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[99] Adenylate kinase (myokinase, ADP phosphomutase)
598
ENZYMES I N PHOSPHATE METABOLISM
[99]
pyrophosphate esters are not split. At 42 ° ATP is hydrolyzed about ten times as fast as ADP. Activators and Inhibitors. The enzyme is optimally activated by Mg at 1 X 10-8 M. There is no activity if Mg is replaced by Na, K, or Ca. Ca inhibits in the presence of Mg. Glutathione is needed for full activity even in fresh enzyme preparations. The ATPase is inhibited by sulfhydryl reagents , p-chloromercuribenzoate producing 96% inhibition at 5 × 10-~ M, and ninhydrin 97% inhibition at 5 X 10-3 M. Iodoacetate is less effective (40% inhibition at
1 X 10-2 M). Effect of pH. The optimal pH range is 7.8 to 8.0. Effect of Temperature. The relationship between temperature and activity follows the Arrhenius equation between 0 ° and 20 °. The optimal temperature for 5-minute tests is 42 °. Activation energy for the hydrolysis of ATP is 29.6 kcal./mole; for the hydrolysis of ADP it is 16.4 kcal./mole. Activity in Relation to Substrate Concentration. The activity/log (S) curve is bell shaped, the optimal substrate concentrations being 1.6 × 10-a M for ATP and 1.4 X 10-8 M for ADP. KATe is 8.6 X 10-4 M; KADp is 3.3 X 10-3 M.
[99] Adenylate Kinase (Myokinase, A D P Phosphomutase) 2 ADP ~ ATP + AMP
By SIDNEY P. COLOWlCK Assay Method Principle. Since this reaction is accompanied by no readily detectable physical or chemical change (e.g., in light absorption, acidity, or labile phosphorus content) it is convenient to convert one of the reaction products to a derivative, the formation of which is readily detectable. For example, one may add an excess of any enzyme system which specifically removes the terminal phosphate from ATP (e.g., hexokinase or ATPase). In the presence of hexokinase and adenylate kinase, the following over-all reaction results: ADP + glucose -* G-6-P + AMP + H +
(1)
When hexokinase is present in excess, the rate of this reaction is proportional to the concentration of adenylate kinase. The resulting reaction may be followed manometrically by measuring CO2 liberation from a bicarbonate buffer, or chemically by measuring the disappearance of
[99]
ADENYLATE KINASE (MYOKINASE, ADP PHOSPttOMUTASE)
599
acid-labile phosphorus. These two procedures, which are essentially procedures for measuring hexokinase activity, are the ones originally used for the detection of myokinase. 1 These and other procedures for hexokinase assay, which should be readily applicable to the assay of adenylate kinase, are described by Crane s and will not be described in detail here. An alternative method is described below for the assay of adenylate kinase. It is based on the detection of A M P formation by the addition of the specific 5r-AMP deaminase of Schmidt. The preparation of the deaminase 3 and its use for the detection of 5-AMP formation 4 are described elsewhere in this treatise. A factor which interferes with the application of this procedure is the difference between the pH optima of the two enzymes involved. Whereas adenylate kinase is optimally active at pH 7.5, the deaminase is essentially inactive at this pH and shows a sharp optimum around pH 6. Although citrate ions shift the pH optimum of deaminase toward the neutral range, 3 they inhibit the activity of adenylate kinase because of its Mg ++ requirement. It is therefore advisable to carry out this assay in two steps, permitting the adenylate kinase reaction to proceed first, then stopping this reaction by addition of a neutral citrate buffer 3 or a strong KC1 solution, 5 and finally adding the deaminase to measure 5'-AMP. The chief advantage of this procedure is that it can be used not only for activity measurements but also for measuring other properties of the adenylate kinase system, such as the equilibrium constant or the effect of activators and inhibitors. It is obvious that certain of such measurements would be difficult or impossible with a one-stage hexokinase assay.
Reagents ADP-MgCI2-Tris mixture. Mix 1 ml. of 0.01 M Na-ADP (Vol. III [118]) with 8 ml. of 0.1 M Tris-HC1 buffer (Vol. I [16]) and 0.5 ml. of 0.1 M MgCI~. Adenylate kinase. Make appropriate dilution in cold H~0 (e.g., dilute crude muscle extract about 1:400). Citrate buffer. 0.1 M pH 6.4, prepared by addition of HC1 to trisodium citrate. Deaminase. Stock solution of fraction 3 (see Vol. II [68]) diluted to contain about 2000 units of deaminase per milliliter. 1 S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148~ 117 (1943). 2 R. K. Crane, Vol. I [33]. 3 See Vol. II [68]. 4 See Vol. I I I [121]. W. J. Bowen and T. D. Kerwin, Arch. Biochem. and Biophys. 49~ 149 (1954).
600
ENZYMES IN P H O S P H A T E
METABOLISM
[99]
Procedure. To 0.15 ml. of the ADP-MgCI.~-Tris mixture in a 3-ml. quartz cuvette, add 0.05 ml. of the diluted adenylate kinase. After 5 minutes at room temperature add 2.8 ml. of the citrate buffer to stop the reaction. Then add 0.03 ml. (60 units) of the deaminase preparation, and measure the optical density at 265 m~ exactly 15 seconds and 30 seconds after mixing. Then continue reading at 1-minute intervals until the rate of change in optical density falls to 0.001 or less per minute. Not more than 10 minutes should be required for complete deamination. To the total optical density change for the period from 15 seconds to the end of the reaction, add the change measured from 15 to 30 seconds, in order to correct approximately for the amount of reaction which occurred in the first 15 seconds after mixing. This cannot be measured directly because the deaminase itself contributes significantly to the initial absorption change at 265 m~. The total optical density change must not exceed 15 % of the actual reading at 265 mu prior to deaminase addition, since the maximum observable change with excess adenylate kinase is only 25% under the condition described here. The amount of 5'-AMP formed is, of course, proportional to the total optical density change; a reading change of 0.09 corresponds to 0.03 micromole of 5'-AS/[P under these conditions. Although the amount of AMP can also be estimated from the initial rate of the deaminase reaction 6 rather than from total optical density change, this procedure is not recommended here because of the possibility that inhibitors or activators of the deaminase might cause false estimates of AMP concentration. Definition of Unit. One unit of enzyme is defined here as that amount which causes the formation of 1 micromole of 5t-AMP per minute under the conditions of the assay. Protein may be determined nephelometrically after precipitation by trichloroacetic acid. 7 Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Tissue Preparations. The onestage hexokinase assay is probably superior to the two-stage deaminase assay for this purpose. The presence of "apyrases," which may directly or indirectly result in dephosphorylation of ADP to form AMP, would lead to falsely high values for adenylate kinase in the deaminase assay but not in the hexokinase assay. The presence of deaminase in the crude extracts would of course invalidate the deaminase assay, but not the hexokinase assay. A potent ATPase in the crude extract could invalidate the hexokinase assay, but the excess of hexokinase added would in most cases be sufficient to "compete" successfully with the ATPase. 6 H. G. Albaum and R. Lipshitz, Arch. Biochem. 27, 102 (1950). T. Biicherp Biochim. et Biophys. Acta 1, 292 (1947).
[99]
ADENYLATEKINASE (MYOKINASE, ADP PHOSFHOMUTASE)
601
Purification Procedure The procedure outlined below is essentially t h a t originally described b y Colowick and Kalckar. ~ F u r t h e r purification by adsorption of impurities on alumina C~, followed b y fractionation with trichloroacetic acid, has been reported by Kalckar. 8 More recently, N o d a and K u b y have announced additional purification as the zinc salt2 However, the original procedure yields a preparation which is sufficiently active and free of interfering proteins for most purposes. Step 1. Preparation of Crude Extract. ~° R a b b i t skeletal muscle is cooled, ground, and extracted twice with 1 vol. of cold 0.03 N K O H 0.002 M Versene. The third extraction is with 0.5 vol. of 0.002 M Versene. Step 2. Acidification and Heating. The combined extracts are acidified with 0.05 volume of 2.0 N hydrochloric acid and heated as rapidly as possible to a temperature of 90 °. After 3 minutes at this temperature, the solution is cooled rapidly and neutralized to p H 6.0 to 6.5 with 2 N sodium hydroxide. A very large precipitate is formed which is removed b y filtration. The resulting filtrate shows about sixfold purification and serves as a convenient source of adenylate kinase for m a n y purposes. I t m a y be stored as a solution in the refrigerator for several weeks without appreciable loss in activity. Information on stability at - 1 5 ° is not available. SUMMARY OF PURIFICATIONPROCEDUREa
Fraction
Total protein~ mg.
Total units, ~M./min.
Specific activity, ~M./min./mg.
1. Crude extract (100 ml.) 2. Filtrate after heating in acid (ca. 100 ml.)
2370 320
2250 1500
0.82 4.68
The specific activity recorded here for the crude extract is about one-tenth of that originally observed. ~,c It should be noted, however, that the adenylate kinase may not be saturated with substrate under the conditions of the present assay, in which the ADP concentration is 8 X 10-~ M, as compared with 1.3 X 10-2 M in the original assay, c b S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). c H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 8 H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 9L. Noda and S. A. Kuby, Federation Proc. 14, 261 (1955). 10The use of alkaline Versene for extraction is primarily for the purpose of isolating 3-phosphoglyceraldehyde dehydrogenase (see Vol. I [60]). The preparation of adenylate kinase was a by-product in this particular case. In the original studies, distilled water was used for extraction.
602
ENZYMES IN PHOSPHATE METABOLISM
[99]
Step 3. Concentration by Salting Out. F o r convenience in storage, or for removal of nucleotides and other small molecules f r o m fraction 2, the a d e n y l a t e kinase m a y be salted out b y adding a m m o n i u m sulfate to 0.8 saturation. T h e resulting precipitate m a y be filtered off and stored in the cold as a paste or dissolved in the minimal volume of water. N o purification results f r o m this step.
Properties Stability. 1 T h e m o s t r e m a r k a b l e p r o p e r t y of the a d e n y l a t e kinase of muscle is its resistance t o w a r d acid and heat. I n 0.1 N hydrochloric acid at 100 ° , its half-life is almost 30 minutes. Distribution. E a r l y studies 1 on distribution were in error b e c a u s e it was not realized t h a t the stability of the enzyme varies with the source. When various tissues were assayed after boiling with 0.1 N HC1, it a p p e a r e d t h a t muscle was the m a j o r source, none of the e n z y m e being found elsewhere, except for small a m o u n t s in brain and heart. Hence the n a m e m y o k i n a s e was adopted. L a t e r it was shown t h a t when other tissues such as liver 11 and yeast 12 are assayed w i t h o u t boiling in acid, the e n z y m e is readily detected. I t was therefore suggested 13 t h a t the n a m e adenylate kinase would be more appropriate. A s y s t e m a t i c reinvestigation of distribution and stability of the e n z y m e would be desirable. I t is not y e t clear whether the adenylate kinase a c t i v i t y found in h e a r t , ' brain, I and spleen 14 after boiling in HC1 represents all the e n z y m e present in those tissues or just t h a t r e m n a n t which survives the acid t r e a t m e n t . Intracellular Distribution and Function. T h e adenylate kinase is present in the m i t o c h o n d r i a of liver ~5 and muscle tissue. ~6 T h e e n z y m e is p r e s u m a b l y necessary whenever A M P is to serve as a p h o s p h a t e acceptor or A D P as a p h o s p h a t e donor. Evidence for a specific role of the e n z y m e in the relaxation of contracted muscle has been reviewed b y Bailey. ~7 Specificity. Until recently, little had been known concerning the specificity of this e n z y m e except t h a t I D P would not serve as subs t r a t e in place of ADP.lS T h e recent studies of L i e b e r m a n et al., 19 S t r o m -
1: A. V. I/:otel'nikova, Chem. Abstr. 43, 6263 (1949). 12R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittleman, Arch. Biochem. 18, 139 (1948). 1~S. P. Colowick, in "The Enzymes" (Sumner and Myrb$ick, eds.), Vol. II, Part A, p. 148, Academic Press, New York, 1951. 14E. M. Uyeki, Federation Proc. 14, 295 (1955). 15p. Siekevitz and V. R. Potter, J. Biol. Chem. 200, 187 (1953). 16A. Kityakara and J. W. Harman, J. Exptl. Med. 97, 553 (1953). ~7K. Bailey in "The Proteins" (Neurath and Bailey, eds.), Vol. II, Part B, pp. 1053-5, Academic Press, New York, 1954. is A. Kleinzeller, Biochem. J. 36, 729 (1942). 19I. Lieberman, A. Kornberg, and E. S. Simms, J. Am. Chem. Soc. 76, 3608 (1954)
[99]
ADENYLATEKINASE (MYOKINASE, ADP PHOSPHOMUTASE)
603
inger et al. 2° and others (see in footnote 3 Strominger et al. 2°) have revealed the existence of enzyme systems in yeast and animal tissues which might be termed "nucleoside monophosphate kinases." According to Heppel and Strominger (personal communication) there may be two enzymes involved in liver, one of which is specific for A T P as phosphate donor: A T P + X M P --~ ADP + X D P and the other of which is specific for AMP as acceptor: X T P + AMP --* ADP + X D P In both cases, X may be adenosine, guanosine, uridine, or cytidine. However, according to Lieberman et al., 19 the muscle enzyme with which we are concerned here works only with adenine nucleotides. Activators and Inhibitors. The adenylate kinases of muscle 5,8 and liver 15 are Mg++-activated. Activating effects of Ca ++ have also been described. 5,15 Fluoride 15,~-23 citrate, and Calgon ~ are inhibitors by virtue of their metal-binding action. This is the basis of the finding 2~that fluoride prevents AMP, but not ADP, from functioning as a phosphate acceptor in respiring mitochondria. The adenylate kinase of muscle can be inactivated by warming with H:02 and reactivated by glutathione (GSH) or cysteine. 1 If the enzyme is not subjected to oxidants, glutathione and cysteine are without effect on the activity and need not be added to the assay system. The muscle enzyme is inactivated by commercial pepsin. p H O p t i m u m . Kalckar 8 reported that the muscle enzyme was maximally active at pH 7.5. Bowen and Kerwin 5 also report a value of about 7.5 for assays in the absence of 5/[g++ but find an optimum at pH 6 when Mg ++ is present. Since the latter figures were obtained by the deaminase " r a t e assay" instead of the "extent assay," the value found by Kalckar would appear to be the more reliable figure for the pH optimum of the enzyme. Equilibrium Constant. Kalckar s showed that the adenylate kinase reaction could be demonstrated in either direction. Difficulty was experienced by both Kalckar 8 and Bowen and Kerwin ~ in reaching the same equilibrium position from the two directions. Starting with ADP, all investigators ~,s,24 agree that somewhat more than half is utilized at 2oj. L. Strominger, L. A. Heppel, and E. S. Maxwell, Arch. Biochem. and Biophys. 52, 488 (1954). 2I S. S. Barkulis and A. L. Lehninger, J. Biol. Chem. 190, 339 (1951). 22A. V. Kotel'nikova, Chem. Abstr. 45, 198 (1951). 28E. C. Slater, Biochem. J. 53, 521 (1953). 24L. V. Eggleston and R. Hems, Biochem. J. 52, 156 (1952).
604
ENZYMES IN PHOSPHATE METABOLISM
[99]
equilibrium. Eggleston and H e m s ~4 report a constant of 0.444 for this reaction a t p H 7.4 and 25 ° with 0.01 M MgC12. According to Bowen and Kerwin, 5 the a p p a r e n t equilibrium constant increases significantly with M g ++ concentration, because of the f o r m a t i o n of a M g - A T P complex with a higher binding constant t h a n t h a t for the M g - A D P complex. The Adenylic Acid Effect. When the s u b s t r a t e for the reaction is A D P , there is a v e r y rapid falling off in the rate of the reaction with time, because of the a c c u m u l a t i o n of 5-AMP, which appears to be strongly inhibitory to the muscle enzyme. 1.8 A T P also appears to be s o m e w h a t inhibitory with the liver enzyme. 15 Slater 23 points out t h a t the 5 - A M P inhibition is readily observed even when a large excess of hexokinase and glucose is present, which m u s t certainly prevent appreciable back reaction of A M P with A T P in a homogeneous system. I t appears t h a t the A M P inhibition is therefore best interpreted as being due to a v e r y high affinity of A M P for the protein, relative to t h a t of A D P . 25 However, it m u s t be k e p t in mind t h a t A M P would not be expected to be an inhibitor of the e n z y m e when the latter is catalyzing the reaction of AS~[P and A T P . I t therefore appears t h a t Slater's ~3,26 use of A M P as an " i n h i b i t o r " of this e n z y m e in a particulate s y s t e m generating A T P oxidatively f r o m A D P could conceivably lead to erroneous results. The generated A T P m i g h t react with A M P within the particles preferentially to reaction with hexokinase and glucose present externally. This could account for the low values for a p p a r e n t A T P synthesis actually observed b y Slater in such a system. ~5Slater23 presents data indicating that ADP has a high affinity for the enzyme, but the conclusion does not appear to he warranted, since the AMP:ADP ratio was maintained constant as the ADP concentration was varied. From the consideration mentioned in footnote a in the accompanyfng table, as well as from the strong inhibitory effect of AMP, it would seem that ADP may actually have a rather low affinity for the enzyme. 26 E. C. Slater, Nature 166, 982 (1950).
ENZYMES I N PHOSPHATE METABOLISM
[99]
pyrophosphate esters are not split. At 42 ° ATP is hydrolyzed about ten times as fast as ADP. Activators and Inhibitors. The enzyme is optimally activated by Mg at 1 X 10-8 M. There is no activity if Mg is replaced by Na, K, or Ca. Ca inhibits in the presence of Mg. Glutathione is needed for full activity even in fresh enzyme preparations. The ATPase is inhibited by sulfhydryl reagents , p-chloromercuribenzoate producing 96% inhibition at 5 × 10-~ M, and ninhydrin 97% inhibition at 5 X 10-3 M. Iodoacetate is less effective (40% inhibition at
1 X 10-2 M). Effect of pH. The optimal pH range is 7.8 to 8.0. Effect of Temperature. The relationship between temperature and activity follows the Arrhenius equation between 0 ° and 20 °. The optimal temperature for 5-minute tests is 42 °. Activation energy for the hydrolysis of ATP is 29.6 kcal./mole; for the hydrolysis of ADP it is 16.4 kcal./mole. Activity in Relation to Substrate Concentration. The activity/log (S) curve is bell shaped, the optimal substrate concentrations being 1.6 × 10-a M for ATP and 1.4 X 10-8 M for ADP. KATe is 8.6 X 10-4 M; KADp is 3.3 X 10-3 M.
[99] Adenylate Kinase (Myokinase, A D P Phosphomutase) 2 ADP ~ ATP + AMP
By SIDNEY P. COLOWlCK Assay Method Principle. Since this reaction is accompanied by no readily detectable physical or chemical change (e.g., in light absorption, acidity, or labile phosphorus content) it is convenient to convert one of the reaction products to a derivative, the formation of which is readily detectable. For example, one may add an excess of any enzyme system which specifically removes the terminal phosphate from ATP (e.g., hexokinase or ATPase). In the presence of hexokinase and adenylate kinase, the following over-all reaction results: ADP + glucose -* G-6-P + AMP + H +
(1)
When hexokinase is present in excess, the rate of this reaction is proportional to the concentration of adenylate kinase. The resulting reaction may be followed manometrically by measuring CO2 liberation from a bicarbonate buffer, or chemically by measuring the disappearance of
[99]
ADENYLATE KINASE (MYOKINASE, ADP PHOSPttOMUTASE)
599
acid-labile phosphorus. These two procedures, which are essentially procedures for measuring hexokinase activity, are the ones originally used for the detection of myokinase. 1 These and other procedures for hexokinase assay, which should be readily applicable to the assay of adenylate kinase, are described by Crane s and will not be described in detail here. An alternative method is described below for the assay of adenylate kinase. It is based on the detection of A M P formation by the addition of the specific 5r-AMP deaminase of Schmidt. The preparation of the deaminase 3 and its use for the detection of 5-AMP formation 4 are described elsewhere in this treatise. A factor which interferes with the application of this procedure is the difference between the pH optima of the two enzymes involved. Whereas adenylate kinase is optimally active at pH 7.5, the deaminase is essentially inactive at this pH and shows a sharp optimum around pH 6. Although citrate ions shift the pH optimum of deaminase toward the neutral range, 3 they inhibit the activity of adenylate kinase because of its Mg ++ requirement. It is therefore advisable to carry out this assay in two steps, permitting the adenylate kinase reaction to proceed first, then stopping this reaction by addition of a neutral citrate buffer 3 or a strong KC1 solution, 5 and finally adding the deaminase to measure 5'-AMP. The chief advantage of this procedure is that it can be used not only for activity measurements but also for measuring other properties of the adenylate kinase system, such as the equilibrium constant or the effect of activators and inhibitors. It is obvious that certain of such measurements would be difficult or impossible with a one-stage hexokinase assay.
Reagents ADP-MgCI2-Tris mixture. Mix 1 ml. of 0.01 M Na-ADP (Vol. III [118]) with 8 ml. of 0.1 M Tris-HC1 buffer (Vol. I [16]) and 0.5 ml. of 0.1 M MgCI~. Adenylate kinase. Make appropriate dilution in cold H~0 (e.g., dilute crude muscle extract about 1:400). Citrate buffer. 0.1 M pH 6.4, prepared by addition of HC1 to trisodium citrate. Deaminase. Stock solution of fraction 3 (see Vol. II [68]) diluted to contain about 2000 units of deaminase per milliliter. 1 S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148~ 117 (1943). 2 R. K. Crane, Vol. I [33]. 3 See Vol. II [68]. 4 See Vol. I I I [121]. W. J. Bowen and T. D. Kerwin, Arch. Biochem. and Biophys. 49~ 149 (1954).
600
ENZYMES IN P H O S P H A T E
METABOLISM
[99]
Procedure. To 0.15 ml. of the ADP-MgCI.~-Tris mixture in a 3-ml. quartz cuvette, add 0.05 ml. of the diluted adenylate kinase. After 5 minutes at room temperature add 2.8 ml. of the citrate buffer to stop the reaction. Then add 0.03 ml. (60 units) of the deaminase preparation, and measure the optical density at 265 m~ exactly 15 seconds and 30 seconds after mixing. Then continue reading at 1-minute intervals until the rate of change in optical density falls to 0.001 or less per minute. Not more than 10 minutes should be required for complete deamination. To the total optical density change for the period from 15 seconds to the end of the reaction, add the change measured from 15 to 30 seconds, in order to correct approximately for the amount of reaction which occurred in the first 15 seconds after mixing. This cannot be measured directly because the deaminase itself contributes significantly to the initial absorption change at 265 m~. The total optical density change must not exceed 15 % of the actual reading at 265 mu prior to deaminase addition, since the maximum observable change with excess adenylate kinase is only 25% under the condition described here. The amount of 5'-AMP formed is, of course, proportional to the total optical density change; a reading change of 0.09 corresponds to 0.03 micromole of 5'-AS/[P under these conditions. Although the amount of AMP can also be estimated from the initial rate of the deaminase reaction 6 rather than from total optical density change, this procedure is not recommended here because of the possibility that inhibitors or activators of the deaminase might cause false estimates of AMP concentration. Definition of Unit. One unit of enzyme is defined here as that amount which causes the formation of 1 micromole of 5t-AMP per minute under the conditions of the assay. Protein may be determined nephelometrically after precipitation by trichloroacetic acid. 7 Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Tissue Preparations. The onestage hexokinase assay is probably superior to the two-stage deaminase assay for this purpose. The presence of "apyrases," which may directly or indirectly result in dephosphorylation of ADP to form AMP, would lead to falsely high values for adenylate kinase in the deaminase assay but not in the hexokinase assay. The presence of deaminase in the crude extracts would of course invalidate the deaminase assay, but not the hexokinase assay. A potent ATPase in the crude extract could invalidate the hexokinase assay, but the excess of hexokinase added would in most cases be sufficient to "compete" successfully with the ATPase. 6 H. G. Albaum and R. Lipshitz, Arch. Biochem. 27, 102 (1950). T. Biicherp Biochim. et Biophys. Acta 1, 292 (1947).
[99]
ADENYLATEKINASE (MYOKINASE, ADP PHOSFHOMUTASE)
601
Purification Procedure The procedure outlined below is essentially t h a t originally described b y Colowick and Kalckar. ~ F u r t h e r purification by adsorption of impurities on alumina C~, followed b y fractionation with trichloroacetic acid, has been reported by Kalckar. 8 More recently, N o d a and K u b y have announced additional purification as the zinc salt2 However, the original procedure yields a preparation which is sufficiently active and free of interfering proteins for most purposes. Step 1. Preparation of Crude Extract. ~° R a b b i t skeletal muscle is cooled, ground, and extracted twice with 1 vol. of cold 0.03 N K O H 0.002 M Versene. The third extraction is with 0.5 vol. of 0.002 M Versene. Step 2. Acidification and Heating. The combined extracts are acidified with 0.05 volume of 2.0 N hydrochloric acid and heated as rapidly as possible to a temperature of 90 °. After 3 minutes at this temperature, the solution is cooled rapidly and neutralized to p H 6.0 to 6.5 with 2 N sodium hydroxide. A very large precipitate is formed which is removed b y filtration. The resulting filtrate shows about sixfold purification and serves as a convenient source of adenylate kinase for m a n y purposes. I t m a y be stored as a solution in the refrigerator for several weeks without appreciable loss in activity. Information on stability at - 1 5 ° is not available. SUMMARY OF PURIFICATIONPROCEDUREa
Fraction
Total protein~ mg.
Total units, ~M./min.
Specific activity, ~M./min./mg.
1. Crude extract (100 ml.) 2. Filtrate after heating in acid (ca. 100 ml.)
2370 320
2250 1500
0.82 4.68
The specific activity recorded here for the crude extract is about one-tenth of that originally observed. ~,c It should be noted, however, that the adenylate kinase may not be saturated with substrate under the conditions of the present assay, in which the ADP concentration is 8 X 10-~ M, as compared with 1.3 X 10-2 M in the original assay, c b S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). c H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 8 H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 9L. Noda and S. A. Kuby, Federation Proc. 14, 261 (1955). 10The use of alkaline Versene for extraction is primarily for the purpose of isolating 3-phosphoglyceraldehyde dehydrogenase (see Vol. I [60]). The preparation of adenylate kinase was a by-product in this particular case. In the original studies, distilled water was used for extraction.
602
ENZYMES IN PHOSPHATE METABOLISM
[99]
Step 3. Concentration by Salting Out. F o r convenience in storage, or for removal of nucleotides and other small molecules f r o m fraction 2, the a d e n y l a t e kinase m a y be salted out b y adding a m m o n i u m sulfate to 0.8 saturation. T h e resulting precipitate m a y be filtered off and stored in the cold as a paste or dissolved in the minimal volume of water. N o purification results f r o m this step.
Properties Stability. 1 T h e m o s t r e m a r k a b l e p r o p e r t y of the a d e n y l a t e kinase of muscle is its resistance t o w a r d acid and heat. I n 0.1 N hydrochloric acid at 100 ° , its half-life is almost 30 minutes. Distribution. E a r l y studies 1 on distribution were in error b e c a u s e it was not realized t h a t the stability of the enzyme varies with the source. When various tissues were assayed after boiling with 0.1 N HC1, it a p p e a r e d t h a t muscle was the m a j o r source, none of the e n z y m e being found elsewhere, except for small a m o u n t s in brain and heart. Hence the n a m e m y o k i n a s e was adopted. L a t e r it was shown t h a t when other tissues such as liver 11 and yeast 12 are assayed w i t h o u t boiling in acid, the e n z y m e is readily detected. I t was therefore suggested 13 t h a t the n a m e adenylate kinase would be more appropriate. A s y s t e m a t i c reinvestigation of distribution and stability of the e n z y m e would be desirable. I t is not y e t clear whether the adenylate kinase a c t i v i t y found in h e a r t , ' brain, I and spleen 14 after boiling in HC1 represents all the e n z y m e present in those tissues or just t h a t r e m n a n t which survives the acid t r e a t m e n t . Intracellular Distribution and Function. T h e adenylate kinase is present in the m i t o c h o n d r i a of liver ~5 and muscle tissue. ~6 T h e e n z y m e is p r e s u m a b l y necessary whenever A M P is to serve as a p h o s p h a t e acceptor or A D P as a p h o s p h a t e donor. Evidence for a specific role of the e n z y m e in the relaxation of contracted muscle has been reviewed b y Bailey. ~7 Specificity. Until recently, little had been known concerning the specificity of this e n z y m e except t h a t I D P would not serve as subs t r a t e in place of ADP.lS T h e recent studies of L i e b e r m a n et al., 19 S t r o m -
1: A. V. I/:otel'nikova, Chem. Abstr. 43, 6263 (1949). 12R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittleman, Arch. Biochem. 18, 139 (1948). 1~S. P. Colowick, in "The Enzymes" (Sumner and Myrb$ick, eds.), Vol. II, Part A, p. 148, Academic Press, New York, 1951. 14E. M. Uyeki, Federation Proc. 14, 295 (1955). 15p. Siekevitz and V. R. Potter, J. Biol. Chem. 200, 187 (1953). 16A. Kityakara and J. W. Harman, J. Exptl. Med. 97, 553 (1953). ~7K. Bailey in "The Proteins" (Neurath and Bailey, eds.), Vol. II, Part B, pp. 1053-5, Academic Press, New York, 1954. is A. Kleinzeller, Biochem. J. 36, 729 (1942). 19I. Lieberman, A. Kornberg, and E. S. Simms, J. Am. Chem. Soc. 76, 3608 (1954)
[99]
ADENYLATEKINASE (MYOKINASE, ADP PHOSPHOMUTASE)
603
inger et al. 2° and others (see in footnote 3 Strominger et al. 2°) have revealed the existence of enzyme systems in yeast and animal tissues which might be termed "nucleoside monophosphate kinases." According to Heppel and Strominger (personal communication) there may be two enzymes involved in liver, one of which is specific for A T P as phosphate donor: A T P + X M P --~ ADP + X D P and the other of which is specific for AMP as acceptor: X T P + AMP --* ADP + X D P In both cases, X may be adenosine, guanosine, uridine, or cytidine. However, according to Lieberman et al., 19 the muscle enzyme with which we are concerned here works only with adenine nucleotides. Activators and Inhibitors. The adenylate kinases of muscle 5,8 and liver 15 are Mg++-activated. Activating effects of Ca ++ have also been described. 5,15 Fluoride 15,~-23 citrate, and Calgon ~ are inhibitors by virtue of their metal-binding action. This is the basis of the finding 2~that fluoride prevents AMP, but not ADP, from functioning as a phosphate acceptor in respiring mitochondria. The adenylate kinase of muscle can be inactivated by warming with H:02 and reactivated by glutathione (GSH) or cysteine. 1 If the enzyme is not subjected to oxidants, glutathione and cysteine are without effect on the activity and need not be added to the assay system. The muscle enzyme is inactivated by commercial pepsin. p H O p t i m u m . Kalckar 8 reported that the muscle enzyme was maximally active at pH 7.5. Bowen and Kerwin 5 also report a value of about 7.5 for assays in the absence of 5/[g++ but find an optimum at pH 6 when Mg ++ is present. Since the latter figures were obtained by the deaminase " r a t e assay" instead of the "extent assay," the value found by Kalckar would appear to be the more reliable figure for the pH optimum of the enzyme. Equilibrium Constant. Kalckar s showed that the adenylate kinase reaction could be demonstrated in either direction. Difficulty was experienced by both Kalckar 8 and Bowen and Kerwin ~ in reaching the same equilibrium position from the two directions. Starting with ADP, all investigators ~,s,24 agree that somewhat more than half is utilized at 2oj. L. Strominger, L. A. Heppel, and E. S. Maxwell, Arch. Biochem. and Biophys. 52, 488 (1954). 2I S. S. Barkulis and A. L. Lehninger, J. Biol. Chem. 190, 339 (1951). 22A. V. Kotel'nikova, Chem. Abstr. 45, 198 (1951). 28E. C. Slater, Biochem. J. 53, 521 (1953). 24L. V. Eggleston and R. Hems, Biochem. J. 52, 156 (1952).
604
ENZYMES IN PHOSPHATE METABOLISM
[99]
equilibrium. Eggleston and H e m s ~4 report a constant of 0.444 for this reaction a t p H 7.4 and 25 ° with 0.01 M MgC12. According to Bowen and Kerwin, 5 the a p p a r e n t equilibrium constant increases significantly with M g ++ concentration, because of the f o r m a t i o n of a M g - A T P complex with a higher binding constant t h a n t h a t for the M g - A D P complex. The Adenylic Acid Effect. When the s u b s t r a t e for the reaction is A D P , there is a v e r y rapid falling off in the rate of the reaction with time, because of the a c c u m u l a t i o n of 5-AMP, which appears to be strongly inhibitory to the muscle enzyme. 1.8 A T P also appears to be s o m e w h a t inhibitory with the liver enzyme. 15 Slater 23 points out t h a t the 5 - A M P inhibition is readily observed even when a large excess of hexokinase and glucose is present, which m u s t certainly prevent appreciable back reaction of A M P with A T P in a homogeneous system. I t appears t h a t the A M P inhibition is therefore best interpreted as being due to a v e r y high affinity of A M P for the protein, relative to t h a t of A D P . 25 However, it m u s t be k e p t in mind t h a t A M P would not be expected to be an inhibitor of the e n z y m e when the latter is catalyzing the reaction of AS~[P and A T P . I t therefore appears t h a t Slater's ~3,26 use of A M P as an " i n h i b i t o r " of this e n z y m e in a particulate s y s t e m generating A T P oxidatively f r o m A D P could conceivably lead to erroneous results. The generated A T P m i g h t react with A M P within the particles preferentially to reaction with hexokinase and glucose present externally. This could account for the low values for a p p a r e n t A T P synthesis actually observed b y Slater in such a system. ~5Slater23 presents data indicating that ADP has a high affinity for the enzyme, but the conclusion does not appear to he warranted, since the AMP:ADP ratio was maintained constant as the ADP concentration was varied. From the consideration mentioned in footnote a in the accompanyfng table, as well as from the strong inhibitory effect of AMP, it would seem that ADP may actually have a rather low affinity for the enzyme. 26 E. C. Slater, Nature 166, 982 (1950).