[CANCER RESEARCH 34, 3058-3061, November 1974] Kinetic Regulation of Adenylate Kinases from Muscle, Liver, and Hepatoma1

Tapas K. Pradhan, Wayne E. Criss,2and Harold P. Morris Department of Obstetrics and Gynecology, University ofFlorida College ofMedicine, Gainesville, Florida 32610 [T. K. P., W.E.C.], and Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 20001 [H. P. M.]

SUMMARY rat tissues (8—15,17,25). Major AK activity is localized in the outer mitochondnial compartment in liver and kidney, Homogeneous preparations of the major adenylate ki but is found predominantly in the cytoplasm of skeletal nases (EC 2.7.4.3) from rat liver, skeletal muscle, and Mor muscle, brain, and fast-growing hepatomas. The liver form ris hepatoma 3924A have been compared kinetically. At of AK responds to hormonal and dietary manipulations; concentrations of 50 mr@i,most citric acid-cycle interme the muscle and hepatoma forms do not. Measurements of diates activated the enzyme from each tissue. The activa tissue levels of ATP, ADP, and AMP, and calculation of tion constants of citrate for liver, muscle, and hepatoma the adenylate charge and an AK reaction parameter (Q), adenylate kinases were 0.09, 0.26, and 15.8 mM, respec indicate that AK in liver is near equilibrium, while the AK tively. It is likely that citrate could effect the in vivo func in fast-growing hepatomas is not near equilibrium. Thus, a tioning of liver adenylate kinase. potential loss of the enzymatic portion of the adenylate Apparent Michaelis constants were 1.9, 6.2, and 18.0 energy-charge system in neoplasia may have serious effects mM for adenosine 5'-monophosphate and were 7.0, 10.0, upon a tumor tissue's ability to maintain a normal homeo and 33.0 mM for adenosine 5'-triphosphate (ATP) with static internal environment. liver, muscle, and hepatoma enzymes, respectively. Citrate Therefore, we have isolated and purified to homogeneity decreased the Km (ATP) with liver and muscle aden the major AK's from rat liver, skeletal muscle, and the ylate kinases; it decreased the Km (adenosine 5'-monophos fast-growing Morris hepatoma 3924A. This and the fol phate) with only the muscle adenylate kinase. lowing report (14) compare the kinetic and physical protein Most Hill plot-slope values (for ATP) were near 1.5, in parameters of the 3 enzymes. dicating partial ligand-ligand cooperativity. Only in the presence of citrate, and with ATP as the variable substrate, was the slope value greater than two for liver adenylate ki MATERIALS AND METHODS nase. Several kinetic studies, in which modulation of the pun Liver, muscle, and tumor AK's from rats were prepared fled enzymes by nucleoside 5'-diphosphates, free fatty and purified according to the scheme as previously de acids, mercurial reagents, and detergent, indicate that all scribed (15). The final specific activities of liver, muscle, three adenylate kinases have distinct and unique kinetic and tumor AK's were 700, > 1000, and 390 units/mg pro properties. tein, respectively. Electrophoresis of the purified enzymes by polyacrylamide disc gel electrophoresis revealed only I protein band for each enzyme; this band contained all of the INTRODUCTION enzymatic activity. Sedimentation equilibrium analysis by analytical ultracentnifugation revealed 1 peak for each en The adenylate energy change is postulated to be the cen zyme. tral energy-mediating system of mammalian cells (1—4,16, The assay system routinely consisted of 50 mr@ttriethanol 20). The adenylate system is composed of AK,3 ATP, amine-HCI buffer at pH 7.0, 15 mM ATP, 22.5 mM AMP ADP, and AMP. The components of this system are cen (or 20 mM ADP), and 15 m@iMgCl2 in 1 ml final volume. tnal to numerous metabolic pathways including: glycolysis, Upon observations of the high Km value for tumor AK, we gluconeogenesis, lipogenesis, lipolysis, oxidative phos used 60 mM each of ATP, AMP, and Mg2@ in the assay for phorylation, RNA, DNA, and protein metabolism, and tumor AK. The concentrations of the modulators and re membrane transport. agents used in the assay system are indicated in “Results.― We have identified several different electrophoretic The assay system was preincubated in a 37°shaker bath forms of AK (EC 2.7.4.3) in various normal and neoplastic for 15 mm. One hundred sI of purified AK preparation (I 5) were then added to start the reaction. The assay sys ‘Thisresearch was supported by Grants CA-l 1818, CA-l0729, and tem was subsequently incubated for 15 mm at 37°.The CA-l0906 from the NIH, and Grant F73-UF4 from the Florida Division reaction was stopped by the addition of 1 ml of cold 1.5 N of the American Cancer Society. perchloric acid and immediate immersion in ice. One hun 2Recipient of NIH Research Career Development Award CA-70I87. 3The abbreviation used is: AK, adenylate kinase. dred sl of the reaction mixture were spotted on Whatman ReceivedApril IS, 1974:accepted August 2, 1974. 3MM filter paper previously moistened with 0.05 M citrate

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buffer, pH 3.3. The spotted paper was placed on a Savant flat-plate electrophoresis system using 0.05 M citrate buf 15 fer, pH 3.3, as electrolytes, and 10 mm were allowed to 10 attain equilibrium. High-voltage electrophoresis was car ned out at 1500 V for 3 hr at 2°.The paper was blotted 5 and dried for 1 hr at 80°.Adenine nucleotide spots were J@T@TT detected with Mineralight UVS-l2, cut into several 5 10 pieces, and placed into vials that contained 3 ml of 0.75 M NH4HCO3 solution. The absorbancy was determined in a Beckman (Acta II) spectrophotometer at 259-nm wave 1@ length. The concentrations of nucleotides were calculated from the molar extinction coefficient at 15.4 x 106 at 259 nm, and AK activity was expressed as smoles of adenine nucleotide produced per ml of assay medium per mm at I I I I I 37°.All reported data are averages from 6 or more repeti tive determinations. The various reagents were prepared by dissolving in 50 mM tniethanolamine-HCI buffer and the pH was adjusted to 7.0. All chemicals were purchased as described in previous papers (8—13,15). All the modulators were obtained from Sigma Chemical Co., St. Louis, Mo., while all of the nu cleotides were purchased from P-L Biochemicals, Inc., Milwaukee, Wis. I I 0.05 0.1 RESULTS 1/Citrate (mM) Chart 1. Double-reciprocal plots of final velocity minus initial velocity Effect of Citric Acid-Cycle Intermediates. Most citric divided by initial velocity versus final citrate concentration for muscle AK acid-cycle intermediates (at 50 mM) activated the liver, (A), liver AK (B), and hepatoma AK (C). tumor, and muscle AK's (Table 1). Liver AK was acti vated by citrate, isocitrate, malate, fumarate, a-ketoglu Michaelis Constants. The apparent Michaelis constants tarate, and cis-aconitate. Tumor enzyme was activated by (ATP and AMP) of liver, muscle, and tumor AK's were all intermediates. The activity of muscle AK was enhanced determined in the presence and absence of citrate (Table 2). by citrate, isocitrate, oxaloacetate, a-ketoglutarate and The values ranged from Km (AMP) of 1.9 for liver AK to cis-aconitate. Km (ATP) of 38.0 for tumor AK. Citrate decreased the Activation Constant. A double-reciprocal plot of final Km (AMP) from 18 to 10 for tumor AK and from 6.2 to velocity minus initial velocity divided by initial velocity 3.3 for muscle AK; it decreased the Km (ATP) of 7.0 to versus final citrate concentration was linear and produced 4.2 for liver AK and from 10.0 to 4.0 for muscle AK. There activation constant (K6) values of citrate for tumor AK, fore, it appears that citrate may not activate the various liver AK, and muscle AK of 15.8, 0.09, and 0.26 m@t, re tissue forms of AK by exactly the same mechanism. spectively (Chart I). With a K6 value of less than 0. 1 mr@i Hill Plots Hill plotting with varying concentrations of for liver AK and with measured values of0.2 m@ifor in vivo ATP and AMP for liver, tumor, and muscle AK's in the concentrations of citrate in liver tissue, it is likely that ci presence and absence of citrate yielded various slope values trate may influence the activity of this form of liver AK. (Table 3). There was indication of cooperative interaction for liver enzyme (with ATP as variable substrate) when ci trate was not present in the assay. In the absence of citrate, Table I the Hill plot slope was 2. 1; in the presence of citrate, it was Effect of citric acid-cycle intermediates 1.45. We did not observe any other n values greater than Treatment included preincubation of nucleotides and intermediates in 1.55 in any of the studies, although all n values (with ATP 50 mMtriethanolamine-HCI buffer at pH 7.0 for 15mm at 37°,afterwhich as variable substrate) was near 1.5, indicating some degree they were assayed. Final concentration of each intermediate was 50 mM. Stimulation is expressed as mean ±SE. (%) of control activity. of partial cooperative ligand interaction. Effect of Modulators. Various kinetic studies with dif AKCitrate+IntermediatesLiver AKTumor AKMuscle ferent modulators have been performed with liver, tumor, and muscle AK's (Table 4). Only the nucleoside 5'-diphos 4Isocitrate+34±6+51±5+19±3Malate+40166 ± 9+25 ±4+48 ± phates stimulated the 3 enzymes, and the stimulation was 30Fumarate+30 ±5+22 ± only in the direction of ADP production. Other modulators 30Succinate0+27 ±6+ 18 ± had no effect on any of the enzymes under these assay con 30Oxaloacetate0+61 ± ditions. Nucleotides that were without effect included 2a-Ketoglutarate+17±4+21±4+12±2cis-Aconitate+28 ±6+ 10 ± GTP, CTP, UTP, TTP, 2'-dGTP, 2'-dCTP, 2'-dATP, ±4+30 ±5+31 ±4 2'-dAMP, GMP, CMP, IMP, TMP, UMP, and adeno sine 3',S'-cyclic phosphate.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. T. K. Pradhan et a!. Table 2 InhibitorConstant(K1)for MercurialReagents.The Comparison of values for Michaelis constants (K,,,) inhibitor constants for mercurial reagents indicate that, Treatment included preincubation of the nucleotides and citrate in 50 for p-hydroxymercuriphenylsulfonate, the K1 values de mM triethanolamine-HCI buffer at pH 7.0 for 15 mm at 37°,after which termined in the absence of citrate were similar for each they were assayed. The final citrate concentration in the reaction system was 10 mM. Km values are in m@ units. AK (Table 5). However, upon the addition of citrate, K1 values decreased from 30 to 11 mM for liver AK but re ATPAMP—Citrate+Citrate—Citrate+CitrateLiverAK7.04.21.92.0Tumor mained constant for tumor and muscle AK. The K, values of p-chloromercuribenzoate for liver and muscle enzyme are similar in the absence of citrate, whereas, upon the addi tion of citrate, K1 values for liver and muscle enzymes were AK33.038.018.010.0MuscleAK10.04.06.23.3 increased 20-fold.

Table 3 DISCUSSION Cooperative interaction upon substrate binding Treatment included preincubation of nucleotides and citrate in 50 mM It has been observed that citric acid-cycle intermediates triethanolamine-HCI buffer at pH 7.0 for IS mm at 37°,after which they activate several key pathway enzymes including glycogen were assayed. The final citrate concentration in the reaction system was 10 synthetase (23, 26), phosphofructokinase (18, 22, 24, 27), mM. hexokinase (19), and AK (25). The effect ofcitrate on all of PlotsForATPForAMP—Citrate+Citrate—Citrate+CitrateLiverAKSlope ofHill the above-mentioned enzymes have in common an involve ment of a metal or chelation phenomenon as a portion of the modulator and/or substrate complex (12, 25). Because all of these enzymes play key roles in regulating their respec tive metabolic pathways, the in vivo concentrations of di valent cations, citrate, and ATP could effectively coordi TumorAK 1.43 1.55 1.0 1.19 nate intracellular pathway flux. Therefore, it is possible 1.430.75 1.08 MuscIeAK2.1 1.421.45 1.500.51 that the evolution of a particular common mode of acute regulation could result in the control of different metabolic Table 4 pathways in a variety of cell types; control would be de Efftctofmodulators pendent upon the enzyme (or isozyme) species that existed Treatment included preincubation of all nucleotides in 50 mM trietha in that cell type and that enzyme's susceptibility to “chela nolamine-HCI buffer at pH 7.0 for 15 mm at 37°in the presence of tion control.― modulators (final concentrations were 5 mM). Stimulation is expressed as If chelation control is considered to be an acute intra ±(%) of control activity. cellular regulatory parameter, it is obvious from our cur ModulatorsLiverAKGDP+40+62+27CDP+15+46+10UDP+12+37+37TDP+18+48+18AKTumor AKMuscle rent studies that this type of regulation of AK activity would have greater importance in normal liver tissue than in skeletal muscle or fast-growing hepatomas. The activa tion constant of citrate was 0.09, 0.26, and 15.8 mr@tfor liver, skeletal muscle, and hepatoma tissues, respectively. The concentration of citrate is approximately 0.2 m@i in rat liver [albeit not reported for Morris hepatoma 3924A Effect of Free Fatty Acids and Detergent. Free fatty acids (6)J. The very high versus very low activity of AK activ irreversibly inhibited each of the 3 purified AK's. Satu ities in liver hepatomas is supported by the previously cal rated fatty acids did not alter the activity of the enzymes, culated reaction parameter (Q) of 1.42 and 2.06 (3.78 in whereas the unsaturated fatty acid (oleic acid) inhibited the enzymatic activity in all cases. Sodium lauryl sulfate inhibited all 3 enzymes. The denaturation of the enzyme Table5 proteins by detergent was not reversible. K1 values for so Inhibitor constant (K1) values dium lauryl sulfate with liver, tumor, and muscle AK's Treatment included preincubation of nucleotides and additives in 50 msi triethanolamine-HCI buffer at pH 7.0 for 15 mm at 37°,after which they were 0.7, 0.25, and 0.31 mr@i,respectively. were assayed. The final concentration of citrate in the assay system was 10 Effect of Mercurial and Sulfhydryl Reagents. Mercurial mM. The K1 values are expressed in m@ units. reagents inhibited the activity of each enzyme. Glutathione containing I —SH group and 2 free —COOH groups in phenylsulfonateP-Chloromercuribenzoate—Citrate hibited each enzyme to varying degrees. Ethylmercurithio salicylate had no effect on liver or muscle AK, but it did —Citrate+CitrateLiverEnzymesP-Hydroxymercuri +Citrate • inhibit tumor AK. Dithiothreitol had no effect on liver or tumor AK, but it inhibited muscle AK. It appears that AK 11 2.5 each enzyme seems to have a slightly different degree of TumorAK 25 30 >0.25 >2.5 MuscIeAK30 35 400.62 0.52> >2.5 sulfhydryl lability.

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ascites form) for Cells. Cancer Res., 33: 56—64,1973. 12. Criss, W. E. Metabolite and Hormonal Control of Energy Metabo n [AMP [ATP] lism in Experimental Hepatomas. In: K. W. McKerns (ed), Hor mones and Cancer, New York: Academic Press, Inc., in press. ‘@- [ADP]2 13. Criss, W. E., Litwack, G., Morris, H. P., and Weinhouse, S. Adeno sine Triphosphate:Adenosine Monophosphate Phosphotransferase liver and tumor, respectively (8, 9). Therefore, it is possible Isozymes in Rat Liver and Hepatomas. Cancer Res., 30: 370-375, that citrate chelation control may regulate liver AK and 1970. thus may affect the adenylate charge in liver tissue but, if 14. Criss, W. E., Pradhan, T. K., and Morris, H. P. Physical Protein so, it is unlikely that the hepatoma tissue is subject to the Regulation of Adenylate Kinases from Muscle, Liver, and Hepa same form of regulation. toma. Cancer Res., 34: 3062-3065, 1974. 15. Criss, W. E., Sapico, V., and Litwack, G. Rat Liver Adenosine Tn phosphate:Adenosine Monophosphate Phosphotnansferase Activ ACKNOWLEDGMENTS ity. I. Purification and Physical and Kinetic Characterization of Adenylate Kinase III. J. Biol. Chem., 245: 6346-6351, 1970. 16. Derr, R. F., and Zieve, L. Adenylate Energy Charge: Relation to We acknowledge the technical assistance of Dorothy Fitzgerald, Willis Guanylate Energy Charge and the Adenylate Equilibrium Constant. Parker, Charity Jackson, and Louise Lawson. Biochem. Biophys. Res. Commun., 49: 1385-1390, 1972. 17. Filler, R., and Cniss, W. E. Development of Adenylate Kinase Iso REFERENCES zymes in Rat Liver. Biochem. J., 122: 553—555,1971. 18. Kemp, R. G., and Knebs, E. G. Binding of Metabolites by Phospho 1. Atkinson, D. E. Biological Control at the Molecular Level. Science, fructokinase. Biochemistry, 6: 423—434, 1967. 150:851-875,1965. 19. Kosow, D. P., and Rose, I. A. Activators of Yeast Hexokinase. J. 2. Atkinson, D. E. Regulation of Enzyme Activity. Ann. Rev. Biochem., Biol.Chem.,246:2618-2625,1971. 35:85-118,1966. 20. Krebs, H. A. Gluconeogenesis. The Croonian Lecture, Proc. Roy. 3. Atkinson, D. E. The Energy Charge of the Adenylate Pool as a Regu Soc. London Sen. B, 159: 545-564, 1964. latory Parameter. Interaction with Feedback Modifiers. Biochemis 21. Matsuhashi, M., Matsuhashi, S., and Lynen, F. Regulation of Acetyl try, 70: 4030-4034, 1968. Coenzyme A Carboxylase. Biochem. Z., 340: 263-289, 1964. 4. Atkinson, D. E. Regulation of Enzyme Function. Ann. Rev. Micro 22. Newsholme, E. A., and Randle, P. J. Regulation of Glucose Uptake biol., 23:47-68, 1969. by Muscle. Biochem. J., 93: 641-651, 1964. 5. Atkinson, D. E., Hathaway, J. A., and Smith, E. C. Kinetics of Regu 23. Piras, M. M., Bindstein, E., and Piras, R. Regulation of Glycogen latory Enzymes. Kinetic Order of Yeast DPN Isocitrate Dehydroge Metabolism in the Adrenal Gland. I. Kinetic and Regulatory Prop nase Reaction and a Model for the Reaction. J. Biol. Chem., 240: erties of Glycogen Synthetase. Arch. Biochem. Biophys., 139: 121- 2682-2690,1965. 129, 1970. 6. Barnett, D., Tassopoulos, C. N., and Fraser, T. R. Citrate and Re 24. Pogson, C. I., and Randle, P. J. Control of Rat Heart Phosphofructo lated Intermediates in Liver during Experimental Diabetes, Con kinase by Citrate and Other Regulators. Biochem. J., 100: 683-693, trasted with Starvation. Hormone Metab. Res., 4: 257-266, 1972. 1966. 7. Cohen, P. F., and Colman, R. F. DPN Dependent Isocitrate Dehy 25. Pradhan, T. K., and Cniss, W. E. Modulation of Mitochondnial Aden drogenase from Pig Heart. Characterization of the Active Substrate ylate Kinase by Citric-Acid-Cycle Intermediates. European J. Bio and Modes of Regulation. Biochemistry,ii: 1501-1508, 1972. chem.,43:54I-547, 1974. 8. Criss,W. E. Rat Liver Adenosine Triphosphate: Adenosine Mono 26. Sevall, J. S., and Kin, K. H. Regulation of Hepatic Glycogen Syn phosphate Phosphotransferase Activity. II. Subcellular Localization thetase of Rana catesbeiana. Significance of Citrate Activation with of Adenylate Kinase Isozymes. J. Biol. Chem., 245: 6352—6356, Reference to Insulin Activation. J. Biol. Chem., 246: 7250-7255, 1970. 1971. 9. Criss, W. E. Relationship of ATP:AMP Phosphotransferase Iso 27. Underwood, A. H., and Newsholme, E. A. Some Properties of Phos zymes to Tissue Respiration. Arch. Biochem. Biophys., /44: 138- phofructokinase from Kidney Cortex and Their Relation to Glucose 142,1972. Metabolism. Biochem. J., 104: 296-299, 1967. 10. Criss, W. E. Control of the Adenylate Charge in Morris “Minimal28. Vagelos, P. R., Alberts, A. W., and Martin, D. B. Studies on the Me Deviation― Hepatomas. Cancer Res., 33: 51-56, 1973. tabolism of Activation of Acetyl Coenzyme A Carboxylase by Citrate. 11. Criss, W. E. Control of the Adenylate Charge in Novikoff Ascites J. Biol. Chem., 238: 533-540, 1963.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Kinetic Regulation of Adenylate Kinases from Muscle, Liver, and Hepatoma

Tapas K. Pradhan, Wayne E. Criss and Harold P. Morris

Cancer Res 1974;34:3058-3061.

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