[CANCER RESEARCH 33, 51—56,January1973J Control of the Adenylate Charge in the Morris “Minimal Deviation― 1 WayneE. Criss Department ofObstetrics and Gynecology, University ofFlorida CollegeofMedicine, Gainesville,Florida 32601 SUMMARY adenylate kinase reaction) and the energy charge of the adenylate system were performed. The Q's for liver tissue and The activity of several enzymes that contribute to the groups of highly and poorly differentiated hepatomas were cellular adenylate energy charge and the change in the total 1.42, 1.88, and 2.06, respectively. The calculated adenylate pool of adenine nucleotides were measured in groups of highly charges were 0.87 in normal liver tissue, 0.89 in a group of and poorly differentiated Morris “minimal-deviation―highly differentiated hepatomas, and 0.90 in a group of poorly hepatomas. differentiated hepatomas. The activities of several substrate-level phosphorylating It would appear that the poorly differentiated hepatomas enzymes were determined: cytoplasmic adenylate kinase, have acquired the potential to maintain a high energy charge pyruvate kinase, and phosphoglycerate kinase; mitochondrial without the need for large mitochondrial involvement. adenylate kinase and nucleoside diphosphokinase. Tabulation of these enzymatic measurements were made by setting up a ratio of cytoplasmic to mitochondrial nonoxidative INTRODUCTION phosphorylation potential. The ratio was 0.44 in normal liver tissue, 0.50 in the group of highly differentiated hepatomas, The adenylate system, consisting of AK2 (EC 2.7.4.3), and 22.2 in the group of poorly differentiated hepatomas. AMP, ADP, and ATP, has been compared to an The activities of several enzymes that are involved in the electrochemical storage cell in its ability to accept, store, and transportation of reducing equivalents across the supply energy (7). The energy charge of the adenylate system mitochondrial membrane were made: cytoplasmic malate may be a central signal in the control of glycolysis, dehydrogenase, glyceraldehyde phosphate dehydrogenase, gluconeogenesis, lipogenesis, and terminal oxidative lactate dehydrogenase, glucose 6-phosphate dehydrogenase, phosphorylations (10—12, 28, 29, 47). Atkinson has defined the energy charge as one-half of the average number of flavin adenine dinucleotide- and nicotinamide adenine anhydride-bound phosphate groups per adenosine moiety. dinucleotide (NAD@)-glycerophosphate dehydrogenase; When only AMP is present, there is a net charge of zero or mitochondrial malate dehydrogenase, flavin adenine complete discharge. When only ATP is present, there is a net dinucleotide- and NAD@-glycerophosphate dehydrogenases. Cytoplasmic NAD@-malate dehydrogenase was over 500 units charge of 1. Atkinson and his colleagues have shown that certain key enzymes that participate in ATP-regenerating in normal liver and less than 100 units in the group of pathways show plots of enzyme activity against energy charge poorly differentiated hepatomas. Cytoplasmic NAD @- with negative slopes that increase with charge ; certain enzymes glycerophosphate dehydrogenase was 120 units in liver that participate in biosynthetic or other ATP-utilizing tissue and about 20 units in the group of poorly differentiated pathways show plots of enzyme activity against energy charge hepatomas. NAD@-malate dehydrogenase is a key enzyme in with positive slopes that increase with charge (for reviews, see the malate-aspartate shuttle system; NAD@-glycerophosphate Refs. 8, 9, 11, and 26). Thus, the energy charge of the dehydrogenase is a key enzyme in the a-glycerophosphate adenylate system could provide the cell with a very sensitive shuttle system. intracellular regulatory control mechanism. Comparison of the ratio of the activities of NAD@-g1ycero Obviously, many enzymes and certain metabolites make phosphate dehydrogenase versus NAD@-lactate dehydrogenase varying contributions to the energy charge within a cell. allows for the examination of the competing hydrogen Compartmentation would probably be an important factor in accepting systems of phospholipid synthesis versus anaerobic determining location and levels of the components of the metabolism. The ratio was 0.5 in liver tissue and 0.1 in the adenylate pool (ATP, ADP, AMP) and raises the possibility of group of poorly differentiated hepatomas. a distinct energy charge in distinct subcellular units. In the Measurement of adenosine mono-, di-, and triphosphates cytoplasm, substrate-level phosphorylating enzymes such as and calculations of Q (a reaction parameter based on the 2 The abbreviations used are: AK, adenylate kinase; PGK, 1 This work was supported by NIH Grants CA-10439, CA-10906-08, phosphoglycerate kinase; PK, pyruvate kinase; MDH, malate and CA-i 1818; Grant F71UF from the Florida Division of the dehydrogenase;GADPH, glyceraldehyde 3-phosphate dehydrogenase; American Cancer Society; and Grant P-202 from the National Division GPDH, glycerophosphatedehydrogenase;LDH, lactate dehydrogenase; of the American CancerSociety. G6PDH, glucose 6-phosphate dehydrogenase; NDK, nucleoside ReceivedApril 28, 1972; acceptedOctober 3, 1972. diphosphokinase. JANUARY 1973 51 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. Wayne E. Criss hexokinase-glucokinase , phosphofructokinase , PGK , PK, etc., 370 in a Beckman Model DU spectrophotometer equipped would directly contribute to the cytoplasmic adenylate charge. with a Gilford recorder. All measurements were continuously The energy charge within the mitochondria would depend monitored at 340 nm by following the oxidation and/or upon the efficiency of the oxidation-reduction shuttle reduction of the pyridine nucleotides. systems of malate-aspartate (59, 60), oxaloacetate (22), MDH (L-malate:NAD@ oxidoreductase, EC 1.1.1.37) was a-glycerophosphate (58), the coupling to the adenine measured as in the work of Skilleter et a!. (49). @ nucleotides, and transport of ADP inward and AlP outward GADPH (D -glyceraldehyde 3-phosphate :NAD oxido (27, 34). It has also become apparent that there is a close link reductase, EC 1.2.1 .12) was measured by the method of between the nicotinamide and adenine nucleotide systems in Furfine and Velick (20). the cell (52, 60). Now, with the postulate that the adenine FAD-GPDH [L-glycerol 3-phosphate:(acceptor) oxidore nucleotides contribute directly to the metabolic control of the ductase, EC 1.1.99.5] activity was assayed with an oxygen Pasteur effect (1 1, 19, 39) and with one of the most notable electrode (23). and controversial features of the neoplastic cell being the NAD @-GPDH (L -glycerol 3-phosphate :NAD@ oxidore inability of the Pasteur effect completely to inhibit its ductase, EC 1.1 .1.8) was measured as by White and Kaplan glycolysis (4, 42, 54, 56), it becomes an important and (58). challenging problem to investigate the contribution of the NAD@-LDH (L-lactate:NAD@ oxidoreductase, EC 1.1.1.27) adenylate charge to metabolic regulation (especially of activity was determined by the method of Pesce et a!. (41). glycolysis) in the neoplastic cell. NADP@-G6PDH (D-glucose 6-phosphate :NADP@ oxido This paper and the following one (16) report the changes in reductase, EC 1.1 .1.49) was measured as described by Criss the levels of several enzymes that contribute to the cellular and McKerns (18). adenylate energy charge and the change in the total cellular NDK (ATP:nucleoside diphosphate phosphotransferase, EC pool of adenine nucleotides in Morris “minimal-deviation― 2.7.4.6) was determined by the method of Ratliff et a!. (44) hepatomas and in Novikoff hepatomas (solid and ascites). with GDP as the phosphate acceptor and coupling the production ofADP as described by Adam (1). AK (ATP:AMP phosphotransferase, EC 2.7.4.3) activity was MATERIALS AND METHODS measured as described by Criss et al. (17). PGK (ATP:3-phospho-D -glycerate l-phosphotransferase, EC Animals and Tumors. Normal male adult rats (CFN) were 2.7.2.3) was determined by the method of Rao and Oesper purchased from Carworth Farms, New City, N. Y.; maintained (43) with the production of ADP as described by Adam (1). on laboratory chow; and sacrificed when approximately 250 g. PK (ATP:pyruvate phosphotransferase, EC 2.7.1.40) All tumors, except the Novikoff hepatoma, were transplanted activity was measured as by Reynard et a!. (45). in Bethesda, and the rats were shipped to our laboratories. The Preparation of Cellular Homogenates for Nucleotide Assay. rats were maintained on laboratory chow until the tumors The rats were kified by cervical dislocation; the liver or tumor were 1 to 3 cm in diameter. The origin and properties of many was exposed, clamped, and removed with pre-nitrogen-cooled of the Morris “minimal-deviation―hepatomas have been Wollenburger tongs and immersed into liquid nitrogen through described (35—37). The tumors were examined histologically which CO had been bubbled. The procedure was completed in by Dr. David Meranze of the Fels Research Institute. 13 sec. The frozen tissue was powdered with a pre-nitrogen Reagents and Materials. Biochemical reagents and glassware CO-cooled mortar and pestle, weighed, and added to 5 were purchased from Fisher Scientific Co., Fairlawn, N. J.; A. volumes of cold 5% HC1O4. The tissue was then homogenized H. Thomas Co., Philadelphia, Pa.; Scientific Products, in a Potter-Elvehjem homogenizer with motor-driven pestle at Evanston, Ill.; Mallinckrodt Chemical Works, McGraw, Ill.; and 00 with acetone and Dry Ice. The extract stood