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An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds: Application to the ATPase assay in the presence of phosphocreatine
ANALYTICAL
BIOCHEMISTRY
69, 26 I-267
An Improved the Presence
Assay
the Presence OHNISHI,
of Inorganic
of Extralabile
Application
TSUYOSHI
(1975)
ROBERT
Phosphate
Phosphate
to the ATPase
in
Compounds:
Assay
in
of Phosphocreatine S. GALL,~
AND
MICHAEL
Biophysics Laboratory. Department of Anesthesiology, Hahnemann Medical College. Philadelphia, Pennsylvania
L. MAYER’ 19102
Received March 25, 1975; accepted May 29. 1975 Two methods of determining inorganic phosphate in the presence of labile phosphate compounds using the catalyst polyvinylpyrrolidone (PVP) are described. The first method provides a simple assay of ATPase activity at room temperature without deproteinization. The second method minimizes the decomposition of phosphocreatine during the assay to 1%. An application to the measurement of myofibrillar ATPase in the presence of phosphocreatine is given, as well as the advantages of the methods.
Of the various methods to measure ATPase (adenosine triphosphatase) by assaying liberated inorganic phosphate, the Fiske-SubbaRow method (1) is the most widely used. This method, however, has various shortcomings, among them the instability of extralabile phosphate compounds during the assay and a lack of stability in optical density of molybdenium blue. Methods available to overcome these problems (2,3) tend to complicate the assay procedure. By applying the catalyzed phosphorus method (4) to the determination of ATPase of myofibrils (5). we have found that we can overcome most of the problems of previous methods without complication. In this communication we will describe detaiis of our method and its application to the measurement of ATPase in the presence of phosphocreatine. MATERIALS I. Cornmercid Reagents
To keep the method simple, we have used commercially prepared reagents manufactured by American Monitor Corp.’ According to com’ Students in the College Accelerated Program. Institute for Human Resource Development, Hahnemann Medical College. ” Available from Fisher Scientific, Pittsburgh. Pa. 261 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
262
OHNISHI,
GALL
AND
MAYER
parry manuals (4) these are: 1. Molybdute preservative contains 0.016 M tetrasodiumethylenediaminetetraacetate (EDTA-Na,) solution. Ammonium molybdate is dissolved in this solution (to produce ammonium molybdate reagent) to reduce turbidity in the acid phase. 2. Reductant catalyst contains 0.172 M hydroxylamine in 0.001 M phosphorus-free polyvinylpyrrolidone3 (PVP), with 0.0875 M sulfuric acid. The solution is stable indefinitely at room temperature. 3. Color developer contains 6.47 M sodium hydroxide with 0.05 M total carbonate concentration. This is also stable indefinitely at room temperature, but it must be kept from prolonged exposure to air to avoid formation of insoluble carbonate. II. Other Chemicals
Ammonium molybdate and phosphate standards were also purchased from Fisher Scientific, Pittsburgh, Pa. HEPES (N-2-hydroxyethyl piperazine-l\r’-2-ethane-sulfonic acid) was purchased from Calbiochem (Los Angeles, California). ATP, phosphocreatine, CPK (creatine phosphokinase), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO.). Phosphocreatine was purified by the method of Wong (6) to ensure against inorganic phosphate contamination. Myofibrils were prepared from rabbit skeletal muscle by the method previously described (5). ASSAY I. Basic Assay
PROCEDURES
(Kit Method)
This method is used in assaying inorganic phosphate in the presence of ATP. Exactly 5.0 ml of an assay mixture containing water, ammonium molybdate reagent (4% ammonium molybdate in molybdate preservative), and reductant catalyst in a 1: 2: 3 ratio, respectively, is added to 1.0 ml of the sample. After a waiting time of 2 min, 0.5 ml of color developer is added while the solution is mixed on a Vortex mixer. The absorbance is then read at any time between 10 and 40 min on a spectrophotometer. Absorbance can be read at any wavelength between 550 and 720 nm, with maximum sensitivity at 720 nm (the ratio of the absorbance readings at 550, 600, and 720 nm is 0.5 :0.7: 1, respectively). The entire assay is done at room temperature (25”C), though it may also be run at 0°C.
3 Available from Sigma Chemical Co., St. Louis, MO.
ATPase II. Modified
ASSAY IN LABILE
PHOSPHATE
263
Assay (LAP Method)
This procedure is used if extralabile phosphate compounds such as phosphocreatine are present. Though basically the same as above, the following changes are necessary: a) 2% ammonium molybdate reagent must be used; b) waiting time must be reduced to 30 set; c) temperature up to the addition of color developer must be reduced to 0°C. RESULTS
1. Efect of Concentration of Ammonium Molybdate on Color Development The first problem to be solved in developing the assay was to find the best concentration of ammonium molybdate for developing a stable color of molybdenium blue. At room temperature, a 4% ammonium molybdate reagent worked best in yielding a stable color as seen in Fig. 1. Color is stable between 10 and 40 min after the addition of color developer (change is less than & l%), while with a 2% reagent, color declines steadily at higher concentrations of Pi. II. Effect of Concentration on Linearity
of Ammonium
Molybdate
As shown in Fig. 2A, a good linear relationship between Pi concentrations and absorbance is obtained using the kit method (4% ammonium molybdate reagent at 25°C). By using a cuvette with a l-mm optical path, we have confirmed that the linearity is maintained up to a concentration of 6 mM. With a 2% ammonium molybdate reagent at 25”C, a good linear relationship can also be obtained as long as the time between 06
I
0
IO
,
I
20
I
1
30
I
I
40
TIME (mmutes)
FIG. 1. Time course
of color development for both high phosphate concentrations (750 PM, upper curves) and low phosphate concentrations (200 PM, lower curves). Results obtained using 2% (0) and 4% (0) ammonium molybdate reagents at 25°C. Readings taken at 600 nm.
264
CA) II:=
OHNISHI,
g 0.5 3 0.4 d 4 0.2 0.1 2 0.3
GALL
AND MAYER
(8)
5B 0.4 :: 0.3 9 0.2 0.1 g 0.5 k 0
250
500 pi (PM)
750
FIG. 2. Linearity of the method. (A) Linearity curves for 2% monium molybdate reagents at 25°C with a 2-min waiting time. (B) (0) ammonium molybdate reagent at 0°C with a 30-set waiting inorganic phosphate, concentration of which is indicated on the assay. Values taken 20 min after the addition of color developer.
(0) and 4% (0) amLinearity curve for 2% time. One milliliter of abscissa was used for
the addition of the color developer and the absorbance reading is kept constant (20 min in Fig. 2A). In the LAP method, linearity is maintained between 0 and 750 PM (Fig. 2B). Again reducing the optical path, we found linearity maintained up to 1 mM Pi. III. Efect
of the Assay on the Decomposition of ATP
We tested the stability of ATP in the kit method and found that the decomposition of ATP is maintained well below 1% of the total ATP present. IV. Efect of Varying Conditions on the Decomposition of Phosphocreatine
By using the kit method and extending the waiting time to 10 min, we were able to obtain 99-100% decomposition of phosphocreatine (Fig. 3). Hydrolysis can be accelerated by putting the sample in a water bath (37°C). However, using the LAP method, decomposition of phosphocreatine can be kept to a minimum of 1% of the total phosphocreatine (the point at 30 set of the lowest curve, Fig. 3). V. Comparison of the LAP Method to the Calorimetric Determination of Creatine
To test the accuracy of our method, we compared it to the Hughes method for the calorimetric determination of creatine (7). The experiment was prepared as follows: 0.2 ml of CPK (activity of 90-100 Sigma
ATPase
ASSAY
IN
LABILE
265
PHOSPHATE
FIG. 3. Effect of temperature, waiting time, and concentration of ammonium molybdate on the decomposition of 1 mM phosphocreatine. Results obtained using 2% (-j and 4% (---) ammonium molybdate reagents at 0°C (0) and 25°C (0).
units/ml), 0.2 ml of myofibrils (40 mg of protein/ml), and 10 ~1 of 20 mM ADP were added to 6.8 ml of buffer solution (70 mM HEPES buffer, pH 7.0, 50 mM KCl, and 2 mM MgCl,) and incubated in a water bath (37°C). By adding 0.8 ml of 6 mM phosphocreatine, we started the reaction. At 5, 10, and 30 min, an aliquot of 1 ml was pipetted out and transferred to a test tube in ice containing 5 ml of the assay mixture used in the LAP method. Another aliquot of 1 ml was pipetted out simultaneously and tested for creatine. The results (Fig. 4) show that the assay of Pi and creatine are in good agreement. DISCUSSION
Previous to the development of this assay, the Fiske-SubbaRow method (1) was the most widely used method for determining inorganic phosphate. The present method has the following advantages over that method. When used to determine the activity of an enzyme (e.g., ATPase),
0
FIG. Values
IO
20 TIME (mmutesl
30
4. Comparison of methods of phosphate determination and creatine determination. obtained for P, (0) and creatine (0). See text for details of experiment.
266
OHNISHI,
GALL
AND
MAYER
deproteinization is unnecessary; the highly alkaline color developer solubilizes the protein. In our assay, the molybdenum blue is stable for a period of 30 min, thus reading at a specific time is not necessary. Maximum color development, however, is still achieved in a reasonable amount of time, 10 min after the addition of color developer. In certain ATPase experiments, the medium contains chemicals such as oxalate, EDTA, or EGTA (ethyleneglycol-bis(-aminoethyl ether)N, N’-tetraacetic acid), which may interfere with color development (8). However, since this method uses the catalyst PVP and a high concentration of EDTA, the assay is free from interference. The Fiske-SubbaRow method has been used to estimate the total amount of phosphocreatine (9) and for the measurement of CPK activity (10) because its relatively high molybdate concentration and high acidity accelerate the hydrolysis of phosphocreatine (9). However, it cannot be used to measure free Pi in the presence of phosphocreatine. The present method, however, with a lower acidity and lower molybdate concentration, is more flexible, and can be used for any of these purposes: a) Estimation of phosphocreatine by hydrolysis (kit method with extended waiting time). b) Measurement of CPK activity by hydrolysis (kit method with extended waiting time). c) Measuring ATPase activity in the presence of phosphocreatine (LAP method). Besides these advantages over the Fiske-SubbaRow method, the present method offers greater ease in determining the amount of phosphocreatine hydrolyzed by enzymes than the Hughes method (7). Disadvantages in that method are: a) Making use of a-naphthol, an unstable compound. This instability results in varying readings for a given amount of creatine from assay to assay. The present assay, however, has been giving consistent readings; thus one may eliminate making a standard curve each time. b) Making use of diacetyl, a terribly odoriferous compound. Note added in proof. The ammonium molybdate reagent dissolved in EDTA-Na, according to original method (4) starts precipitating two weeks after preparation. We have found that if ammonium molybdate is dissolved in redistilled water and filtered through No. 2 filter paper, it can be stored at room temperature almost indefinitely. For the sake of better preservation, the following method of preparing the assay mixture may be used: One part of ammonium molybdate solution (8% for kit and 4% for LAP; dissolved in water as mentioned above), two parts of 16 rnM EDTA-Na,, and three parts of reductant catalyst.
ACKNOWLEDGMENTS The authors thank Dr. H. L. Price of the Department of Anesthesiology and Dr. V. P. Satinsky of the Institute of Human Resource Development at Hahnemann Medical College for their encouragement. This work was supported in part by NIH Grant HE 05692-08 to Dr. Satinsky.
ATPase
ASSAY IN LABILE
PHOSPHATE
267
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 0.
Fiske. C. H., and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375. Lowry, 0. H., and Lopez, J. A. (1946) J. Biol. Chem. 162, 421. Martin, J. B.. and Doty, D. M. (1949) Anal. Chem. 21, 965. Denny, J. W. (1968) A Manual of Sequential Automated Enzymes, American Monitor Corp., Indianapolis, Ind. Ohnishi, T., Pressman, G. S., and Price, H. L. (1974) Biochem. Biophys. Rrs. Comttucn. 57, 3 16. Wong, T. (1969) Anal. Biochem. 27, 218. Hughes, B. P. (1962) C/in Chim. Acta 7, 597. Ohnishi, T., Gall, R. S., and Mayer, M. L.. to be published. Leloir, L. H., and Cardini, C. H. (1957) in Methods in Enzymology (Colowick, S. P. and Kaplan. N. O., eds.), Vol. 3, p. 840. Academic Press, New York. Noda, L.. Kuby, S., and Lardy, H. (1957) in Methods in Enzymology (Colowick. S. P. and Kaplan, N. O., eds.), Vol. 2, p. 605, Academic Press, New York.
BIOCHEMISTRY
69, 26 I-267
An Improved the Presence
Assay
the Presence OHNISHI,
of Inorganic
of Extralabile
Application
TSUYOSHI
(1975)
ROBERT
Phosphate
Phosphate
to the ATPase
in
Compounds:
Assay
in
of Phosphocreatine S. GALL,~
AND
MICHAEL
Biophysics Laboratory. Department of Anesthesiology, Hahnemann Medical College. Philadelphia, Pennsylvania
L. MAYER’ 19102
Received March 25, 1975; accepted May 29. 1975 Two methods of determining inorganic phosphate in the presence of labile phosphate compounds using the catalyst polyvinylpyrrolidone (PVP) are described. The first method provides a simple assay of ATPase activity at room temperature without deproteinization. The second method minimizes the decomposition of phosphocreatine during the assay to 1%. An application to the measurement of myofibrillar ATPase in the presence of phosphocreatine is given, as well as the advantages of the methods.
Of the various methods to measure ATPase (adenosine triphosphatase) by assaying liberated inorganic phosphate, the Fiske-SubbaRow method (1) is the most widely used. This method, however, has various shortcomings, among them the instability of extralabile phosphate compounds during the assay and a lack of stability in optical density of molybdenium blue. Methods available to overcome these problems (2,3) tend to complicate the assay procedure. By applying the catalyzed phosphorus method (4) to the determination of ATPase of myofibrils (5). we have found that we can overcome most of the problems of previous methods without complication. In this communication we will describe detaiis of our method and its application to the measurement of ATPase in the presence of phosphocreatine. MATERIALS I. Cornmercid Reagents
To keep the method simple, we have used commercially prepared reagents manufactured by American Monitor Corp.’ According to com’ Students in the College Accelerated Program. Institute for Human Resource Development, Hahnemann Medical College. ” Available from Fisher Scientific, Pittsburgh. Pa. 261 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
262
OHNISHI,
GALL
AND
MAYER
parry manuals (4) these are: 1. Molybdute preservative contains 0.016 M tetrasodiumethylenediaminetetraacetate (EDTA-Na,) solution. Ammonium molybdate is dissolved in this solution (to produce ammonium molybdate reagent) to reduce turbidity in the acid phase. 2. Reductant catalyst contains 0.172 M hydroxylamine in 0.001 M phosphorus-free polyvinylpyrrolidone3 (PVP), with 0.0875 M sulfuric acid. The solution is stable indefinitely at room temperature. 3. Color developer contains 6.47 M sodium hydroxide with 0.05 M total carbonate concentration. This is also stable indefinitely at room temperature, but it must be kept from prolonged exposure to air to avoid formation of insoluble carbonate. II. Other Chemicals
Ammonium molybdate and phosphate standards were also purchased from Fisher Scientific, Pittsburgh, Pa. HEPES (N-2-hydroxyethyl piperazine-l\r’-2-ethane-sulfonic acid) was purchased from Calbiochem (Los Angeles, California). ATP, phosphocreatine, CPK (creatine phosphokinase), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO.). Phosphocreatine was purified by the method of Wong (6) to ensure against inorganic phosphate contamination. Myofibrils were prepared from rabbit skeletal muscle by the method previously described (5). ASSAY I. Basic Assay
PROCEDURES
(Kit Method)
This method is used in assaying inorganic phosphate in the presence of ATP. Exactly 5.0 ml of an assay mixture containing water, ammonium molybdate reagent (4% ammonium molybdate in molybdate preservative), and reductant catalyst in a 1: 2: 3 ratio, respectively, is added to 1.0 ml of the sample. After a waiting time of 2 min, 0.5 ml of color developer is added while the solution is mixed on a Vortex mixer. The absorbance is then read at any time between 10 and 40 min on a spectrophotometer. Absorbance can be read at any wavelength between 550 and 720 nm, with maximum sensitivity at 720 nm (the ratio of the absorbance readings at 550, 600, and 720 nm is 0.5 :0.7: 1, respectively). The entire assay is done at room temperature (25”C), though it may also be run at 0°C.
3 Available from Sigma Chemical Co., St. Louis, MO.
ATPase II. Modified
ASSAY IN LABILE
PHOSPHATE
263
Assay (LAP Method)
This procedure is used if extralabile phosphate compounds such as phosphocreatine are present. Though basically the same as above, the following changes are necessary: a) 2% ammonium molybdate reagent must be used; b) waiting time must be reduced to 30 set; c) temperature up to the addition of color developer must be reduced to 0°C. RESULTS
1. Efect of Concentration of Ammonium Molybdate on Color Development The first problem to be solved in developing the assay was to find the best concentration of ammonium molybdate for developing a stable color of molybdenium blue. At room temperature, a 4% ammonium molybdate reagent worked best in yielding a stable color as seen in Fig. 1. Color is stable between 10 and 40 min after the addition of color developer (change is less than & l%), while with a 2% reagent, color declines steadily at higher concentrations of Pi. II. Effect of Concentration on Linearity
of Ammonium
Molybdate
As shown in Fig. 2A, a good linear relationship between Pi concentrations and absorbance is obtained using the kit method (4% ammonium molybdate reagent at 25°C). By using a cuvette with a l-mm optical path, we have confirmed that the linearity is maintained up to a concentration of 6 mM. With a 2% ammonium molybdate reagent at 25”C, a good linear relationship can also be obtained as long as the time between 06
I
0
IO
,
I
20
I
1
30
I
I
40
TIME (mmutes)
FIG. 1. Time course
of color development for both high phosphate concentrations (750 PM, upper curves) and low phosphate concentrations (200 PM, lower curves). Results obtained using 2% (0) and 4% (0) ammonium molybdate reagents at 25°C. Readings taken at 600 nm.
264
CA) II:=
OHNISHI,
g 0.5 3 0.4 d 4 0.2 0.1 2 0.3
GALL
AND MAYER
(8)
5B 0.4 :: 0.3 9 0.2 0.1 g 0.5 k 0
250
500 pi (PM)
750
FIG. 2. Linearity of the method. (A) Linearity curves for 2% monium molybdate reagents at 25°C with a 2-min waiting time. (B) (0) ammonium molybdate reagent at 0°C with a 30-set waiting inorganic phosphate, concentration of which is indicated on the assay. Values taken 20 min after the addition of color developer.
(0) and 4% (0) amLinearity curve for 2% time. One milliliter of abscissa was used for
the addition of the color developer and the absorbance reading is kept constant (20 min in Fig. 2A). In the LAP method, linearity is maintained between 0 and 750 PM (Fig. 2B). Again reducing the optical path, we found linearity maintained up to 1 mM Pi. III. Efect
of the Assay on the Decomposition of ATP
We tested the stability of ATP in the kit method and found that the decomposition of ATP is maintained well below 1% of the total ATP present. IV. Efect of Varying Conditions on the Decomposition of Phosphocreatine
By using the kit method and extending the waiting time to 10 min, we were able to obtain 99-100% decomposition of phosphocreatine (Fig. 3). Hydrolysis can be accelerated by putting the sample in a water bath (37°C). However, using the LAP method, decomposition of phosphocreatine can be kept to a minimum of 1% of the total phosphocreatine (the point at 30 set of the lowest curve, Fig. 3). V. Comparison of the LAP Method to the Calorimetric Determination of Creatine
To test the accuracy of our method, we compared it to the Hughes method for the calorimetric determination of creatine (7). The experiment was prepared as follows: 0.2 ml of CPK (activity of 90-100 Sigma
ATPase
ASSAY
IN
LABILE
265
PHOSPHATE
FIG. 3. Effect of temperature, waiting time, and concentration of ammonium molybdate on the decomposition of 1 mM phosphocreatine. Results obtained using 2% (-j and 4% (---) ammonium molybdate reagents at 0°C (0) and 25°C (0).
units/ml), 0.2 ml of myofibrils (40 mg of protein/ml), and 10 ~1 of 20 mM ADP were added to 6.8 ml of buffer solution (70 mM HEPES buffer, pH 7.0, 50 mM KCl, and 2 mM MgCl,) and incubated in a water bath (37°C). By adding 0.8 ml of 6 mM phosphocreatine, we started the reaction. At 5, 10, and 30 min, an aliquot of 1 ml was pipetted out and transferred to a test tube in ice containing 5 ml of the assay mixture used in the LAP method. Another aliquot of 1 ml was pipetted out simultaneously and tested for creatine. The results (Fig. 4) show that the assay of Pi and creatine are in good agreement. DISCUSSION
Previous to the development of this assay, the Fiske-SubbaRow method (1) was the most widely used method for determining inorganic phosphate. The present method has the following advantages over that method. When used to determine the activity of an enzyme (e.g., ATPase),
0
FIG. Values
IO
20 TIME (mmutesl
30
4. Comparison of methods of phosphate determination and creatine determination. obtained for P, (0) and creatine (0). See text for details of experiment.
266
OHNISHI,
GALL
AND
MAYER
deproteinization is unnecessary; the highly alkaline color developer solubilizes the protein. In our assay, the molybdenum blue is stable for a period of 30 min, thus reading at a specific time is not necessary. Maximum color development, however, is still achieved in a reasonable amount of time, 10 min after the addition of color developer. In certain ATPase experiments, the medium contains chemicals such as oxalate, EDTA, or EGTA (ethyleneglycol-bis(-aminoethyl ether)N, N’-tetraacetic acid), which may interfere with color development (8). However, since this method uses the catalyst PVP and a high concentration of EDTA, the assay is free from interference. The Fiske-SubbaRow method has been used to estimate the total amount of phosphocreatine (9) and for the measurement of CPK activity (10) because its relatively high molybdate concentration and high acidity accelerate the hydrolysis of phosphocreatine (9). However, it cannot be used to measure free Pi in the presence of phosphocreatine. The present method, however, with a lower acidity and lower molybdate concentration, is more flexible, and can be used for any of these purposes: a) Estimation of phosphocreatine by hydrolysis (kit method with extended waiting time). b) Measurement of CPK activity by hydrolysis (kit method with extended waiting time). c) Measuring ATPase activity in the presence of phosphocreatine (LAP method). Besides these advantages over the Fiske-SubbaRow method, the present method offers greater ease in determining the amount of phosphocreatine hydrolyzed by enzymes than the Hughes method (7). Disadvantages in that method are: a) Making use of a-naphthol, an unstable compound. This instability results in varying readings for a given amount of creatine from assay to assay. The present assay, however, has been giving consistent readings; thus one may eliminate making a standard curve each time. b) Making use of diacetyl, a terribly odoriferous compound. Note added in proof. The ammonium molybdate reagent dissolved in EDTA-Na, according to original method (4) starts precipitating two weeks after preparation. We have found that if ammonium molybdate is dissolved in redistilled water and filtered through No. 2 filter paper, it can be stored at room temperature almost indefinitely. For the sake of better preservation, the following method of preparing the assay mixture may be used: One part of ammonium molybdate solution (8% for kit and 4% for LAP; dissolved in water as mentioned above), two parts of 16 rnM EDTA-Na,, and three parts of reductant catalyst.
ACKNOWLEDGMENTS The authors thank Dr. H. L. Price of the Department of Anesthesiology and Dr. V. P. Satinsky of the Institute of Human Resource Development at Hahnemann Medical College for their encouragement. This work was supported in part by NIH Grant HE 05692-08 to Dr. Satinsky.
ATPase
ASSAY IN LABILE
PHOSPHATE
267
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 0.
Fiske. C. H., and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375. Lowry, 0. H., and Lopez, J. A. (1946) J. Biol. Chem. 162, 421. Martin, J. B.. and Doty, D. M. (1949) Anal. Chem. 21, 965. Denny, J. W. (1968) A Manual of Sequential Automated Enzymes, American Monitor Corp., Indianapolis, Ind. Ohnishi, T., Pressman, G. S., and Price, H. L. (1974) Biochem. Biophys. Rrs. Comttucn. 57, 3 16. Wong, T. (1969) Anal. Biochem. 27, 218. Hughes, B. P. (1962) C/in Chim. Acta 7, 597. Ohnishi, T., Gall, R. S., and Mayer, M. L.. to be published. Leloir, L. H., and Cardini, C. H. (1957) in Methods in Enzymology (Colowick, S. P. and Kaplan. N. O., eds.), Vol. 3, p. 840. Academic Press, New York. Noda, L.. Kuby, S., and Lardy, H. (1957) in Methods in Enzymology (Colowick. S. P. and Kaplan, N. O., eds.), Vol. 2, p. 605, Academic Press, New York.