 pg protein. Method B. I n this case, cyclic [3H]AMPwas added to the homogenizing medium a t saturating concentrations up to 0. 2 p M a t 0 â€Å"C, centrifugation was carried out immediately and cyclic [3H]AMP bound measured directly on the extr act. Cyclic [3H]AMP bound to the proteins, under either condition, was determined after different incubation times at 0 â€Å"C: the reaction mixtures were then diluted to 3 m l with cold buffer (20mM TrisHC1, 10mM MgCl,, pH 7. 5) and passed through cellulose acetate Millipore filters (0. 45 pm).The filters were washed with 25ml of the same buffer, dried and counted in i 0 ml scintillation fluid, in a Packard Tri-Carb liquid scintillation spectrometer. Results were expressed as pmol cyclic AMP bound/mg protein ; the concentration of endogenous unlabelled cyclic AMP has been always taken into account for the estimation of the specific activity of cyclic [3H]AMP present in the incubation medium. Incubation Procedures The animals were killed by decapitation. The diaphragms were rapidly removed, freed from connective tissue, cut to small pieces, pooled and divided into equal parts. 200-250 mg tissue were preincubated in 2. ml Krebs-Ringer-bicarbonate buffer pH 7. 4, gas phase (95O/, O, , 5O//, CO,) for 30 min a t 37 â€Å"C, in the absence or presence of 10 mM theophylline. Incubations were then performed in the absence or presence of epinephrine (5 pM) for varying periods of time. Extraction of the Tissue Standard Binding Assays for Cyclic A M P Two methods have been deviced to extract the tissue and estimate the binding of exogenous cyclic [3H]AMP to the extracted proteins, both slightly modified from the method defined by Walton and Garren [15]. Method A . The tissue was homogenized a t 0 â€Å"C in 3 ml of one of the following solutions: 20 mM TrisHCl buffer pH 7. or 20 mM sodium acetate pH 7. 5 or 4 mM EDTA pH 6. 0. Theophylline (10 mM) was always present in the various homogenizing media in order to minimize any degradation of cyclio AMP by phosphodiesterase present in diaphragm extracts. A first centrifugation was carried out for 5 min a t 3000 x g , followed by a second one a t 50000 x g for 30min. The supernatants will be referred to as Tris extract, ac etate extract and EDTA extract. Assay for Cyclic-AMP Levels For cyclic AMP assay, the tissue was homogenized in 3 ml cold 7 trichloroacetic acid and centrifuged for 30 min a t 50000 xg.After addition of 0. 1 ml N HC1, the supernatants were extracted 7-8 times with twice their volume of cold ether and evaporated to dryness. Total levels of cyclic AMP in the tissue trichloroacetic acid extract were determined according to Gilman using a protein b a s e and the heatstable inhibitor prepared from rabbit skeletal muscle [161. I n some instances, cyclic AMP content was also evaluated in the Tris and acetate extracts. Proteins were precipitated by trichloroacetic acid and extracts processed as described above. Proteins in the extracts were determined according to Lowry et al. 18] using bovine serum albumin as a standard. RESULTS AXD DISCUSSION Total Cyclic-AMP Levels in Rat Diaphragm. Effects of Epinephrine and Theophylline In order to study the cyclic AMP binding capacity of rat diaphragm proteins and its possible rnodification under the influence of epinephrine, it seemed necessary to test the first effect of the catecholamine, viz. the rise in the tissue cyclic AMP level under our experimental conditions. Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 1. Total cyclic A H P levels in trichloroacetic acid extracts of rat diaphragm.Effect of epinephrine and theophylline R a t diaphragms (200-250 mg) were preincubated for 30 rnin a t 37 â€Å"C in 2. 5 ml Krebs-Ringer-bicarbonate buffer (0, 95 °/0-C0, 50/0) in the absence or presence of 10mM theophylline, Incubation was then performed for 5 rnin with or without 5 pM epinephrine. The tissue was then homogenized in 7O/, trichloroacetic acid for cyclic AMP assay as described under Methods. Levels of cyclic AMP were expressed as pmol cyclic AMP/100mg wet tissue and as pmol cyclic AMP/mg soluble protein (as estimated by the Lowry procedure in the Tris extract.Values are means f S. E. M. of 5 di fferent experiments Incubation condit,ions Total cyclic AMP TheoDhvlline EDineDhrine pmo1/100 mg pmol/mg wet tissue soluble protein 41 f 8. 0 20. 5 f 4. 7 104 & 1. 1 52 & 0. 47 93 f 4. 5 46 & 2 350 f 21 170 f 10. 7 179 Table 3. Distribution of cyclic [3H]AMP-bindingfractions i n different hom. ogenutes from rat diaplwagms incubated with or without epinephrine Preincubation and incubation conditions as described in Table 2. Tissues were homogenized in 3 ml 20mM TrisHCI, p H 7. 5, 4 mM EDTA or 20 mM sodium acetate pH 7. and centrifuged for 5 rnin at 3000 x g, the supernatants were centrifuged once more at 500OOxg for 30 min yielding extract 1 and pellet 1. The sediment of the first centrifugation was resuspended in 1. 5 ml of the corresponding buffer and centrifuged at 500OOxg for 30 min giving extract 2 and pellet 2. Binding activity for cyclic rSH]AMP was measured in each fraction as described in the text under method A and was expressed as pmol cyclic AMP bound/l00 mg wet tissue Fr action Cyclic AMP bound in EDTA Acetate Tris extract extract, extract, 5 yM noepinoepino epinephrine nephrine nephrine – + + + + – :Lhrine pmo1/100 mg wet tissue Extract 1 Extract 2 Pellet 1 Pellet 2 15. 70 1. 47 0. 76 1. 49 14. 90 1. 54 0. 83 1. 50 15. 30 1. 35 0. 80 1. 10 9. 40 0. 80 0. 44 0. 39 Table 2. Cyclic A M P levels in different extracts obtained from epinephrine-treated and untreated rat diaphragms Preincubation with 10 mM theophylline and incubation conditions in the absence or presence of 5 pM epinephrine as in Table 1. Diaphragms were homogenized in three different solutions: cold 7O/, trichloroacetic acid, Tris-HC1 pH 7. 5 or acetate p H 7. 5 as described under methods.Centrifugation was carried out for 30 rnin at 50000 x g. Soluble Tris extract, acetate extract and their corresponding sediments were deproteinized by 7 o/o trichloroacetic acid before cyclic AMP assay Incubation with epinephrine None 5wM Total cyclic AMP in Trichloroacetic 20 mM acetate a cid extract pellet 57 280 – 20 mM Tris extract pellet 48 218 9. 5 26 extract pellet 45 242 pmo1/100 mg wet tissue – 8. 5 8. 3 As shown in Table 1, epinephrine (5 pM) in the absence of theophylline increases (by a factor of 2. 5) the total cyclic AMP content of rat diaphragm extracted by trichloroacetic acid.Theophylline alone (10 mM) had a stimulating effect, double; when both compounds were used together, the rise in cyclic AMP levels was 8- t o 9-fold, reaching 350pmol cyclic AMP/100 mg wet tissue. When cyclic AMP was assayed in either acetate or Tris extracts after deproteinization with trichloroacetic acid the values obtained were identical t o those found when the diaphragms were directly extracted with trichloroacetic acid ; hence almost none of the cyclic nucleotide in these extracts was associatcd with membrane-bound fractions (Table 2). Eur. J. Biochem. 0 (1973) Location of Cyclic AMP-Binding Fractions Table 3 shows the distribution of cyclic AMP binding activ ity in various fractions of three rat diaphragm homogenates measured by method A : in all cases more than goo/, of this activity was recovered in the 50000 x g supernatant, almost no cyclic AMP binding occurred in the pellets. Preincubation of the diaphragm with epinephrine did not modify the percentage distribution of the radioactive nucleotide between the supernatants and the pellets, hence subsequent experiments have been performed on the soluble extracts.On the other hand, in the case of epinephrine-treated diaphragms, less exogenous labelled cyclic AMP (about 50-60 °/0) was bound to the various fractions, indicating a decrease in the binding capacity of the extract as compared to the untreated diaphragm. Dilution by endogenous cyclic AMP cannot explain the effect of epinephrine, since allowance was made for this parameter (see Methods) ; the phenomenon was consistently reproducible and will be further substantiated and discussed below.The binding capacities of the various ext racts for cyclic E3H]AMP have also been verified in the absence of any free endogenous cyclic AMP after removal of the latter by filtration through Sephadex G 50 (1x 37 cm) columns, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7. 5 a t 4 â€Å"C. I n these experiments, the detail of which w l not be reported in i l the present manuscript, the effect of epinephrine was still observed, when binding was measured on the main protein peak emerging with the void volume of the columns. When the corrections outlined in the 180 Intracellular Titration of Cyclic AMP-Receptor Protein Binding Z A 0. 51 / 0 20 40 60 Time ( m i n ) l / f r e e cyclic AMP (nM-‘) l / f r e e cyclic A M P (nM-‘) Fig. 1. The time wurse and cyclic-AMP-concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method A ) . (A) Diaphragms were incubated for 30 min in the presence of 10 mM theophylline and extracted with Tris HCI buffer (method A). Cyclic AMP binding was estimated in the presence of various concentrations of cyclic E3H]AMP: 20nM ( 0 – 0 ) ; 60nM ( – ) 0 0 ; SO& (A-A); 100 nM ( –) #-. , a t 0 â€Å"C. The react,ion mixtures contained in a final volume of 2. 5 ml, 20 mM Tris-HC1 buffer, pH 7. , 10 mM MgCI,, 6. 5 mM theophylline. The reaction was initiated by the addition of 930 pg protein. At the indicated times, aliquots were pipetted, immediately diluted with cold 30 mM Tris-HC1buffer pH 7. 5,lO mM MgCl, and passed on the Millipore filters. Filters were washed with the same buffer, dried and counted. Binding activity is expressed as pmol cyclic AMP bound/mg protein. (B) Data obtained from similar experiments where binding for cyclic AMP was performed a t 0 â€Å"C, for 1 h, in the presence of cyclic [aHIAMP ranging from 12 nM to 110 &I. Double-reciprocal plot, according to Klotz [25] Fig. 2.Cyclic-AMP-Concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method B ) . Binding assays were carried out as described under method B. Various concentrations of cyclic [3H]AMP ranging from 12nM to 200 nM were added directly to the homogenizing medium for preparing extracts from epinephrine treated (A-A) and untreated (0-0) rat diaphragms. Aliquot,s of the extracts were filtered through Millipore filters, dried and counted. Double-reciprocal plot, according to Klotz [25] present paper were applied to these figures, the results were essentially identical to those obtained with the unfiltered extracts.Specificity. Kinetics and Concentration Dependence of Exogenous Cyclic-AMP Binding in the Extracts Specificity of cyclic AMP binding has been assessed by dilution experiments of cyclic [3H]AMP (100 nM) with unlabelled nucleotides (adenine, AMP, ATP, cyclic AMP) a t molar concentrations equalling up t o 100 times cyclic [3H]AMP concentrations. I n no case, except with unlabelled cyclic AMP, the amount of radioactive material bound to proteins by either method A or B was significantly reduced (the details of these experiments are not reported).When various concentrations of cyclic [3H]AMP were added to diaphragm extracts (after homogenization and centrifugation) and the binding reaction (method A) carried out for different incubation times at 0 â€Å"C (Fig. I), it appears that saturation was obtained at a concentration of 80 nM for the cyclic nucleotide which essentially coincides with previously published data [14-161 and that binding equilibrium was reached a t p H 7. 5 and 0 â€Å"C after less than 60 min incubation. It has also been verified that with the protein concentration used (70-150 pg in 250 pl) binding of cyclic AMP was directly proportional to the amount of added proteins.From a reciprocal plot of cyclic AMP binding versus cyclic AMP concentration (inset of Fig. I), an apparent Kd of 33 nM can be calculated. When similar experiments were performed by adding various concentrations of cyclic [3H]AMP into the homogenizing medium (method B) and using diaphra gms which have been incubated in the presence and absence of epinephrine, the double-reciprocal plots of Fig. 2 were obtained. The apparent Kd values calculated with this method (45 nM) are in the same range as with method A.I n addition this figure shows that epinephrine treatment of the diaphragms does not modify this Kd but decreases the amount of exogenous cyclic AMP which can be bound to the extract proteins. By comparing exogenous cyclic AMP binding values obtained with methods A and B, it appears (Table 4) that when cyclic [3H]AMPwas added to the Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 4. Comparison of exogenous binding of cyclic [SII]AMP to diaphragm extracts by method A or method B. Rat diaphragms were incubated with theophylline in the absence or presancc of 5 p M epinephrine.Extracts in Tris-HC1 were prepared as described under method A for subsequent binding of cyclic [3H]AMP (100 nM), 1 h, a t 0 â€Å"C. A second series of extracts wer e prepared in the same way but in the prescnce of 100 nM cyclic [3H]ABIP in the homogenizing medium (method R); binding of cyclic [3H]AMP was measured in a n aliquot immediately after centrifugation at 0 â€Å"C (about 1 h after the end of incubation). Values are expressed as pmol bound cyclic AMP/mg protein. Numerals within brackets indicate number of experiments Method Cvclic A P bound with M 5 pM epinephrine no epinephrine pmol/mg protein 4 f 0. 22 (9) 4. 80 5 0. 2 (5) 181 6 t e . ;? 4 Q Q E A B 2 f 0. 13 (9) 3 f 0. 19 (5) 0 I I I 30 60 90 * Time (rnin) homogenization medium (extract B) higher binding values were obtained both with epinephrine-treated and untreated diaphragms, than with method A. This demonstrates that some additional binding of endogenous cyclic AMP occurred during the homogenization and fractionation procedures, which tends to decrease the amount of unoccupied binding sites available for exogenous cyclic [3H]AMP. Hence method B has been currently used to measu re exogenous cyclic AMP binding, since the values obtained with this method seem to reflect intracellular conditions more accurately.Fig. 3. Time course of cyclic [3H]AMP binding in extracts from rat diaphragms incubated in the absence or presence of theophylline orland epinephrine. Half rat diaphragms were preincubated in the absence (m, A ) or in the presence ( 0 , 0 ) of 10 m31 theophylline for 30 min at 37 â€Å"C. Epinephrine (5 pM) was added ( A , 0 )and incubation continued for 5min. Tissue was homogenized in 1. 5 ml Tris-HC1 buffer containing 200 nnf cyclic [3H]AMP and centrifuged at 5000xg for 10 min at 0 â€Å"C.Binding of cyclic [3H]AMP was measured in aliquots of the supernatant at the times indicated, through Millipore filtration, t = 0 corresponds to the onset of the extraction. Results are expressed as pmol cyclic AMP bound/ mg protein (without correction for cyclic AMP exchange) Effect of Theophylline and Epinephrine Treatment on the Binding of Exogenous Cyclic [3H ]AMP by Diaphragm Extracts Fig. 3 shows the results of a typical experiment in which diaphragms have been incubated in the absence or presence of theophylline and epinephrine. Homogenization has been performed according to method B, the centrifugation time of the homogenate kept to a inimum (10 min), and the binding capacity for cyclic [3H]AMP determined a t different times. As may have been expected, this cyclic [3H]AMP binding (which measures the residual binding capacities of the extracts) was, in the course of the whole titration period, inversely related t o the amount of endogenous cyclic AMP present in the relevant extracts (see Table 1). Hence the agents which increase the intracellular cyclic AMP level appear to decrease the amount of binding sites available for exogenous cyclic [3H]AMP, probably through an increase of endogenous cyclic AMP binding to the receptors.I n order to titrate endogenous binding of cyclic AMP accurately, experiments were designed to estiEm. J. Bioc hem. 40 (1973) mate the total binding capacities of the extracts through complete exchange of endogenously bound cyclic AMP with cyclic [3H]AMP, and also to estimate the actual amount of exchange occurring in the extracts between endogenous bound unlabelled cyclic AMP and exogenous cyclic [3H]AMP during the titration period. A precise knowledge of these two parameters is required for the determination of the binding sites occupied by endogenous cyclic AMP at the moment where the tissues are homogenized.Cyclic-AM P Exchange and Determination of Maximal Binding Capacities Total cyclic AMP exchange has been measured under the conditions defined by Wilchek et al. [19] for parotid gland and skeletal muscle : extracts from both treated and untreated diaphragms were f i s t incubated at 0 â€Å"C with cyclic [3H]AMP (100 nM) under binding conditions of method A and then allowed t o exchange with 1 pM unlabelled cyclic AMP at 20 â€Å"C in the presence of 100p. M ATP and 10mM MgCl,. Fig. 4 shows that almost complete exchange of the bound labelled nucleotide occurred within 30 min, 182Intracellular Titration of Cyclic AMP-Receptor Protein Binding 0 10 20 30 40 50 60 Time (min) 70 80 90 Fig. 4. Exchange of bound cyclic [SHIAMP. Extracts were prepared from epinephrine-treated ( + o ) and untreated (0-0) rat diaphragms. Binding of cyclic [3H]AMP was carried out a t 0 â€Å"C in a volume of 2. 5 ml with 500 pg proteins, and 100 nM cyclic r3H]AMP in Tris-HC1 buffer, MgCl, and theophylline a t the concentrations described for the standard binding assay. After 1-h incubation, 1 pM unlabelled cyclic AMP and 100 pM ATP were added and the mixture allowed to stand at 20 °C.At the different times indicated in the figure, aliquots corresponding t o 50 pg protein were pipetted, rapidly diluted with 20 mM Tris-HC1 buffer, 2. 5 mM MgC1, p H 7 5 and filtered through Millipore filters. The filters . were washed with the same buffer, dried and counted. Results are expressed as pmol/ mg protein 0 30 60 90 120 Time (rnin) 180 240 – Total binding capacities of the proteins could thus be measured by incubating the extracts first with 100 nM unlabelled cyclic AMP a t 0 â€Å"C and carrying on the exchange reaction in the presence of 1 pM cyclic I13H]AMP at 20 â€Å"C for 1-2 h ; the values obtained averaged 8. -9. 5 pmol cyclic [3H]AMP/mg soluble protein, both with epinephrine-treated and untreated diaphragms. These results were confirmed by direct assay of bound cyclic AMP: the extracts have been fully saturated with unlabelled 1pM cyclic AMP and filtered as described. After washing the Millipore filters, bound cyclic AMP was extracted by cold 7 O/, trichloroacetic acid and the cyclic nucleotide was directly assayed according to Gilman [16]. The average value was 9. 8 f 0. 4 pmol cyclic AMP bound per mg protein, which is of the same order of magnitude as the amount of bound cyclic [3H]AMP calculated above.Previously published data are in close agreement wi th these values. Walton and GarFen [15] reported maximal binding capacities of 9. 8 pmol/mg protein for adrenal extracts, whereas Gilman [l6] found a total binding of 12pmol/mg protein in muscle extracts. The values for maximal cyclic AMP binding are very low as compared t o the total endogenous cyclic AMP present in the extract (46 pmol/mg protein with the theophylline-treated diaphragm and 170 pmol/mg protein with the epinephrine theophylline-treated diaphragm).It must be added that the binding proteins, saturated with cyclic AMP or not, were almost completely retained on the Millipore filters, and that endogenous cyclic AMP, not Fig. 5. T i m e course of cyclic A M P exchange under binding (0 â€Å"C) and exchange (20 â€Å"C} conditions. Extracts were prepared from epinephrine treated (0,A ) and untreated ( 0 , A) r a t diaphragms. Binding of cyclic AMP was performed as described in Fig. 2 in the presence of 100 nM cyclic AMP for 60 min at 0 â€Å"C. A t the end of the bindin g reaction 1 pM cyclic [3H]AMP was added t. the different extracts, in the absence (A, A ) or presence ( 0 , 0 ) of l00p. M ATP. The reaction mixtures were maintained a t 0 â€Å"C for 2 h and then at 20 â€Å"C (arrow) for 2 more hours. At the different times indicated on the figure, aliquots corresponding t o 70 pg protein were pipetted and treated as in Fig. 4. Results are expressed as cyclic rH]AMP bound in pmol/mg protein. bound to these fractions, was quantitatively recovered in the Millipore filtrates after trichloroacetic acid extraction. The extent t o which this â€Å"free† cyclic AMP may or not be bound to other proteins is presently not known.Cyclic-AMP Exchange under Binding Conditions The extent of cyclic AMP exchange under binding conditions (0 â€Å"C, 1 h, 100 nM cyclic AMP) must be controlled if corrections for simultaneous exchange have to be applied t o binding data: extracts of rat diaphragms treated with theophylline and theophylline epinephrine were first saturated with 1 O O n M unlabelled cyclic AMP (binding conditions) and then exchanged with 1 pM cyclic [3H]AMP but a t 0 â€Å"C. After 2 h, the temperature was raised to 20 â€Å"C and completion ofthe exchange measured after 1-2 h further incubation.Fig. 5 shows that a t 0 â€Å"C, within 1h incubation time, which are the conditions described above for the binding assay, about 200/, of total sites were exchangeable. Under these conditions, ATP and Mg ions slightly increase the exchange velocity. I n addition, this figure confirms that a t 20 â€Å"C total exchange capacities were identical for epinephrine-treated and untreated diaphragms ; hence initial + + Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser 183 Table 5. Relationship between intracellular cyclic A M P levels and cyclic AM P binding in extracts from diaphragm incubated under various conditions Diaphragms were incubated with or without 10 mM theophylline for 30 min at 37 â€Å"C, 5 pM epin ephrine was added where indicated and incubation continued for varying times. From each incubation, half a diaphragm was extracted by trichloroacetic acid for cyclic AMP estimation. The other half was homogenized with Tris-HC1buffer lOOnM cyclic [3H]AMP(method B) for exogenous cyclic AMP binding after 1 h a t 0 â€Å"C; maximal binding capacities were determined in the same extracts a t 20 â€Å"C in the presence of 1 pM cyclic [3H]AMP under conditions described for cyclic A P exchange.R. esults are expressed as pmol cyclic AMP/mg M protein. Endogenous binding values were calculated as the difference between maximal binding capacities ( A )and exogenousbinding ( B ) and corrected for the 200/, exchange + Incubation conditions Theophylline 10 mM Epinephrine 5t*M Time Cyclic AMP Total level Maximal binding Exogenous capacity binding (a) (b) Endogenous binding (a-b) corrected min pmol/mg protein – – – + + + + + + 0 2 10 30 5 5 20. 5 52 43 38 46 170 f 4. 7 & 0. 47 f2 f 10. 7 9. 6 f 0. 9 9. 4 f 0. 1 9. 20 9. 40 8. 9 5 0. 73 8. 9 & 0. 85 5. 35 f0. 40 4. 50 f 0. 133 4. 40 4. 70 4. 46 f 0. 20 2. 7 f0. 224 5. 31 6. 13 6 5. 5 5. 53 7. 77 differences in residual binding capacities reflect variations in the degree of saturation of the receptor proteins by endogenous cyclic AMP, rather than modifications of their maximal binding capacity. 1 Titration o Endogenous Cyclic-AMP Binding in Rat f Diaphragm. Effects of Theophylline and Epinephrine Since total binding capacities of the receptor proteins in the extracts and the amount of exogenous cyclic [3H]AMP bound by these extracts after homogenization may be estimated, it appears possible to calculate endogenous cyclic AMP bound in the intact organs, correcting for a 2001, exchange during the titration period.Table 5 summarizes the results of a series of experiments where diaphragms have been incubated under conditions which modify endogenous levels of cyclic AMP :in every case, half of the diaphragm was extracted with cold trichloroacetic acid (see Methods) for the assay of intracellular cyclic AMP levels: the second half was extracted according to method B for the estimation of exogenous cyclic [3H]AMP binding and of total cyclic AMP binding capacities. The endogenous cyclic AMP bound was calculated from the latter experimental data.This table definitely establishes that the average values obtained for the intracellular binding of endogenous cyclic AMP in the intact organ seem to correlate with its cyclic AMP levels. A reciprocal plot of intracellular binding versus intracellular cyclic AMP concentrations (Fig. 6) shows that this correlation fits simple saturation kinetics very accurately. I n the unstimulated diaphragm (no theophylline nor epinephrine added to the incubation medium) about 50 °/, of the available binding sites are occupied by endogenous cyclic AMP; this Eur. J. Biochem. 40 (1973) -0. 002 I 0. 002 l/Free cyclic AMP (nM-‘) 0 0. 004 . Fig. 6. Reciprocal plot of intracellular cyclic A M P levels and cyclic A M P binding in rat-diaphragm extracts. Data arc obtained from experiments performed as described in Table 5 and replotted according t o the Klotz equation. The intercept on the y axis yields a n estimate of the number of binding sites and the x intercept provides a n estimation of the intracellular apparent dissociation constant. Statistical analysis of the data were performed according to Cleland [26] using a Wang electronic calculator alue increases to almost goo/,, when the diaphragms have been fully stimulated with both theophylline and epinephrine. Various treatments with one of the agonists alone cause endogenous bindings ranging between these two extreme values. The apparent Kd value for intracellular binding according to this plot was estimated to 330 nM f 50, as compared to the apparent Kd (33-45 nM) when binding was assayed in the extracts (Fig. l and 2). Hence a difference of about one order of magnitude appears to obtain between the Kd values calculated within the cell and the 84 Intracellular Titration of Cyclic AMP-Receptor Protein Binding same constant measured with diaphragm homogenates. The double-reciprocal plot may also be used to calculate the intracellular maximal binding capacities, from its intercept with the ordinate axis. A value of 8. 9 pmol/mg protein was found which coincides with the values measured in the extracts by total cyclic [3H]AMP exchange. This discrepancy between the intracellular Kd and the Kd measured in vitro in a variety of tissue extracts including diaphragm may a t first sight seem surprising.It has however repeatedly been pointed out that cyclic AMP concentration even in the unstimulated cell was far in excess of the concentration which should result in almost maximal stimulation of protein kinases and compartmentalization of the nucleotide within the cell has usually been postulated to explain this contradiction [8,9,20]. The present work shows that despite these high intracellular concentrations of cyclic AMP, protein kinases could indeed not be fully activated, since under the same conditions, the receptor proteins appear not to be fully saturated with cyclic AMP. Concluding RemarksAs might have been expected from Equation (1) (if this reaction truly reflects intracellular conditions) a rise in cyclic AMP should be paralleled by an increase in the amount of cyclic AMP bound to receptor protein in the cell. The results reported show this indeed to be the case in the isolated rat diaphragm: when this tissue is stimulated by various agents which increase the level of cyclic AMP the amount of protein receptors endogenously saturated by cyclic AMP (R cyclic AMP) rises, as indicated in our experiments by a decrease in their ability to bind exogenously added cyclic [3H]AMP after tissue extraction.Maximal binding capacities for cyclic AMP do not seem to be affected under any circumstance. A parallel approach t o the study of this problem has been undertaken by Corbin et al. [12] and Soderling et al. [13] who investigated in adipose tissue under various stimulatory conditions, the state of activation of the catalytic subunit (C) by assaying the cyclic AMP dependence of the protein kinase in tissues extracts. These authors demonstrated that under well-defined xperimental conditions, there was a quantitative relationship between the intracellular level of cyclic AMP and the amount of the active C unit which could be separated from the complex protein kinase RC. However in their experiments high concentrations of NaCl had to be added to the extracts, since in its absence R and C tended to reassociate almost immediately, indicating that cyclic AMP is no longer bound to its receptor protein (R). The situation in various other tissue xtracts has been found to be analogous, except with skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. T his seems also to be the case for the diaphragm, since under the conditions of the present work, it has been possible to titrate for R * cyclic AMP in the crude extracts even in the absence of high salt concentrations : acccurate estimations of intracelM a r binding of cyclic AMP have been obtained and correlated with the absolute amounts of the nucleotide present in the stimulated and unstimulated cell.The binding seems t o obey simple saturation kinetics but the apparent Kd of this binding is about10 times higher as compared with the crude extracts. These results may be explained by cyclic AMP compartmentalization within the cell ; in this case, however, the simple saturation kinetics would indicate that the various pools of the cyclic nucleotide attain equilibrium very rapidly.Or else, if cyclic AMP within the cell is not compartmentalized, and if the reaction described by Equation (1) may be applied, without any modification, to intracellular equilibria, a decrease in the appare nt Kd could be merely a consequence of the dilution (about 10-fold) of the protein components during extraction of the tissue, while cyclic AMP concentrations are maintained by the addition of exogenous cyclic [3H]AMP.However these two hypotheses are certainly oversimplified, since they do not take into account factors like the intracellular concentration of the heat-stable kinase inhibitor [21,22], ATP or Mg2+ [19,23], which are known to affect cyclic AMP binding either in crude extracts or with purified protein kinase preparations. It seems impossible to decide at present which of these interpretations is most likely to reflect true intracellular conditions. It is noteworthy that the apparent Kd estimated is close to the intracehlar cyclic AMP concentration of the nstimulated tissue, a fact which should account for maximal sensitivity of the regulatory mechanisms under physiological conditions. Hormonal controls at the level of cyclic AMP-receptor protein interaction have hitherto never been described; the data reported above provide a suitable means for investigating such problems. The authors are very much indebted to Mrs Ginette Delarbre for her excellent technical assistance and to Mrs Marie-ThBrBse Crosnier for preparing the manuscript. The present work has been performed thanks to two official grants of the C. N. R. S. Paris, France: ERA No 33 and ATP No 429. 914), to a grant obtained from the D. G. R. S. T. 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