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Fluorimetric determination of carbamoyl phosphate
ANALYTICAL
BIOCHEMISTRY
129.80-87
Fluorimetric RONALDJ.
(1983)
Determination
of Carbamoyl
Phosphate
A. WANDERS, CARLOVAN ROERMUND, COR LOF,ANDALFRED
J.MEIJER
Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, P.O. Box 20151. 1000 HD Amsterdam, The Netherlands Received July 5, 1982 A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay,production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present. KEY WORDS: carbamoyl phosphate; omithine carbamoyl transferase; citrulline synthesis; aspartate carbamoyl transfer%.
The list of intermediates involved in urea biosynthesis was completed in 1955 when Jones et al. (1) identified compound X (2) as carbamoyl phosphate1 From the time of its discovery, it was recognized that solutions of carbamoyl phosphate are unstable both at acid and at alkaline pHs (for a review, see Ref. (3)). At an acid pH, the decomposition of carbamoyl phosphate generates ammonia, carbon dioxide, and inorganic phosphate (4-6), whereas at an alkaline pH, cyanate and phosphate are produced (4-6). The instability of carbamoyl phosphate is presumably one of the reasons why so little information about the intracellular concentrations of this compound is available. Three methods have been described for the determination of carbamoyl phosphate in tissue extracts. In the first (7,8), carbamoyl phosphate is quantitatively converted into stable [‘4C]carbamoyl-L-aspartate after reaction with [14C]aspartate and purified aspartate carbamoyltransferase (EC 2.1.3.2). The labeled compound is separated from [ “C]aspartate by column chromatography. With this method, the carbamoyl phosphate content of blood plasma (7) and Escherichia coli cells (8) has been determined. In the second method (9- 13), purified ornithine carbamoyltransferase (EC 2.1.3.3) and
0003-2697/83/030080-08$03.00/0 CopyrigJa 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
[‘4C]ornithine are used for the conversion of carbamoyl phosphate into stable [i4C]citrulline. This is followed by separation of [ “C]omithine and [‘4C]citrulline using cation-exchange chromatography (9- 13). In the third method, carbamoyl phosphate is determined after its direct chemical conversion to urea or hydroxyurea ( 14,15). In the present paper, a rapid fluorimetric method for the determination of very small amounts of carbamoyl phosphate is described. In this assay, chromatographic procedures are not required. MATERIALS
AND METHODS
Isolation of mitochondria. Rat liver mitochondria were isolated from male Wistar rats (200-250 g) by the method of Hogeboom (16), as described by Myers and Slater (17), using 250 mM mannitol, 5 mM Tris-HCl and 0.5 mrvt ethylene glycol bis(&aminoethyl ether) iV,N,N’,N’-tetraacetic acid (EGTA) (final pH 7.4) as the isolation medium. The final pellet (about 150 mg protein) was suspended in 34 ml 250 mM mannitol and kept on ice. Incubation conditions. Mitochondria (3-5 mg protein/ml) were incubated at 25°C in a standard reaction medium containing the following components: 100 mM KCl, 50 mM
80
DETERMINATION
OF CARBAMOYL
PHOSPHATE
81
neutralized extracts in liquid nitrogen (about 5 min). Fluorimetric determination of carbamoyl phosphate. Carbamoyl phosphate was measured fluorimetrically using an Eppendorf photometer with a fluorescence attachment (Type 1030; primary filter, 366 nm, secondary filter, >400 nm) equipped with a linear Servogor recorder (Model 2 10). To a l-cm2 quartz cuvette containing 2.00 ml Buffer A [consisting of 50 mM triethanolamine-HCl, 10 mM MgS04, 5 mM ethylenediaminetetraacetic acid (EDTA), and 75 mM KCl; pH 7.41, 5 ~1NADP+ (5 mg/ml), 5 ~1 1 M glucose, 10 ~1 0.1 M ADP, 5 ~1 glucose-6-phosphate dehydrogenase (NADP+) from yeast (5 mg/ ml; 140 IU/mg; diluted 1:5 with distilled water), a sample of 75-100 ~1 was added. When a constant recording signal was obtained, 5 ~1 hexokinase (10 mg/ml, 140 IU/mg; diluted 1:5 with distilled water) was added. After completion of the reaction, the recorder pen was reset to the baseline and 5 ~1 carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) was added (700 U/mg enzyme protein; 1 mg lyophilized material dissolved in 250 ~1 Buffer A). Calibration was done with the internal standard procedure (18), using carbamoyl phosphate standard solutions freshly prepared each day and kept on ice in Buffer A. The concentrations of the carbamoyl phosphate standard solutions were determined spectrophotometrically on a ZeissPMQ-II spectrophotometer equipped with a logarithmic Servogor recorder, using the same reaction mixture as described for the fluorimetric assay with the exception that a higher concentration of NADP was added (final concentration in cuvette 0.4 mM). In some experiments, carbamoyl phosphate was determined calorimetrically as citrulline; in that case, samples containing carbamoyl phosphate were incubated at 25’C for 30 min in a medium containing 50 mM TrisHCl, 20 mM ornithine, 2 mM EDTA, and 1 ’ Abbreviations used: EGTA, ethylene glycol bi@aminoethyl ether) N,N,N’,N’-tetraacetic acid; Mops, 4- U/ml ornithine carbamoyl transferase from moruholine .orooanesulfonic acid. -Streptococcus faecaks (final pH 7.5). Reac.
Tris-HCl, 1 mM EGTA’, 1 mM ATP, 5 mM potassium phosphate, 20 mM succinate, 10 mM ammonium chloride, 16.6 mM potassium bicarbonate, and 2.5 &ml rotenone. The final pH was 7.4. Ornithine was present where indicated. Incubations were carried out at 25°C in a thermostatically controlled reaction vessel with vigorous stirring under a continuous stream of 95% 02/5% CO*. Extraction procedurefor mitochondrial carbamoyl phosphate. For the determination of intramitochondrial carbamoyl phosphate, we used the silicone oil centrifugation technique to separate the mitochondria from their suspending medium. For this purpose, Eppendorf centrifuge tubes ( 1.6 ml) were filled with 0.25 ml of a solution containing 7% (w/v) perchloric acid, 0.5 M mannitol, and 20 mM EDTA, followed by an 0.4-ml layer of silicone oil (Wacker AR 200:AR 20 = 270:60 (v/v); density 1.033 g/ml at 25’C). The tubes were precooled in an ice-water bath. At the time desired, a 0.5- to 0.8-ml sample was drawn from the mitochondrial incubation mixture, layered on top of the silicone oil, and centrifuged at 12 OOOg for 60 s in an Eppendorf centrifuge (Model 3200). After careful removal of the supematant and rinsing of the wall of the tube above the oil with cold distilled water to remove any residual extramitochondrial fluid, the mitochondrial pellet in the acid layer was resuspended with a glass rod in order to obtain full extraction of the mitochondria. After removal of the denatured protein by centrifugation in the cold, a sample (0.2 ml) was taken from the acid phase below the oil, neutralized with cold 2 M KOH/0.2 M 4-morpholinepropanesulfonic acid (Mops) to pH 7.00 + 0.25, and immediately frozen in liquid nitrogen. All operations were carried out in ice water as quickly as possible to minimize the time elapsing between the start of the centrifugation and final freezing of the
82
WANDERS
lions were stopped with perchloric acid (final concentration 3.5% (w/v)) and citrulline was determined in the protein-free acid extracts. Citrulline determination. Citrulline was determined according to the method of Pierson (15) in 4-ml polypropylene tubes in a final volume of 1 ml. For each series of determinations, a calibration curve consisting of 10 points (O-150 nmol) was constructed with a stock solution of L-citrulline (1 mM), stored frozen. All determinations were carried out at least in duplicate. Protein determination. Protein was determined exactly as described by Cleland and Slater (19), using egg albumin as a standard. Enzymes and reagents. Carbamate kinase and omithine carbamoyltransferase (as lyophilized powders) and dilithium carbamoyl phosphate were obtained from Sigma Chemical Co. (St. Louis, Missouri). Other enzymes and nucleotides were obtained from Boehringer GmbH (Mannheim, FRG). All other reagents were of analytical grade. RESULTS
Principle of the Method Carbamate kinase catalyzes the reversible conversion of carbamate and ATP to carbamoyl phosphate and ADP. The equilibrium position of this reaction lies far towards carbamoyl phosphate and ADP formation (K, = 24.2 X lo3 in the presence of 10 mM MgC12 (20)). Consequently, quantitative determination of carbamoyl phosphate is only possible if the reaction products are removed. This can be achieved by coupling the carbamate kinase reaction with the hexokinase and glucose-6phosphate dehydrogenase reactions (20). An additional advantage of the coupled system is that conversion of carbamoyl phosphate can be measured directly by following the appearance of NADPH spectrophotometrically or fluorimetrically. The validity of the method was first investigated with dilithium-carbamoyl phosphate standard solutions (see Materials and Meth-
ET
AL.
ods). Carbamoyl phosphate was measured spectrophotometrically at 340 nm, using the carbamate kinase/hexokinase/glucose-6phosphate dehydrogenase system, and also calorimetrically as citrulline after incubation with omithine and omithine carbamoyltransferase. The agreement between the two methods was 99.2 f 1.2% (means +- SEM of 10 determinations) with carbamoyl phosphate concentrations ranging from 20 to 200 nmol/ml. The levels of carbamoyl phosphate in animal tissues are usually too low to be determined spectrophotometrically. We therefore set up a fluorimetric assay procedure, since a loo-fold gain in sensitivity is achieved by measuring changes in fluorescence rather than in absorbance of NAD(P)H. Standard curves were found to be linear for amounts of carbamoyl phosphate corresponding to concentrations in the cuvette of O-5 pM (results not shown). Since most tissues contain appreciable amounts of ATP (IQ this nucleotide must first be removed before carbamoyl kinase is added (Fig. 1). This did not obstruct sensitive and quantitative measurement of carbamoyl phosphate, as is shown in Fig. 1. Reactions were usually fast, being completed in 5- 10 min, with hardly any drift.
Stability of Carbamoyl Phosphate Instability of carbamoyl phosphate, especially at low and high pH values (4-6), presents a serious difficulty, since deproteinizalion of tissue usually occurs via acidification. Indeed, variable recoveries of carbamoyl phosphate have been reported, ranging from low (36%, Ref. (9)) to very high (98%, Ref. (8)) (see also under Discussion). Other authors (7, 10-13) reported a recovery between these two extremes. In order to find optimal conditions for tissue extraction, we studied the decomposition of dilithium-carbamoyl phosphate as a function of the acidity of the medium and the incubation time, at two temperatures (0 and 25°C). At 25°C hydrolysis
DETERMINATION
z
OF CARBAMOYL
PHOSPHATE
hexokinase
FIG. 1. Fhtorimetric determination of carbamoyl phosphate. Rat liver mitochondria (3.1 mg protein/ ml) isolated from rats fed a normal diet were incubated in the standard reaction medium in the absence of omithine. After 4 min, a 0.5-m] sample was drawn from the incubation mixture, subjected to siliconeoil centrifugation, and further handled as described under Materials and Methods. 0.075 ml of the neutralized perchloric acid extract of the pellet was added to the cuvette containing the reaction mixture described under Materials and Methods. When temperature equilibration was complete, hexokinase was added. After completion of the reaction and readjustment of the recorder, carbamate kinase was added. Calibration was carried out with an internal standard.
83
84
WANDERS
of carbamoyl phosphate was found to be dependent upon the concentration of perchloric acid (in agreement with the data of Halmann et al. (5)) and to increase with the time of incubation (Fig. 2A). If, however, the incubation was carried out in an ice-water bath, hydrolysis occurred to a limited extent only (Fig. 2B). In the light of these experimental findings, a protocol for the termination of the reactions was set up which minimized hydrolysis of carbamoyl phosphate (see Materials and Methods). In the experiment shown in Fig. 3, the results of this procedure with regard to the recovery of carbamoyl phosphate during sample preparation was tested. Isolated mitochondria were incubated for 10 min in the standard reaction medium plus 10 mM or-
ET AL.
nmol carbamoyl
phosphate
added
RG. 3. Recovery of carbamoyl phosphate during preparation of perchloric acid extracts of mitochondria. Mitochondria (4.5 mg protein/ml) were incubated at 25°C in the standard reaction medium plus 10 mM omithine, but without ammonia and bicarbonate. After 4 min, 0.5ml samples were withdrawn from the incubation mixture and subjected to silicone-oil centrifugation. After removal of the top layer, different volumes (O-25 ~1) of a stock solution of carbamoyl phosphate prepared in cold Buffer A were added to the acid phase underneath the oil with a Hamilton syringe. Samples were further handled as described under Materials and Methods. Carbamoyl phosphate was determined fluorimetrically. Each value rep resents the mean of two experiments with the same prep aration; measurements were carried out in triplicate.
nithine in the absence of ammonia and bicarbonate. Under these conditions, intramitochondrial carbamoyl phosphate is expected to be very low and, indeed, no carbamoyI phosphate could be detected (Fig. 3). Samples 01 ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 were taken from the incubation mixture and 0 60 120 0 60 120 time ImId centrifuged through silicone oil as described FIG. 2. Stability of carbamoyl phosphate. A stock so- under Materials and Methods. After careful lution of lithium carbamoyl phosphate was prepared by removal of the supernatant, different amounts dissolving the compound in an ice-cold medium con- of a cold standard solution of carbamoyl taining 100 mM KC], 50 mM Tris-HCl, 1 mM EGTA, 1 phosphate were added to the acid phase unmM ATP, 5 mM potassium phosphate, 20 mM succinate, 0- to 25and 10 mM ammonium chloride, final pH 7.4. At zero derneath the oil, using a Hamilton ~1 syringe. After resuspending the pellet in the time, 0.4 ml of the stock solution was added to polypropylene Micromedic tubes (4 ml) containing 1.6 ml of the acid with a glass rod, samples were prepared same medium plus perchloric acid resulting in the fol- as described under Materials and Methods. lowing final concentrations: 0, 0%; A, 3.5%; 0, 7%; 0, Added carbamoyl phosphate was completely 10.5%; and x, 14% (w/v) HClO,. Incubations were carried out at 25°C (Fig. 2A) and 0°C (Fig. 2B). After 30, 60, recovered (Fig. 3). and 120 mitt, 0.4 ml samples were taken, neutralized Mitochondrial Content of immediately with cold 2 M KOH/0.2 M Mops, and frozen Carbamoyl Phosphate in liquid nitrogen. The concentration of carbamoyl phosphate was determined as citrulline after reaction with ornithine and omithine transcarbamylasc. Determination via the spectrophotometric assayinvolving carbamate kinase gave almost identical values (not shown).
Using the fluorimetric assay, the mitochondrial content of carbamoyl phosphate was determined in mitochondria isolated from rats
DETERMINATION
OF
CARBAMOYL
fed a high-protein diet for 1 day and incubated with succinate (to generate ATP), NH3, and bicarbonate. Zero time values for intramitochondrial carbamoyl phosphate were below the limit of detection (~0.05 nmol/mg mitochondrial protein, not shown). After 4 min, in the absence of ornithine, intramitochondrial carbamoyl phosphate reached a level of 22 nmol/mg protein. In the presence of 3 mM omithine, carbamoyl phosphate levels decreased dramatically to a value of 0.06 nmol/mg protein (Table 1). When norvaline [which inhibits omithine carbamoyltransferase in a competitive manner with respect to omithine (21,22)] was added in the presence of omithine, citrulline production progressively decreased with increasing inhibitor concentrations; at the same time, intramitochondrial carbamoyl phosphate increased to about 10 nmol/mg protein at the highest concentration of inhibitor used (Table 1). Recovery of carbamoyl phosphate in this experiment was tested as described in the preceding paragraph and was almost complete (98%). DISCUSSION
Current procedures for a quantitative determination of low concentrations of carbamoyl phosphate, in which carbamoyl phosphate is converted to radioactively labeled carbamoyl aspartate or citrulline, are laborious and contain several sources of error [for a discussion, see Ref. (23)]. This is mainly due to the chromatographic procedures involved, which are necessary for prior removal of endogenous (unlabeled) aspartate or ornithine and to separate the labeled compounds. For removal of endogenous aspartate or omithine, the protein-free perchloric acid extracts must be applied onto a cation-exchange column in the acid form and a considerable amount of time is needed before the samples are neutralized. This may be one of the reasons why incomplete recovery has usually been reported (7, 9- 13). In this context, it is of interest to note that Christophersen and Finch (8) using the carbamoyl aspartate assay, reported complete recovery for carbamoyl phosphate in E. co/i
PHOSPHATE
85
extracts that contained very low amounts of endogenous aspartate, by omission of this step. In our present paper, a simple and sensitive method for the quantitative determination of carbamoyl phosphate is described. Chromatographic procedures are not required. Recovery of carbamoyl phosphate was almost complete. Since our method does not involve conversion of carbamoyl phosphate to either citrulline (with omithine) or carbamoyl aspartate (with aspartate), endogenous omithine or aspartate do not interfere with the assay. We have checked the possible complication of contaminating ornithine carbamoyltransferase or aspartate carbamoyltransferase activity present in the hexokinase, glucosed-phosphate dehydrogenase and carbamate kinase preparations used; this was not the case (not shown). The lack of interference by omithine is illustrated in Fig. 3, which shows excellent recovery of carbamoyl phosphate in the presence of omithine. The only drawback of our method is that its sensitivity is determined by the amount of ATP present relative to that of carbamoyl phosphate. In our hands, 0.1 nmol of carbamoyl phosphate could still be detected in the presence of a loo-fold excess of ATP. Beyond this ratio, however, the precision of the assay decreased markedly. In practice this limitation may become important whenever ATP present in the preparation under investigation is present in large excess over carbamoyl phosphate. For example, total intrahepatic ATP in the rat is about 2.5 pmol/g wet wt (24). According to Raijman (lo), intrahepatic carbamoyl phosphate is 110 nmol/ g wet wt. This amount can easily be detected with our method. Of course, such values only represent total intrahepatic carbamoyl phosphate. If hepatocytes are subjected to a rapid fractionation of mitochondria, such as the digitonin fractionation procedure (24), the distribution of carbamoyl phosphate across the mitochondrial membrane (in the intact hepatocyte) can be studied. Since rat liver mitochondrial ATP represents about 30% of the total cell ATP (24), the detection limit of in-
86
WANDERS
ET
TABLE
AL.
I
THECARBAMOYLPHOSPHATECONTEN? OF IS~LATEDRATLIVERMITOCHONDR~A INCUBATED UNDER DIFFERENT CONDITIONS
Additions Omithine 0 3 3 3 3 3
(mM) Norvaline
Citrulhne production (nmol . min-’ . mg-‘)
0 0 4 10 20 50
Intramitochondrial carbamoyl phosphate (nmol . mg-’ mitochondrial protein at 4 min)
0 13.1 11.0 8.8 7.5 4.3
22.1 0.06 0.85 1.9 5.9 9.9
Note. Mitochondria (4.0 mg protein/ml) were incubated exactly as described under Materials and Methods. Further additions are indicated in the table. Reactions were terminated after 4 min according to the procedure described under Materials and Methods. The rate of citrulline production was calculated from the measured citrulline concentrations found in perchloric acid extracts of samples taken from the incubation mixture at 2 and 4 min. Each value represents the mean of duplicate incubations with the same mitochondtial preparation; measurements were carried out in triplicate.
tramitochondrial carbamoyl phosphate is ‘/loo X i/3 X 2500 = 8 nmol/g wet wt, which equals 0.15 nmol/mg mitochondrial protein, using the generally accepted value of 60 mg mitochondrial protein/g wet wt of liver. With our assay, the concentration of carbamoyl phosphate in isolated rat liver mitochondria could easily be measured. The high concentrations of carbamoyl phosphate in mitochondria incubated with NH3 and bicarbonate, in the absence of ornithine (Table l), are in agreement with similar high values reported by Cohen et al. (25) and by Williamson et al. (26). The intramitochondrial carbamoyl phosphate concentration in the presence of ornithine (0.06 nmol/mg protein, Table 1) is lower than the value of 3-4 nmol/ mg protein reported by Cohen et al. (23, who estimated intramitochondrial carbamoyl phosphate as the difference between the measured intramitochondrial citrulline content and the measured sum of intramitochondrial carbamoyl phosphate plus citrulline after conversion of carbamoyl phosphate into citrulline. However, our low value of intramitochondrial carbamoyl phosphate is more in agreement with the value of 0.5 nmol/mg mitochondrial protein reported by Williamson
et al. (26); these authors determined
carbamoyl phosphate as phosphate after acid hydrolysis, correcting for intramitochondrial phosphate as calculated from the extramitochondrial phosphate concentration and the measured pH gradient across the mitochondrial inner membrane. It is likely that the differences in these intramitochondrial concentrations of carbamoyl phosphate are caused by differences in the magnitude of the flux through carbamoyl-phosphate synthetase. Cohen et al. (27) have recently shown that, in the presence of ornithine, intramitochondrial carbamoyl phosphate starts to accumulate when flux through carbamoyl-phosphate synthetase exceeds a value of about 40 nmol/mg mitochondrial protein. With the method described in this paper, we are currently investigating the reason for the large discrepancy in reported values for intrahepatic carbamoyl phosphate which range from 0.4 (11) to 110 (10) nmol/g wet wt. ACKNOWLEDGMENT This study was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO) under the auspices of the Netherlands Foundation for Fundamental Medical Research (FUNGO).
DETERMINATION
OF CARBAMOYL
REFERENCES 1. Jones, M. E., Spector, L., and Lipmann, F. (1955) J. Amer. Chem. Sot. 11,819~820. 2. Grisolia, S., and Cohen, P. P. (1951) J. Biol. Chem. 198, 561-571. 3. Kennedy, J. (1976) in The Urea Cycle (Grisolia, S., Baguena, R., and Mayor, F., eds.), pp. 39-54, Wiley, New York. 4. Jones, M. E., and Lipmann, F. (1960) Proc. Nut. Ad. Sci. USA 46, 1194-1205. 5. Halmann, M., Lapidot, A., and Samuel, D. (1962) J. Chem. Sot. 1944-1957. 6. Allen, C. M., and Jones, M. E. (1964) Biochemistry 3, 1238-1247. 7. Herzfeld, A., Hager, S. E., and Jones, M. E. (1964) Arch. Biochem. Biophys. 107, 544-549. 8. Christopherson, R. I., and Finch, L. R. (1976) Anal. Biochem. 73, 342-349. 9. Williams, L. G., Bemhardt, S. A., and Davis, R. H. (1971) J. Biol. Chem. 246, 973-978. 10. Raijman, L. (1974) B&hem. J. 1X$225-232. 11. Tatibana, M. and Shigesada, K. (I 976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 301-313, Wiley, New York. 12. Shigesada, K., Aoyagi, K., and Tatibana, M. (1978) Eur. J. Biochem. 85, 385-391. 13. Huisman, W. H., and Becker, M. A. (1980) Anal. Biochem. 101, 160-165. 14. Anderson, P. M., Wellner, V. P., Rosenthal, G. A., and Meister, A. (1970) in Methods in Enzymology
15. 16. 17. 18. 19. 20.
PHOSPHATE
87
(Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 235-243, Academic Press, New York. Pierson, D. L. (1980) J. Biochem. Biophys. Methods 3, 3 l-37. Hogeboom, G. H. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 16-19, Academic Press, New York. Myers, D. K., and Slater, E. C. (1957) B&hem. J. 67, 558-572. Williamson, J. R., and Corkey, B. E. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 13, pp. 434-5 13, Academic Press, New York. Cleland, K. W., and Slater, E. C. (1953) Biochem. J. 53, 547-556. Marshall, M., and Cohen, P. P. (1966) J. Biol. Chem. 241, 4197-4208.
2 1. Marshall, M., and Cohen, P. P. (1972) J. Biol. Chem. 247, 1654-1668. 22. Lusty, C. J., Jilka, R. L., and Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036. 23. Jones, M. E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 4, pp. 17491757, Verlag Chemie, Weinheim. 24. Akerboom, T. P. M., Van der Meer. R., and Tager. J. M. (1979) Tech. Metub. Res. B205, l-33. 25. Cohen, N. S., Cheung, C. W., and Raijman, L. (1980) J. Biol. Chem. 255, 10248-10255. 26. Williamson, J. R., Steinman, R., Coil, K.. and Rich, T. (1981) J. Bid. Chem. 256, 7287-7297. 27. Cohen, N. S.. Cheung, C. W., Kyan, F. S., Jones, E. E., and Raijman, L. (1982) J. Biol. Chem. 257, 6898-6907.
BIOCHEMISTRY
129.80-87
Fluorimetric RONALDJ.
(1983)
Determination
of Carbamoyl
Phosphate
A. WANDERS, CARLOVAN ROERMUND, COR LOF,ANDALFRED
J.MEIJER
Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, P.O. Box 20151. 1000 HD Amsterdam, The Netherlands Received July 5, 1982 A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay,production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present. KEY WORDS: carbamoyl phosphate; omithine carbamoyl transferase; citrulline synthesis; aspartate carbamoyl transfer%.
The list of intermediates involved in urea biosynthesis was completed in 1955 when Jones et al. (1) identified compound X (2) as carbamoyl phosphate1 From the time of its discovery, it was recognized that solutions of carbamoyl phosphate are unstable both at acid and at alkaline pHs (for a review, see Ref. (3)). At an acid pH, the decomposition of carbamoyl phosphate generates ammonia, carbon dioxide, and inorganic phosphate (4-6), whereas at an alkaline pH, cyanate and phosphate are produced (4-6). The instability of carbamoyl phosphate is presumably one of the reasons why so little information about the intracellular concentrations of this compound is available. Three methods have been described for the determination of carbamoyl phosphate in tissue extracts. In the first (7,8), carbamoyl phosphate is quantitatively converted into stable [‘4C]carbamoyl-L-aspartate after reaction with [14C]aspartate and purified aspartate carbamoyltransferase (EC 2.1.3.2). The labeled compound is separated from [ “C]aspartate by column chromatography. With this method, the carbamoyl phosphate content of blood plasma (7) and Escherichia coli cells (8) has been determined. In the second method (9- 13), purified ornithine carbamoyltransferase (EC 2.1.3.3) and
0003-2697/83/030080-08$03.00/0 CopyrigJa 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
[‘4C]ornithine are used for the conversion of carbamoyl phosphate into stable [i4C]citrulline. This is followed by separation of [ “C]omithine and [‘4C]citrulline using cation-exchange chromatography (9- 13). In the third method, carbamoyl phosphate is determined after its direct chemical conversion to urea or hydroxyurea ( 14,15). In the present paper, a rapid fluorimetric method for the determination of very small amounts of carbamoyl phosphate is described. In this assay, chromatographic procedures are not required. MATERIALS
AND METHODS
Isolation of mitochondria. Rat liver mitochondria were isolated from male Wistar rats (200-250 g) by the method of Hogeboom (16), as described by Myers and Slater (17), using 250 mM mannitol, 5 mM Tris-HCl and 0.5 mrvt ethylene glycol bis(&aminoethyl ether) iV,N,N’,N’-tetraacetic acid (EGTA) (final pH 7.4) as the isolation medium. The final pellet (about 150 mg protein) was suspended in 34 ml 250 mM mannitol and kept on ice. Incubation conditions. Mitochondria (3-5 mg protein/ml) were incubated at 25°C in a standard reaction medium containing the following components: 100 mM KCl, 50 mM
80
DETERMINATION
OF CARBAMOYL
PHOSPHATE
81
neutralized extracts in liquid nitrogen (about 5 min). Fluorimetric determination of carbamoyl phosphate. Carbamoyl phosphate was measured fluorimetrically using an Eppendorf photometer with a fluorescence attachment (Type 1030; primary filter, 366 nm, secondary filter, >400 nm) equipped with a linear Servogor recorder (Model 2 10). To a l-cm2 quartz cuvette containing 2.00 ml Buffer A [consisting of 50 mM triethanolamine-HCl, 10 mM MgS04, 5 mM ethylenediaminetetraacetic acid (EDTA), and 75 mM KCl; pH 7.41, 5 ~1NADP+ (5 mg/ml), 5 ~1 1 M glucose, 10 ~1 0.1 M ADP, 5 ~1 glucose-6-phosphate dehydrogenase (NADP+) from yeast (5 mg/ ml; 140 IU/mg; diluted 1:5 with distilled water), a sample of 75-100 ~1 was added. When a constant recording signal was obtained, 5 ~1 hexokinase (10 mg/ml, 140 IU/mg; diluted 1:5 with distilled water) was added. After completion of the reaction, the recorder pen was reset to the baseline and 5 ~1 carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) was added (700 U/mg enzyme protein; 1 mg lyophilized material dissolved in 250 ~1 Buffer A). Calibration was done with the internal standard procedure (18), using carbamoyl phosphate standard solutions freshly prepared each day and kept on ice in Buffer A. The concentrations of the carbamoyl phosphate standard solutions were determined spectrophotometrically on a ZeissPMQ-II spectrophotometer equipped with a logarithmic Servogor recorder, using the same reaction mixture as described for the fluorimetric assay with the exception that a higher concentration of NADP was added (final concentration in cuvette 0.4 mM). In some experiments, carbamoyl phosphate was determined calorimetrically as citrulline; in that case, samples containing carbamoyl phosphate were incubated at 25’C for 30 min in a medium containing 50 mM TrisHCl, 20 mM ornithine, 2 mM EDTA, and 1 ’ Abbreviations used: EGTA, ethylene glycol bi@aminoethyl ether) N,N,N’,N’-tetraacetic acid; Mops, 4- U/ml ornithine carbamoyl transferase from moruholine .orooanesulfonic acid. -Streptococcus faecaks (final pH 7.5). Reac.
Tris-HCl, 1 mM EGTA’, 1 mM ATP, 5 mM potassium phosphate, 20 mM succinate, 10 mM ammonium chloride, 16.6 mM potassium bicarbonate, and 2.5 &ml rotenone. The final pH was 7.4. Ornithine was present where indicated. Incubations were carried out at 25°C in a thermostatically controlled reaction vessel with vigorous stirring under a continuous stream of 95% 02/5% CO*. Extraction procedurefor mitochondrial carbamoyl phosphate. For the determination of intramitochondrial carbamoyl phosphate, we used the silicone oil centrifugation technique to separate the mitochondria from their suspending medium. For this purpose, Eppendorf centrifuge tubes ( 1.6 ml) were filled with 0.25 ml of a solution containing 7% (w/v) perchloric acid, 0.5 M mannitol, and 20 mM EDTA, followed by an 0.4-ml layer of silicone oil (Wacker AR 200:AR 20 = 270:60 (v/v); density 1.033 g/ml at 25’C). The tubes were precooled in an ice-water bath. At the time desired, a 0.5- to 0.8-ml sample was drawn from the mitochondrial incubation mixture, layered on top of the silicone oil, and centrifuged at 12 OOOg for 60 s in an Eppendorf centrifuge (Model 3200). After careful removal of the supematant and rinsing of the wall of the tube above the oil with cold distilled water to remove any residual extramitochondrial fluid, the mitochondrial pellet in the acid layer was resuspended with a glass rod in order to obtain full extraction of the mitochondria. After removal of the denatured protein by centrifugation in the cold, a sample (0.2 ml) was taken from the acid phase below the oil, neutralized with cold 2 M KOH/0.2 M 4-morpholinepropanesulfonic acid (Mops) to pH 7.00 + 0.25, and immediately frozen in liquid nitrogen. All operations were carried out in ice water as quickly as possible to minimize the time elapsing between the start of the centrifugation and final freezing of the
82
WANDERS
lions were stopped with perchloric acid (final concentration 3.5% (w/v)) and citrulline was determined in the protein-free acid extracts. Citrulline determination. Citrulline was determined according to the method of Pierson (15) in 4-ml polypropylene tubes in a final volume of 1 ml. For each series of determinations, a calibration curve consisting of 10 points (O-150 nmol) was constructed with a stock solution of L-citrulline (1 mM), stored frozen. All determinations were carried out at least in duplicate. Protein determination. Protein was determined exactly as described by Cleland and Slater (19), using egg albumin as a standard. Enzymes and reagents. Carbamate kinase and omithine carbamoyltransferase (as lyophilized powders) and dilithium carbamoyl phosphate were obtained from Sigma Chemical Co. (St. Louis, Missouri). Other enzymes and nucleotides were obtained from Boehringer GmbH (Mannheim, FRG). All other reagents were of analytical grade. RESULTS
Principle of the Method Carbamate kinase catalyzes the reversible conversion of carbamate and ATP to carbamoyl phosphate and ADP. The equilibrium position of this reaction lies far towards carbamoyl phosphate and ADP formation (K, = 24.2 X lo3 in the presence of 10 mM MgC12 (20)). Consequently, quantitative determination of carbamoyl phosphate is only possible if the reaction products are removed. This can be achieved by coupling the carbamate kinase reaction with the hexokinase and glucose-6phosphate dehydrogenase reactions (20). An additional advantage of the coupled system is that conversion of carbamoyl phosphate can be measured directly by following the appearance of NADPH spectrophotometrically or fluorimetrically. The validity of the method was first investigated with dilithium-carbamoyl phosphate standard solutions (see Materials and Meth-
ET
AL.
ods). Carbamoyl phosphate was measured spectrophotometrically at 340 nm, using the carbamate kinase/hexokinase/glucose-6phosphate dehydrogenase system, and also calorimetrically as citrulline after incubation with omithine and omithine carbamoyltransferase. The agreement between the two methods was 99.2 f 1.2% (means +- SEM of 10 determinations) with carbamoyl phosphate concentrations ranging from 20 to 200 nmol/ml. The levels of carbamoyl phosphate in animal tissues are usually too low to be determined spectrophotometrically. We therefore set up a fluorimetric assay procedure, since a loo-fold gain in sensitivity is achieved by measuring changes in fluorescence rather than in absorbance of NAD(P)H. Standard curves were found to be linear for amounts of carbamoyl phosphate corresponding to concentrations in the cuvette of O-5 pM (results not shown). Since most tissues contain appreciable amounts of ATP (IQ this nucleotide must first be removed before carbamoyl kinase is added (Fig. 1). This did not obstruct sensitive and quantitative measurement of carbamoyl phosphate, as is shown in Fig. 1. Reactions were usually fast, being completed in 5- 10 min, with hardly any drift.
Stability of Carbamoyl Phosphate Instability of carbamoyl phosphate, especially at low and high pH values (4-6), presents a serious difficulty, since deproteinizalion of tissue usually occurs via acidification. Indeed, variable recoveries of carbamoyl phosphate have been reported, ranging from low (36%, Ref. (9)) to very high (98%, Ref. (8)) (see also under Discussion). Other authors (7, 10-13) reported a recovery between these two extremes. In order to find optimal conditions for tissue extraction, we studied the decomposition of dilithium-carbamoyl phosphate as a function of the acidity of the medium and the incubation time, at two temperatures (0 and 25°C). At 25°C hydrolysis
DETERMINATION
z
OF CARBAMOYL
PHOSPHATE
hexokinase
FIG. 1. Fhtorimetric determination of carbamoyl phosphate. Rat liver mitochondria (3.1 mg protein/ ml) isolated from rats fed a normal diet were incubated in the standard reaction medium in the absence of omithine. After 4 min, a 0.5-m] sample was drawn from the incubation mixture, subjected to siliconeoil centrifugation, and further handled as described under Materials and Methods. 0.075 ml of the neutralized perchloric acid extract of the pellet was added to the cuvette containing the reaction mixture described under Materials and Methods. When temperature equilibration was complete, hexokinase was added. After completion of the reaction and readjustment of the recorder, carbamate kinase was added. Calibration was carried out with an internal standard.
83
84
WANDERS
of carbamoyl phosphate was found to be dependent upon the concentration of perchloric acid (in agreement with the data of Halmann et al. (5)) and to increase with the time of incubation (Fig. 2A). If, however, the incubation was carried out in an ice-water bath, hydrolysis occurred to a limited extent only (Fig. 2B). In the light of these experimental findings, a protocol for the termination of the reactions was set up which minimized hydrolysis of carbamoyl phosphate (see Materials and Methods). In the experiment shown in Fig. 3, the results of this procedure with regard to the recovery of carbamoyl phosphate during sample preparation was tested. Isolated mitochondria were incubated for 10 min in the standard reaction medium plus 10 mM or-
ET AL.
nmol carbamoyl
phosphate
added
RG. 3. Recovery of carbamoyl phosphate during preparation of perchloric acid extracts of mitochondria. Mitochondria (4.5 mg protein/ml) were incubated at 25°C in the standard reaction medium plus 10 mM omithine, but without ammonia and bicarbonate. After 4 min, 0.5ml samples were withdrawn from the incubation mixture and subjected to silicone-oil centrifugation. After removal of the top layer, different volumes (O-25 ~1) of a stock solution of carbamoyl phosphate prepared in cold Buffer A were added to the acid phase underneath the oil with a Hamilton syringe. Samples were further handled as described under Materials and Methods. Carbamoyl phosphate was determined fluorimetrically. Each value rep resents the mean of two experiments with the same prep aration; measurements were carried out in triplicate.
nithine in the absence of ammonia and bicarbonate. Under these conditions, intramitochondrial carbamoyl phosphate is expected to be very low and, indeed, no carbamoyI phosphate could be detected (Fig. 3). Samples 01 ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 were taken from the incubation mixture and 0 60 120 0 60 120 time ImId centrifuged through silicone oil as described FIG. 2. Stability of carbamoyl phosphate. A stock so- under Materials and Methods. After careful lution of lithium carbamoyl phosphate was prepared by removal of the supernatant, different amounts dissolving the compound in an ice-cold medium con- of a cold standard solution of carbamoyl taining 100 mM KC], 50 mM Tris-HCl, 1 mM EGTA, 1 phosphate were added to the acid phase unmM ATP, 5 mM potassium phosphate, 20 mM succinate, 0- to 25and 10 mM ammonium chloride, final pH 7.4. At zero derneath the oil, using a Hamilton ~1 syringe. After resuspending the pellet in the time, 0.4 ml of the stock solution was added to polypropylene Micromedic tubes (4 ml) containing 1.6 ml of the acid with a glass rod, samples were prepared same medium plus perchloric acid resulting in the fol- as described under Materials and Methods. lowing final concentrations: 0, 0%; A, 3.5%; 0, 7%; 0, Added carbamoyl phosphate was completely 10.5%; and x, 14% (w/v) HClO,. Incubations were carried out at 25°C (Fig. 2A) and 0°C (Fig. 2B). After 30, 60, recovered (Fig. 3). and 120 mitt, 0.4 ml samples were taken, neutralized Mitochondrial Content of immediately with cold 2 M KOH/0.2 M Mops, and frozen Carbamoyl Phosphate in liquid nitrogen. The concentration of carbamoyl phosphate was determined as citrulline after reaction with ornithine and omithine transcarbamylasc. Determination via the spectrophotometric assayinvolving carbamate kinase gave almost identical values (not shown).
Using the fluorimetric assay, the mitochondrial content of carbamoyl phosphate was determined in mitochondria isolated from rats
DETERMINATION
OF
CARBAMOYL
fed a high-protein diet for 1 day and incubated with succinate (to generate ATP), NH3, and bicarbonate. Zero time values for intramitochondrial carbamoyl phosphate were below the limit of detection (~0.05 nmol/mg mitochondrial protein, not shown). After 4 min, in the absence of ornithine, intramitochondrial carbamoyl phosphate reached a level of 22 nmol/mg protein. In the presence of 3 mM omithine, carbamoyl phosphate levels decreased dramatically to a value of 0.06 nmol/mg protein (Table 1). When norvaline [which inhibits omithine carbamoyltransferase in a competitive manner with respect to omithine (21,22)] was added in the presence of omithine, citrulline production progressively decreased with increasing inhibitor concentrations; at the same time, intramitochondrial carbamoyl phosphate increased to about 10 nmol/mg protein at the highest concentration of inhibitor used (Table 1). Recovery of carbamoyl phosphate in this experiment was tested as described in the preceding paragraph and was almost complete (98%). DISCUSSION
Current procedures for a quantitative determination of low concentrations of carbamoyl phosphate, in which carbamoyl phosphate is converted to radioactively labeled carbamoyl aspartate or citrulline, are laborious and contain several sources of error [for a discussion, see Ref. (23)]. This is mainly due to the chromatographic procedures involved, which are necessary for prior removal of endogenous (unlabeled) aspartate or ornithine and to separate the labeled compounds. For removal of endogenous aspartate or omithine, the protein-free perchloric acid extracts must be applied onto a cation-exchange column in the acid form and a considerable amount of time is needed before the samples are neutralized. This may be one of the reasons why incomplete recovery has usually been reported (7, 9- 13). In this context, it is of interest to note that Christophersen and Finch (8) using the carbamoyl aspartate assay, reported complete recovery for carbamoyl phosphate in E. co/i
PHOSPHATE
85
extracts that contained very low amounts of endogenous aspartate, by omission of this step. In our present paper, a simple and sensitive method for the quantitative determination of carbamoyl phosphate is described. Chromatographic procedures are not required. Recovery of carbamoyl phosphate was almost complete. Since our method does not involve conversion of carbamoyl phosphate to either citrulline (with omithine) or carbamoyl aspartate (with aspartate), endogenous omithine or aspartate do not interfere with the assay. We have checked the possible complication of contaminating ornithine carbamoyltransferase or aspartate carbamoyltransferase activity present in the hexokinase, glucosed-phosphate dehydrogenase and carbamate kinase preparations used; this was not the case (not shown). The lack of interference by omithine is illustrated in Fig. 3, which shows excellent recovery of carbamoyl phosphate in the presence of omithine. The only drawback of our method is that its sensitivity is determined by the amount of ATP present relative to that of carbamoyl phosphate. In our hands, 0.1 nmol of carbamoyl phosphate could still be detected in the presence of a loo-fold excess of ATP. Beyond this ratio, however, the precision of the assay decreased markedly. In practice this limitation may become important whenever ATP present in the preparation under investigation is present in large excess over carbamoyl phosphate. For example, total intrahepatic ATP in the rat is about 2.5 pmol/g wet wt (24). According to Raijman (lo), intrahepatic carbamoyl phosphate is 110 nmol/ g wet wt. This amount can easily be detected with our method. Of course, such values only represent total intrahepatic carbamoyl phosphate. If hepatocytes are subjected to a rapid fractionation of mitochondria, such as the digitonin fractionation procedure (24), the distribution of carbamoyl phosphate across the mitochondrial membrane (in the intact hepatocyte) can be studied. Since rat liver mitochondrial ATP represents about 30% of the total cell ATP (24), the detection limit of in-
86
WANDERS
ET
TABLE
AL.
I
THECARBAMOYLPHOSPHATECONTEN? OF IS~LATEDRATLIVERMITOCHONDR~A INCUBATED UNDER DIFFERENT CONDITIONS
Additions Omithine 0 3 3 3 3 3
(mM) Norvaline
Citrulhne production (nmol . min-’ . mg-‘)
0 0 4 10 20 50
Intramitochondrial carbamoyl phosphate (nmol . mg-’ mitochondrial protein at 4 min)
0 13.1 11.0 8.8 7.5 4.3
22.1 0.06 0.85 1.9 5.9 9.9
Note. Mitochondria (4.0 mg protein/ml) were incubated exactly as described under Materials and Methods. Further additions are indicated in the table. Reactions were terminated after 4 min according to the procedure described under Materials and Methods. The rate of citrulline production was calculated from the measured citrulline concentrations found in perchloric acid extracts of samples taken from the incubation mixture at 2 and 4 min. Each value represents the mean of duplicate incubations with the same mitochondtial preparation; measurements were carried out in triplicate.
tramitochondrial carbamoyl phosphate is ‘/loo X i/3 X 2500 = 8 nmol/g wet wt, which equals 0.15 nmol/mg mitochondrial protein, using the generally accepted value of 60 mg mitochondrial protein/g wet wt of liver. With our assay, the concentration of carbamoyl phosphate in isolated rat liver mitochondria could easily be measured. The high concentrations of carbamoyl phosphate in mitochondria incubated with NH3 and bicarbonate, in the absence of ornithine (Table l), are in agreement with similar high values reported by Cohen et al. (25) and by Williamson et al. (26). The intramitochondrial carbamoyl phosphate concentration in the presence of ornithine (0.06 nmol/mg protein, Table 1) is lower than the value of 3-4 nmol/ mg protein reported by Cohen et al. (23, who estimated intramitochondrial carbamoyl phosphate as the difference between the measured intramitochondrial citrulline content and the measured sum of intramitochondrial carbamoyl phosphate plus citrulline after conversion of carbamoyl phosphate into citrulline. However, our low value of intramitochondrial carbamoyl phosphate is more in agreement with the value of 0.5 nmol/mg mitochondrial protein reported by Williamson
et al. (26); these authors determined
carbamoyl phosphate as phosphate after acid hydrolysis, correcting for intramitochondrial phosphate as calculated from the extramitochondrial phosphate concentration and the measured pH gradient across the mitochondrial inner membrane. It is likely that the differences in these intramitochondrial concentrations of carbamoyl phosphate are caused by differences in the magnitude of the flux through carbamoyl-phosphate synthetase. Cohen et al. (27) have recently shown that, in the presence of ornithine, intramitochondrial carbamoyl phosphate starts to accumulate when flux through carbamoyl-phosphate synthetase exceeds a value of about 40 nmol/mg mitochondrial protein. With the method described in this paper, we are currently investigating the reason for the large discrepancy in reported values for intrahepatic carbamoyl phosphate which range from 0.4 (11) to 110 (10) nmol/g wet wt. ACKNOWLEDGMENT This study was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO) under the auspices of the Netherlands Foundation for Fundamental Medical Research (FUNGO).
DETERMINATION
OF CARBAMOYL
REFERENCES 1. Jones, M. E., Spector, L., and Lipmann, F. (1955) J. Amer. Chem. Sot. 11,819~820. 2. Grisolia, S., and Cohen, P. P. (1951) J. Biol. Chem. 198, 561-571. 3. Kennedy, J. (1976) in The Urea Cycle (Grisolia, S., Baguena, R., and Mayor, F., eds.), pp. 39-54, Wiley, New York. 4. Jones, M. E., and Lipmann, F. (1960) Proc. Nut. Ad. Sci. USA 46, 1194-1205. 5. Halmann, M., Lapidot, A., and Samuel, D. (1962) J. Chem. Sot. 1944-1957. 6. Allen, C. M., and Jones, M. E. (1964) Biochemistry 3, 1238-1247. 7. Herzfeld, A., Hager, S. E., and Jones, M. E. (1964) Arch. Biochem. Biophys. 107, 544-549. 8. Christopherson, R. I., and Finch, L. R. (1976) Anal. Biochem. 73, 342-349. 9. Williams, L. G., Bemhardt, S. A., and Davis, R. H. (1971) J. Biol. Chem. 246, 973-978. 10. Raijman, L. (1974) B&hem. J. 1X$225-232. 11. Tatibana, M. and Shigesada, K. (I 976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 301-313, Wiley, New York. 12. Shigesada, K., Aoyagi, K., and Tatibana, M. (1978) Eur. J. Biochem. 85, 385-391. 13. Huisman, W. H., and Becker, M. A. (1980) Anal. Biochem. 101, 160-165. 14. Anderson, P. M., Wellner, V. P., Rosenthal, G. A., and Meister, A. (1970) in Methods in Enzymology
15. 16. 17. 18. 19. 20.
PHOSPHATE
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(Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 235-243, Academic Press, New York. Pierson, D. L. (1980) J. Biochem. Biophys. Methods 3, 3 l-37. Hogeboom, G. H. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 16-19, Academic Press, New York. Myers, D. K., and Slater, E. C. (1957) B&hem. J. 67, 558-572. Williamson, J. R., and Corkey, B. E. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 13, pp. 434-5 13, Academic Press, New York. Cleland, K. W., and Slater, E. C. (1953) Biochem. J. 53, 547-556. Marshall, M., and Cohen, P. P. (1966) J. Biol. Chem. 241, 4197-4208.
2 1. Marshall, M., and Cohen, P. P. (1972) J. Biol. Chem. 247, 1654-1668. 22. Lusty, C. J., Jilka, R. L., and Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036. 23. Jones, M. E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 4, pp. 17491757, Verlag Chemie, Weinheim. 24. Akerboom, T. P. M., Van der Meer. R., and Tager. J. M. (1979) Tech. Metub. Res. B205, l-33. 25. Cohen, N. S., Cheung, C. W., and Raijman, L. (1980) J. Biol. Chem. 255, 10248-10255. 26. Williamson, J. R., Steinman, R., Coil, K.. and Rich, T. (1981) J. Bid. Chem. 256, 7287-7297. 27. Cohen, N. S.. Cheung, C. W., Kyan, F. S., Jones, E. E., and Raijman, L. (1982) J. Biol. Chem. 257, 6898-6907.