A Review of Isozymes in Cancer1

Home , Adenylate kinase, Aldolase A, Aldolase C, DNA polymerase, DNA polymerase I, Galactokinase

Cancer Research

VOLUME31 NOVEMBER 1971 NUMBER11 [CANCER RESEARCH 31, 1523-1542, November 1971] A Review of Isozymes in Cancer1

Wayne E. Criss Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida 32601

TABLE OF CONTENTS postulated role for that particular isozymic system in cellular metabolism. Summary 1523 Introduction 1523 Normal enzyme differentiation 1523 INTRODUCTION Tumor enzyme differentiation 1524 Isozymes 1524 Normal Enzyme Differentiation DNA polymerase 1524 Enzyme differentiation is the process whereby, during the Hexokinase 1525 Fructose 1,6-diphosphatase 1525 development of an organ in an animal, the organ acquires the quantitative and qualitative adult enzyme patterns (122). Key Aldolase 1526 pathway enzymes in several metabolic processes have been Pyruvate kinase 1527 found to undergo enzymatic differentiation. The enzymes LÃ¡clatedehydrogenase 1527 Isocitrate dehydrogenase 1527 involved in nitrogen metabolism, and also in urea cycle Malate dehydrogenase 1528 metabolism (180), are tyrosine aminotransferase (123, 151, Glycerol phosphate dehydrogenase 1529 330, 410), tryptophan pyrrolase (261), serine dehydratase Glutaminase 1529 (123, 410), histidine ammonia lyase (11), and aspartate Aspartate aminotransferase 1530 aminotransferase (337, 388). The enzymes involved in nucleic Adenylate kinase 1531 acid metabolism are DNA polymerase (156, 277) and RNase (52). In glycolysis the enzymes are hexokinase-glucokinase Carbamyl phosphate synthetase 1531 Lactose synthetase 1533 (98, 389), galactokinase 30, aldolase (267, 315), pyruvate Discussion 1533 kinase (73, 386), and lactate dehydrogenase (67, 69). In References 1533 mitochondrial oxidation they are NADH oxidase, succinic oxidase, a-glycero-P oxidase, ATPase, cytochrome oxidase, and flavin content (84, 296). In glycogen metabolism the SUMMARY enzymes involved are UDPG pyrophosphorylase and UDPG glucosyltransferase (19). The enzymes involved in glu- Several distinct isozymic systems which were observed to coneogenesis are glucose 6-phosphatase (79), fructose have alterations during differentiation from the normal to the diphosphatase (18), phosphoenolpyruvate carboxylase (21, neoplastic state have been reviewed. Most of these isozymes 413), and pyruvate carboxylase (17). Most of these enzyme are key pathway or regulatory enzymes of intermediary shifts are simple quantitative changes (296, 410) which have metabolism. The isozymic systems which were reviewed been interpreted as the initiation or termination of a metabolic include DNA polymerase, hexokinase-glucokinase, fructose pathway in that tissue. However, some enzymes undergo shifts 1,6-diphosphatase, aldolase, pyruvate kinase, lactate in their isozymic complements (67, 156, 389), thus making dehydrogenase, isocitrate dehydrogenase, malate the overall metabolic interpretation more or less difficult, dehydrogenase, glycerol phosphate dehydrogenase, depending upon how much is known about each isozymic glutaminase, aspartate aminotransferase, adenylate kinase, form. carbamylphosphate synthetase, and lactose synthetase. The With the same type of reasoning, one can assume that the basic isozymic system was outlined, and alterations which disappearance of a key pathway enzyme in neoplastic tissue occurred in the neoplastic state were discussed in terms of the represents a loss of that particular metabolic pathway to the tissue. The question one can ask is, "Do neoplastic tissues have 1This work was supported by the Florida Division of the American a distinctive and unique enzyme pattern just as the normal Cancer Society (Grant F71UF-1). organ systems in an adult animal have distinctive and unique Received April 21, 1971; accepted June 15, 1971. enzyme patterns?" If so, we should be able to examine key

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pathway enzymes and establish potentially functional and enzymes which exist as multiple forms and which have been potentially obsolete metabolic routes in tumors. found to undergo changes in cancers.

Tumor Enzyme Differentiation ISOZYMES Numerous reviews have been written which reported the measurable levels in neoplastic tissues of nearly all of the DNA Polymerase enzymes involved in glycolysis (47, 149, 192, 287, 397, 399, 405, 406), pentose-P metabolism (149, 192), gluconeogenesis Deoxynucleotidyl transferase (DNA polymerase) activity (47, 192, 397), oxidative phosphorylation (219, 399), urea exists throughout nature in a heterogeneous state (37, 110, cycle metabolism (418), and nucleic acid metabolism (58, 125, 111, 160, 179, 258, 269, 270,421, 425). Purified Escherichia 246, 352, 408). It would appear to be very difficult, if not coli DNA polymerase used native and heat-denatured DNA as impossible, to extrapolate the abnormal enzyme patterns primer and formed a complex with native and heat-denatured found in one type of tumor directly into other types of DNA (34, 160). Calf thymus DNA polymerase was purified as malignant tissues. The patterns may vary within different 2 separate and distinct forms, a replicative deoxynucleotidyl tumor tissues in the same animal species and may also deviate transferase which used only denatured DNA as primer and was in similar tumor types from different animal species. a DNA polymerase and a terminal deoxynucleotidyl Weber (397) has presented a "molecular correlation concept transferase which used short-chain oligodeoxynucleotides or of neoplasia" which was based principally upon the Morris denatured DNA as primer and was a terminal addition enzyme "minimal-deviation" hepatoma system (247, 254â€”256). This (37-41, 167, 216, 421). The thymus enzymes did not concept is an attempt to make a phenotypic complex with DNA and thus probably differ from E. coli description of the metabolism of a "typical" tumor cell as polymerase. DNA polymerases, as purified from regenerating follows. Patterns of carbohydrate metabolism show a decrease rat liver (248) from nonhistone chromosomal proteins of in gluconeogenic capacity and an increase in glycolytic normal rat liver (141) and from sea urchin embryo (220), capacity in the hepatoma cell. The capacity for the synthesis preferentially used native DNA as primer. Different physical of glycogen and lipid is greatly decreased. The tumor cell can and enzymatic (e.g., substrate specificity) properties were synthesize proteins and nucleic acids at increased rates, while observed with nuclear and mitochondrial DNA polymerases the catabolism of amino acids, proteins, and nucleic acids are from normal rat liver and yeast (157, 162, 242). Therefore, as decreased. Thus, it would appear that the hepatoma cell has a generalization, DNA polymerase activity can be classified streamlined its metabolism in an attempt simply to grow and according to preference for native or denatured substrate. multiply. However, one must continuously keep in mind that With Sephadex gel filtration, DNA polymerase activity from the in vitro measurements of enzymes or enzyme systems, adult rat liver has been separated into 2 fractions (156, upon which most of the above-mentioned reviews and much of 275â€”277). Peak I had a large molecular weight, was isolated the foundation for the molecular correlation concept are from the postmicrosomal supernatant, and worked best with based, are an indication only of the potential activity of heat-denatured DNA as primer. Peak II was smaller, was specific enzymes and their representative metabolic pathways. concentrated in the free ribosomes, and used mainly native Loss of an enzyme may or may not be indicative of a loss of DNA as primer (26, 156). Peak I was the predominant form of the specific pathway in which the enzyme plays a key role. DNA polymerase in malignant tumors and fetal liver. Peak II Also, the presence or even the increase in the activity of a key was the predominant form in adult and regenerating rat liver enzyme (especially from in vitro assays) may not necessarily (275). indicate an increase in the corresponding pathway. However, 3 forms of DNA polymerase from rat liver and The latter statements are particularly true in the situations hepatomas have been observed with phosphocellulose column in which a key enzyme exists in multiple forms and the various chromatography (156, 270, 275-277). Peaks I and II used forms have different modulators. Decreases in one enzyme heat-denatured DNA, and Peak III accepted native DNA as form may be compensated by increases in another enzyme primer. Normal liver and slow-growing hepatomas had similar form, with the net result being an increase, a decrease, or no levels of Peaks I and II and a much smaller level of Peak III. net change in the total activity of that enzyme system. Regenerating and fetal liver and fast-growing hepatomas had Therefore, an examination of the changes in the activity levels higher levels of Peak II than of Peak I and only a very small of one of the isozymic forms of a key enzyme system, a amount of Peak III. DNA polymerase I remained fairly process which occurs naturally during the phenomena of constant, while the II form increased and the III form enzyme differentiation and which was cited earlier in this decreased with increased tissue growth rates. It would appear review, could yield considerable information about the that the predominant form of normal adult rat liver DNA functioning of specific metabolic pathways in the tumor cell, polymerase acts on native DNA but that the major form of especially in isozymic systems where functional modulators hepatoma DNA polymerase is increased and acts and physiological roles of the different isozymes have been predominantly on heat-denatured DNA as primer. established. The purpose of this review is to extend the DNA polymerase has recently been purified from information which is known about total enzyme changes in hypotonically shocked Ehrlich ascites cells (313). At an early cancers. I have attempted to survey certain key pathway stage in the purification, the polymerase used both native and

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1971 American Association for Cancer Research. Â¡sozymes in Cancer heat-denatured DNA as primer. As purification progressed, dependent for its activity on the dietary intake of glucose in activity with native and heat-denatured DNA decreased, and the intact animal; and, in the adult, was induced by insulin the purified enzyme preferred single-stranded synthetic poly- (279, 317, 318). The other hexokinases did not respond in the [d(A-T)] or DNA partially digested by pancreatic DNase for same way to dietary or hormonal change (394) and were template (314). The intracellular location of this DNA inhibited by ADP and glucose-6-P. The hexokinases polymerase is not known, but Roychoudhury and Block (314) predominated in fetal rat liver, but glucokinase became the reported that this enzyme has a different template preference predominant form in the adult rat liver (20, 98, 389, 390). than does the chromatin-bound polymerase of Ehrlich ascites Antibody to rat liver glucokinase reacted with glucokinases cells. It would appear from the multiplicity of substrate from several species but did not inhibit any hexokinase specificities (native, denatured, or synthetic; single- or activity (285). double-stranded primers), the tendency for molecular Glucose phosphorylation has been examined in several aggregation (422), and behavior observed with different malignant tissues (108, 198, 236, 326, 404). Preneoplastic polymerase preparations that the in vivo functions of the livers (from azo dye-fed rats) had normal to decreased various DNA polymerases in normal and malignant tissues levels of glucokinase activity and increased activity of the low remains obscure. Km hexokinases (333, 334). In a series of Morris hepatomas, Just to increase the complexity of the picture, 2 factors Farina et al. (98) and Shatton et al. (334) reported a decrease which inhibit DNA synthesis have been observed and partially in glucokinase and an increase (20- to 40-fold) in hexokinase purified (322, 372). The 2 factors are present in adult rat liver activity with decrease in tissue differentiation. Similarly, Sato cell sap. Factor II is increased in regenerating liver cell sap, et al. (325) examined a group of Morris hepatomas, Yoshida while in hepatomas both factors decrease with increasing sarcomas, and Yoshida ascites hepatomas. They observed all 4 degree of malignancy. The effects of these factors upon the isozymes in the slower-growing and less-deviated tumors and a various purified DNA polymerase preparations have not been loss of hexokinases III and IV in the rapidly growing and reported. highly deviated tumors. Thus, in liver neoplasia, the normal glucose Hexokinase phosphotransferase which is specific for glucose, which is inhibited only by ADP, and the level of which is controlled by Hexokinase (ATP:D-hexose 6-phosphotransferase, EC diet and hormones is replaced by high levels of a glucose 2.7.1.1) is known to exist in from 2 to 6 forms in nature phosphotransferase which is not specific for glucose, which is (116, 117, 128, 129, 161, 174, 175, 378, 379). However, only inhibited by ADP and glucose-6-P, and for which the activity 4 isozymes with hexokinase activity have been observed, level is apparently not responsive to changes in diet or purified, and studied from mammalian tissues (116, 128, 332). hormones. The 4 forms have been designated I, II, III, and IV in order of Recent studies on the control of respiration in mitochondria increasing electrophoretic mobility. Hexokinases I, II, and III have indicated the possibility that hexokinase might play a had low apparent Michaelis constants for glucose (1CT4 to role in regulating the Crabtree effect (117, 176, 249,250, 311, 10~6M), were found predominantly in nonhepatic rat tissues, 383, 415). The relative distribution of hexokinase between and phosphorylated several sugars. They differed from one cytoplasm and mitochondria may be regulated by the another with respect to apparent Km's (glucose and ATP), intracellular levels of glucose-6-P, ATP, and inorganic apparent Kj's (ADP and glucose-6-P), and stability to heat and phosphate. The latter metabolites apparently cause release of proteolytic inactivation. The 3 latter isozymes were similar the mitochondrially bound hexokinase. The soluble with respect to molecular weight, apparent Km (fructose), pH hexokinase is 5 times more susceptible to inhibition by optimum, hexose and nucleotide specificities, and the glucose-6-P than the bound enzyme. Thus, during times of qualitative nature of inhibition by ADP and glucose-6-P, (117, high energy requirement (low ATP), hexokinase would be 128, 136. 174, 239, 240, 332). Isozyme IV was found bound to mitochondria and could aid in the supply of ADP to predominantly in adult mammalian liver; had a high apparent the mitochondria; whereas at times of low energy requirement Km for glucose (1CT2M), phosphorylated mannose, and (high ATP), the enzyme would become solubilized and could deoxyglucose (Km's IO"1 M); and was inhibited by ADP-Mg^ aid only in the supply of glucose-6-P for glycolysis. This kind but not by glucose-6-P (278, 318, 391). The latter isozyme has of reasoning would tend to support the hypothesis that the been separated into 2 active components by electrophoresis flux of glycolytic intermediates is directly related to the (284). observed Crabtree effect in intact tumor cells. Tissues with In the revised report on enzyme nomenclature (93), we high levels of glucokinase (e.g., liver of rat fed high glucose) find the classification of the hepatic glucokinase (EC 2.7.1.2) could not respond with similar mechanisms. as distinct from the other nonhepatic hexokinases (EC 2.7.1.1). The 2 classes are different in many respects. Fructose 1,6-Diphosphatase Glucosamine and A^-acetylglucosamine were not phosphorylated but act as competitive inhibitors of Fructose 1 ,6-diphosphat ases (o-fructose glucokinase; they were phosphorylated as substrates by the l,6-diphosphate:l-phosphohydrolase, EC 3.1.3.11) have been hexokinases (279, 317). Glucokinase was relatively specific for isolated from a large variety of sources, both mammalian and glucose; was inhibited by ADP but not by glucose-6-P; was nonmammalian (5, 42, 107, 109, 241, 289, 293, 299, 312).

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Some of the many properties that the mammalian enzymes major aldolase isozymes in mammalian tissues; the other had in common include limited substrate specificity, observed electrophoretic entities are probably hybrid forms stimulation by EOT A, optimum pH above 7, requirement for (35, 78, 278, 315, 380). Skeletal muscle, heart, and spleen Mg* or Mri*, and inhibition by AMP and/or fructose-1,6-di-P. contain aldolase A, which splits fructose-1,6-di-P at a rate Purified fructose 1,6-diphosphatase from bovine liver and about 50 times faster than it splits fructose-1-di-P (35,278, rabbit liver and kidney were observed to disassociate reversibly 341, 380). Liver contains aldolase B, which splits both into 2 subunits which were composed of 2 nonidentical substrates equally well (35, 278). Aldolase A is inhibited by polypeptide chains with molecular weights of 30,000 and diethylstilbestrol and ATP; aldolase B is not (353, 371). Other 37,000 (59, 206, 293, 344). The rabbit liver and kidney tissues, such as kidney, erythrocytes, lung, and intestine, enzyme are immunologically identical (92). These 2 enzymes, contained a mixture of A and B isozymes; while a 3rd as well as the fructose 1,6-diphosphatase from pig kidney, isozyme, aldolase C, had been found in rabbit brain tissue were desensitized against AMP inhibition by pyridoxical-5'-P (278). Aldolases A, B, and C had different catalytic properties, (205, 207, 224) and were sensitive to cortisone action (397, amino acid compositions, and immunochemical characteristics, 401,402). all reversibly hybridized (35, 120, 278, 340). A 2nd type of mammalian fructose 1,6-diphosphatase has Data from substrate specificity studies have revealed that been found in the skeletal muscle of several species (204,271, oncogenic tissues contained both aldolases A and B (3, 329, 272, 323). A major difference between this muscle form of 360, 380, 404). The aldolase substrate activity ratio fructose 1,6-diphosphatase and the liver or kidney forms was (fructose-1,6-di-P/fructose-1 -P) changed from 1 to as high as 13 that the former showed increased sensitivity to inhibition by in human primary cancer (329). The ratio ranged from 1 to 5 AMP. The muscle enzyme had a KÂ¡(AMP) of 0.1 to 2.0 Â¿(M in a series of well-differentiated Morris hepatomas and was whereby the liver and kidney enzymes had a KÂ¡(AMP of 130 found to be very high, approximately 50, in the poorly to 200 MM)(206, 224, 323, 324, 366, 392). The rabbit muscle differentiated hepatic tumors (3, 404). The activity ratio was and liver enzymes were differentially activated by cystamine considerably greater than 1 (range, 4 to 25) in cancers of liver, (100, 292), had different amino acid composition and lung, and colon (380). Studies with antialdolase A serum molecular weights (99, 205,293), and were electrophoretically revealed total inhibition of muscle aldolase A, no inhibition of and immunologically distinct (92, 100). the adult liver isozyme (B), and a range of inhibitions with the The liver isozyme of fructose 1,6-diphosphatase has been aldolases from either fetal liver or hepatomas (267). The reported in slow-growing tumors, while the AMP-sensitive absolute activity of aldolase in malignant tissues varies from muscle form was observed in fast-growing tumors (324, 365, tumor to tumor, but it would appear that substrate specificity 400). It would appear that the levels of AMP in Ehrlich ascites studies indicate that the predominant aldolase in cancerous tumor cells are high enough to cause suppression of the tissues is aldolase A. AMP-sensitive fructose 1,6-diphosphatase activity in those cells Kawabe et al. (178) isolated in crystalline form the aldolase (323). Also, the normal increase in the liver enzyme upon from rat Rhodamine sarcomas. The properties of this tumor cortisone treatment was considerably reduced in Morris aldolase, including Km values for fructose-1,6-di-P and hepatomas 5123 and 7800 (397). Thus, the AMP-sensitive fructose-1-P, loss of fructose-1,6 ,di-P aldolase activity by enzyme has become the predominant form during the loss of limited hydrolysis with carboxypeptidase A, inhibition by differentiation and the normal response of fructose adenine nucleotides, inhibition by antialdolase A serum, and 1,6-diphosphatase to cortisone has been diminished in electrophoretic characteristics, were indistinguishable from malignant tissue. Loss of fructose 1,6-diphosphatase activity in those of crystalline aldolase A of the rat muscle. The rat tumors could result in loss of the gluconeogenic capacity of skeletal muscle aldolase A was also found to be identical to the malignant tissue. crystallized enzyme from the Novikoff Hepatoma (119) and from an azo dye-induced primary tumor (154). Even in the Aldolase preneoplastic liver or very early stages of hepatocarcinogenesis, antisera studies have showed the levels of muscle aldolase A to Two classes of aldolases (fructose l,6-diphosphate:D- be increased (90). glyceraldehyde 3-phosphate lyase, EC 4.1.2.7) have been The aldolase isozyme pattern in tumors has obviously observed in lower and higher organisms (4, 25, 152, 200, 306, shifted to an enzyme form which splits predominantly 395). Those aldolases found in plant and mammalian tissues fructose-1,6-di-P, a metabolite in the "main" stream of (Class 1) were characterized by the formation of Schiff base glycolysis. This would appear to benefit those cells involved in intermediates which were reduced by borohydride to yield rapid glucose metabolism. However, another area of extreme inactive secondary amine derivatives (152, 200). Aldolases importance is enzyme-structural component interaction. from yeast and bacteria (Class 2) did not form inactive Clarke et al. (68) observed a differential response in the enzyme-substrate products on reduction with borohydride; binding of aldolase A versus aldolase C to tissue components. they were found to be metalloproteins which were sensitive to Aldolase A remained bound, while aldolase C was solubilized chelating agents and were activated by potassium ions (25, from brain tissue components in the presence of lactic acid. 200, 306). Therefore, if there is such a possible feedback circuit (lactic Starch gel and cellulose acetate strip electrophoresis have acid-aldolase-tissue interaction) involved in glycolysis, a shift revealed multiple forms of mammalian aldolase (4, 278). Five in the adlolase isozyme pattern in tumors could have enzyme forms were detected in tissues from man and at least 7 consequence beyond that of just a shift in potential target forms in tissues from other mammals. Apparently, there are 3 substrate.

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Pyruvate Kinase absolute level of pyruvate kinase activity and the observation that nucleoside triphosphate levels are low in hepatomas, it is Multiple forms of pyruvate kinase (ATP:pyruvate possible that the decrease in triphosphate might contribute to dehydrogenase, EC 2.7.1.40) have been observed in various an increase in pyruvate kinase activity and thus an increase in tissues (15, 16, 72, 112, 147, 148, 194, 245, 290, 297). tumor glycolysis. Cellulose acetate electrophoresis has revealed one major and one minor form in rat liver and kidney and a single 3rd form in Lai-tute Dehydrogenase rat skeletal muscle and heart (290, 362). All other rat tissue studies had only 1 of these 3 forms. Tanaka and colleagues LÃ¡clate dehydrogenase (L-lactate:NAD oxidoreductase, EC (368), using block zonal electrophoresis, found 4 1.1.1.27) has been extensively studied and many reviews have electrophoretic pyruvate kinase isozymes from rat liver. been written concerning its multiple forms (61, 164,215,406, Although 1 of these 4 forms was identical with the muscle 412). It has been purified and studied in numerous tissues and form in electrophoretic mobility, the antibody to the muscle in many different animal species (196, 197, 226, 227, 298, form showed no cross-reactivity with the major liver enzyme. 358, 411). There are 2 major types of lÃ¡clate dehydrogenase, The rat liver and muscle isozymes have been purified and each consisting of 4 identical subunits. One type is found characterized. Rat liver isozyme (L) was allosterically activated predominantly in the heart (H) and is inhibited by pyruvale; by fructose-1,6-di-P, whereas muscle isozyme (M) was not the other is in skeletal muscle (M) and is not inhibited by (15). Reynard et al. (305) showed that pyruvate kinase from pyruvate (115). The 2 forms differ in electrophoretic both muscle and liver are inhibited in the forward direction by migration and amino acid composition and are ATP. This may result from Mg* chelation (417). Rat liver immunologically and catalytically distinct. They are under pyruvate kinase activity was 10-fold greater when rats were fed control of separate genes. Three hybrid forms are found in a high-carbohydrate diet then when fed a low-carbohydrate many tissues (totaling 5 isozymes) and have characteristics diet. Activity was low in the liver of the diabetic rat and intermediate of both parental forms (60, 80, 102, 105, 225, increased in the liver when a diabetic rat was treated with 234,281). insulin (202, 401, 402). The muscle enzyme did not seem to Shifts in the lÃ¡clate dehydrogenase isozyme patlerns have be adaptive to diet or hormonal stimuli (369, 401, 402). The been observed during developmenl and under changing major pyruvate kinase isozyme from rat liver and muscle also biological conditions. Increased oxygen tension caused a differed in their reactivity to p-chloromercuribenzoate, in decrease in the M subunits in rat uterus endometrium (67) and molecular weight, and in their neutralization by liver pyruvate human lymphocytes in cullure (144). The M sub unit was kinase antiserum (367, 368). regulated in the rat uterine endometrium where it was low in Using diethylaminoethyl cellulose column chromatography, diestrus and high in estrus, decreased in pseudopregnancy, Farina et al. (98) and Lo et al. (218, 219) reported, from decreased after ovariectomy, and increased in the studies with hepatomas, an increase in total pyruvate kinase estrogen-lreated ovariectomized ulerus (67). The H subunit activity and in the muscle isozyme, but they found a decrease was regulated in adipose lissue where it increased upon fasting in the liver isozyme during increased tumor growth rates and and decreased upon refeeding the fasted animal (259). In fetal degree of dedifferentiation. However, using isoelectrofocusing tissue, the M and H subunits were nearly equal but began to column chromatography, Criss (73) observed a 5th pyruvate take on adult characterislics (e.g., Ihe M subunits increased in kinase isozyme, absent from liver and muscle, but which fetal muscle) near term or shortly after birth (69). Some appeared in hepatomas and increased with loss of differentia patterns shifted during aging, e.g., Ihe H subunil was high in tion. Similarly, Weber (398) and Taylor et al. (370) have the brain and heart tissue of older rats (163). partially purified the pyruvate kinase from the poorly LÃ¡clate dehydrogenase isozyme shifts are frequently differentiated Morris Hepatoma 3924A and found that observed in cancers (22,27, 115, 307, 354,382). Dawson (80) enzyme to have several properties which distinguish it from found an absolule increase in lÃ¡clate dehydrogenase activily the liver and muscle forms. It was more thermally labile, had and an increase in M subunils upon examination of several different electrophoretic migration on cellulose acetate, was human tumors. Increased levels of the M subunit or Ihe M not stimulated by fructose-1,6-di-P, and had some other form of lÃ¡clale dehydrogenase have been observed in breast properties in common with either the muscle or liver enzyme. lumors (146), uterine carcinoma (268), malignant colon (212), Thus, not only does the normal liver pyruvate kinase isozyme a series of carcinomas (213), and Morris "minimal-deviation" decrease, but there is also an increase in absolute pyruvate hepatomas (310). It would appear thai Ihe lÃ¡clale kinase activity and possible evidence for a new species of dehydrogenase in tumors is more like the muscle enzyme, pyruvate kinase in liver tumors. which is Ihe major form found in tissues exhibiting a high rate A comparison study of pyruvate kinases isolated from liver of glycolysis and is not inhibited by pyruvate. and Hepatoma 3924A has been reported (370, 398, 403). Both enzymes phosphorylated several nucleoside Isocitrate Dehydrogenases diphosphates, and both were inhibited by a series of nucleoside triphosphates. These investigators pointed out the The oxidation of threo-D-isocitrate to a-keloglutarate and influence of TTP on pyruvate kinase activity. High levels of C02 occurs by 2 separate pathways, one medialed by a TTP (~1 mM) and low levels of ADP (~0.01 mM) almost NADP-linked dehydrogenase (tfzreo-D-isocilrate:NADP completely inhibited both the liver and hepatoma pyruvate oxidoreductase, EC 1.1.1.42) and the other by an NAD-linked kinase activity. It was speculated that, along with the increased dehydrogenase (i/ireo-D-isocitrate:NAD oxidoreductase, EC

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1.1.1.41) (95, 150, 196, 197, 230, 231, 282, 288). The ascites) and the kinetic characteristics of the NAD-linked NADP-linked isocitrate dehydrogenase was found in both isocitrate dehydrogenase which was partially purified from cytoplasmic and mitochondria! fractions, while the Ehrlich ascites carcinoma. These studies revealed that only the NAD-linked enzyme was localized exclusively in the tumor and brain mitochondria had a predominance of the mitochondria (29, 94, 114, 282). The total quantity of NAD-linked enzyme. And the tumor NAD-linked isocitrate mitochondrial versus cytoplasmic isocitrate dehydrogenase dehydrogenase had positive cooperative homotropic activity varied from tissue to tissue, and the amount of NADP interactions with isocitrate, magnesium ion, and ADP, while versus NAD-linked mitochondrial isocitrate dehydrogenase NADH and NADPH were inhibitory. It would appear that the activity varied within different tissues. In most mammalian decreased level of cytoplasmic NADP-linked isocitrate tissues, the total mitochondrial isocitrate dehydrogenase dehydrogenase (when compared to normal liver) and the activity greatly exceeded the cytoplasmic activity. However, in predominance of the NAD-linked isocitrate dehydrogenase in liver and adrenal tissues, the dominant form was the tumor (also skeletal muscle) mitochondria, the latter enzyme cytoplasmic NADP-linked enzyme (95, 150, 282, 283, 339). responding directly to adenine and pyridine nucleotide levels, With regard to mitochondrial enzymatic activity, the could bring about a highly sensitive regulatory parameter for NADP-linked enzyme was highest in mitochondria from rat citrate oxidation in the tumor cell. heart and kidney; the NAD-linked enzyme was the major mitochondrial form in rat skeletal muscle and brain and in Malate Dehydrogenase locust flight muscle (190, 282); rat liver mitochondria had about equal NAD- and NADP-linked enzyme activities (357). Malate dehydrogenase (L-malate:NAD oxidoreductase, EC Extensive studies have been performed which demonstrate a 1.1.1.37) has been isolated from several different tissues and feedback-inhibitory effect by adenine nucleotides upon organisms and has been found to exist in at least 2 separable isocitrate dehydrogenase activity (7, 9, 10, 29, 63, 140, 230, forms (82, 165, 221, 226,257,327,343,347,373,387,407, 231, 319). The NADP-linked enzymes exhibited classical 413, 419, 424). One isozyme was isolated from mitochondrial Michaelis-Menton kinetics and were noncompetitively extracts, and the other was found in the high-speed inhibited by nucleoside triphosphates. Two molecules of ATP supernatant fraction. Both the mitochondrial and supernatant were required for inhibition (230, 231). ATP and NADH were enzymes have been purified and characterized from the hearts inhibitory, while AMP and ADP were activators of the of tuna (182), chicken (185), cattle (126), pig (113), and from NAD-linked isocitrate dehydrogenase (29, 63, 355). Atkinson ox kidney (85, 86). (8) showed that the NAD-linked enzyme responded directly to The 2 forms from bovine heart had molecular weights the adenylate energy charge in a fashion which was similar to between 64,000 and 72,000 but differed in Chromatographie the response of several other enzymes which are involved in behavior, electrophoretic mobility, pH optima, Michaelis producing ATP for the cell. Thus the mitochondrial constants, and immunological reactivity (126, 346,416). Kitto NAD-linked isocitrate dehydrogenase activity responds to an and Lewis (184) found the molecular weights of both acute intracellular energy control mechanism. supernatant and mitochondrial malate dehydrogenases from The NADP-linked isocitrate dehydrogenases are probably tuna heart to be 67,000 and observed differences in their distinct and separate entities from the NAD-linked enzyme. substrate inhibition by oxaloacetate. The tuna Neurospora (320), Blastocladiella (214), and bovine heart (63) antimitochondrial enzyme serum inhibited the tuna NAD-linked isocitrate dehydrogenases had molecular weights mitochondrial enzyme, failed to inhibit or cross-react with the near 300,000 to 400,000. The heart form was found to supernatant enzyme from tuna, but cross-reacted with the aggregate in the presence of its activator, ADP (62). The mitochondrial malate dehydrogenase of other fish. The 2 NADP-linked enzyme, as purified from pig heart (345), had a malate dehydrogenases from chicken heart had a molecular molecular weight near 61,000 and did not aggregate. It has not weight of 67,000 and were composed of 2 subunits, but they yet been established whether the cytoplasmic and differed in amino acid composition, peptide maps, sensitivity mitochondrial forms of the NADP-linked isocitrate to inhibition by oxaloacetate and malate, and cross-reactivity dehydrogenase are true isozymes (e.g., have subunits in to mitochondria enzyme antisera (185). The pig heart enzymes common). had molecular weights near 70,000, were probably made up of Isocitrate dehydrogenase activity has been examined in 2 identical or very similar subunits, but had different amino several tumors. McLean and Brown (239, 240) observed acid compositions (83, 113, 260, 374, 375). reduced levels of the cytoplasmic NADP-linked enzyme in rat Although the 2 different malate dehydrogenase entities hepatomas when compared with normal rat liver. Although were each found to be homogeneous by ultracentrifugai hepatomas had a measurable level of both mitochondrial analysis and by several other criteria, electrophoresis on starch enzymes (142), and the NAD-linked isocitrate dehydrogenase or polyacrylamide gels revealed multiple enzymatically active was the major form in Ehrlich ascites carcinoma (355, 356), it bands for each major isozyme species (113, 145, 182â€”184, is thought that certain liver tumor mitochondria possess a 207, 375). Electrophoresis of the mitochondrial enzyme from pathway for isocitrate oxidation which is different from that the heart of the chick yielded 5 bands. The heart mitochondria of normal liver (142). of the tuna revealed only 1 malate dehydrogenase band. Evidence supporting this idea is observed from the work of However, the chick heart mitochondrial enzyme and the tuna Stein et al. (355-357) who studied the oxidation of citrate by heart mitochondrial enzyme cross-hybridized (65, 184). mitochondria from several rat tissues (including Ehrlich Gerding and Wolfe (113) observed 3 bands upon

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1971 American Association for Cancer Research. Isozymes in Cancer electrophoresis of the enzyme from the pig heart supernatant. NADP-glycerol-P dehydrogenase has been purified and Thus, malate dehydrogenase is found in vertebrates as 2 major studied from E. coli (187). The enzyme had a molecular distinct and separable entities, 1 in the mitochondria and 1 in weight of 65,000 to 80,000; was substrate specific for the supernatant fraction, each consisting of 2 or more subunits glycerol-3-P, dihydroxyacetone-P, and NADPH; and showed an and possible multiple forms. inhibition of dihydroxyacetone-P reduction by glycerol-3-P. It It is possible that the metabolic significance of the observed was postulated that the function of the NADP-linked molecular heterogeneity of malate dehydrogenases in the cell glycerol-P dehydrogenase in E. coli was to provide glycerol-3-P may be somewhat related to differences in the ability of the for lipid synthesis, and that the feedback inhibition by enzyme to adapt to microenvironmental influences. Kidney glycerol-3-P would act to restrict the intracellular pool size of mitochondrial malate dehydrogenase was highly sensitive to this intermediate. the ionic environment, while the cytoplasmic form was not The NAD-glycerol-P dehydrogenase has been purified and sensitive (86, 186, 348). One of the more important extensively studied from the muscle of rabbit (23, 43, 106, characteristics of the mitochondrial malate dehydrogenase was 385, 423), rat (104), bee (56), and chicken (409), and also the apparent loss of the substrate inhibition (1 mM from chicken liver (393, 409). The muscle enzymes had oxaloacetate) at higher ionic strengths. The substrate molecular weights between 60,000 and 70,000 and were inhibition was also abolished at pH 9.0 or above. The composed of 2 identical subunits (^30,000). The amino acid mitochondrial enzyme appeared to undergo limited compositions of the muscle enzymes were very similar, and a conformational change which unmasked thiol groups. The comparison of peptide maps of the rabbit muscle enzyme with thiol groups were protected by NADH and to a lesser extent those of a rat muscle enzyme showed that both possessed a by ADP (351). Thus the mitochondrial malate dehydrogenase 14C-labeled peptide that contained tyrosine from a would appear to be more susceptible to environmental control trypsin-chymotrypsin digest. The chicken liver and muscle than the cytoplasmic form. NAD-linked glycerol-P dehydrogenases were similar in The activity of malate dehydrogenase in cancerous tissue molecular weights, each had 2 identical subunits, and the was observed to be either similar to or lower in activity than in amino acid compositions were similar (409). However, normal tissues (177, 251, 294, 407). Reese and Huggins (304) differences between the chicken liver and muscle enzymes found high malate dehydrogenase activity in normal and were observed in electrophoretic mobility, peptide maps, lactating rat mammary glands but lower activity in mammary immunological cross-reactivity, and catalytic properties. The cancer. A progressive decrease was observed in hepatomas of binding constants for oxidized and reduced NAD and the Km increasing growth rat (399). The anodic (supernatant) form of values for dihydroxyacetone-P and glycerol-3-P were, malate dehydrogenase predominated in normal breast tissue, respectively, 5-, 25-, 5-, and 100-fold lower for the liver but the predominant form in breast cancer was the cathodic enzyme than for the muscle enzyme. Thus, the 2 (mitochondrial) enzyme (146). Since malate dehydrogenase is NAD-glycerol-P dehydrogenases are probably isozymic in a necessary component of the malate-aspartate shuttle, an character. It has been proposed (409) that the skeletal muscle important series of reactions which allows reducing equivalents enzyme might operate in concert with skeletal muscle lÃ¡clate to enter the mitochondria (214), a decrease in either the dehydrogenase to regenerate NAD during anaerobic glycolysis, cytoplasmic or mitochondrial form could reduce the activity while the liver enzyme might function in triglycÃ©rideand of this shuttle system in tumors. phospholipid biosynthesis. The cytoplasmic NAD glycerol-P dehydrogenase activity has Glycerol-Phosphate Dehydrogenase been examined in several tumors from mice, rats, hamsters, and humans (46, 82, 316, 341). The activity was found to be Nature has provided living cells with glycerol-3-P high in normal liver, reduced in preneoplastic liver, and very dehydrogenases which are linked to NAD or NADP (EC low in hepatomas. It was observed to be either very low or 1.1.1.8) and FAD or a general acceptor (EC 1.1.99.5). It absent in over 30 different tumor types (46, 342). The would appear that the pyridine-linked enzymes are mitochondrial FAD glycerol-P dehydrogenase activity was cytoplasmic and catalyze reversible reactions (23, 24, 385, found to be either similar or slightly elevated in several 393, 423). The flavin-linked enzymes are mitochondrial and neoplastic tissues (45, 153). The greatly reduced levels of catalyze a reaction that is essentially irreversible (309, 350). cytoplasmic glycerol-P dehydrogenase would greatly reduce The flavin-linked enzyme is fairly widely distributed in the ability of the glycero-P shuttle system to provide reducing mammalian cells, insect tissues, and yeast (350). In animal equivalents to tumor mitochondria. Thus, there is evidence tissues, the highest concentration was found in brain and that the glycerol-P shuttle does not occur in tumors (124). skeletal muscle (308). FAD-glycerol-P dehydrogenase has been purified from pig brain and was found to contain 1 mole of GlutaminasÂ« FAD and l g atom of nonheme iron (308). Since the enzyme was observed to be functionally linked to the mitochondrial Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) has cytochrome system, it is thought that it is the been purified and studied from E. coli (135, 137, 138), yeast intramitochondrial portion of the glycero-P shuttle system (1), a pseudomonad (302, 303), dog kidney (328), pig kidney which oxidates a-glycero-P and thus provides reducing (208), and rat kidney and liver (170, 172). It exists both asa equivalents for mitochondrial oxidative phosphorylation phosphate-independent enzyme and a phosphate-dependent (350). enzyme in most mammalian tissues (169). The pig kidney

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1971 American Association for Cancer Research. Wayne E. Criss glutaminase activity (phosphate-dependent or independent similar to the adult kidney phosphate-independent glutaminase glutaminase) was found to be kinetically allosteric and had a as opposed to the adult liver phosphate-independent molecular weight of 150,000 in Tris buffer, 250,000 in glutaminase and is therefore probably not under the regulation phosphate buffer, 750,000 in borate buffer, and 2,000,000 in of the citrate acid cycle metabolites or the dietary changes of phosphate-borate buffer (208, 209). These various forms of the host, but it would be susceptible to product inhibition by pig kidney glutaminase were activated and protected by glutamate (171). Thus the intricate and highly efficient regulatory mechanism (170) NH4 *-Â»â€¢ureaconversion would inorganic and organic acids (363). Katunuma et al. (172) isolated 2 forms of glutaminase from rat kidney. The rat probably not be present in tumors. kidney phosphate-independent glutaminase did not require Because glutaminase is located in the matrix of phosphate-dependent glutaminase required 0.1 M phosphate mitochondria and glutamate is an excellent reductant, it has ./V-acetylglutamate, was inhibited by L-glutamate, was stable to been proposed that this system could provide an excellent heat and p-chloromercuribenzoate, had a Km (glutamine) of source of reducing power for the mitochondrion (199). This ^4 (10~3) M, and was not affected by most citrate acid cycle would help to explain the observation that the mitochondria! metabolites or dietary changes in the host; while rat kidney matrix NAD/NADH ratio is about 9 as compared to the phosphate-dependent glutaminase required 0.1 M phosphate whole-cell NAD/NADH ratio of 700 (414). The mechanism buffer for activity, was not activated by malÃ©ateorcarbonate, would be of significant value to liver or kidney cells with a was labile to heat and p-chloromercuribenzoate (stable if high rate of glucose metabolism, but the mechanism would phosphate present), had Km (glutamine 4 (10~2 M), and was probably not be present in tumor cells which have greatly induced by a high-protein diet (172). reduced levels of glutaminase activity or a predominance of The phosphate-independent glutaminases from rat liver and the kidney phosphate-independent glutaminase which is kidney have been purified and compared (170). Rat liver readily feedback inhibited by glutamate. phosphate-independent glutaminase did not require phosphate for activity, had a molecular weight near 135,000, was labile Aspartate Aminotransferase to heat and p-chloromercuribenzoate, was resistant to the action of lipase, had a Km (glutamine) from IO"1 to IO"3 M Multiple forms of aspartate aminotransferase depending on the level of the activator, was activated by (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.10) are several citrate acid cycle metabolites, and was induced by a present in rat liver, heart, brain, and skeletal muscle; in pig high-protein diet. The rat kidney phosphate-independent heart and liver; in bovine heart and liver; and in human liver, glutaminase had a molecular weight near 50,000, was labile to heart, and kidney (33, 48-50, 89, 103). These forms are the action of lipase, and differed from the liver distributed between the cytosol and mitochondria of most of phosphate-independent glutaminase in most of the other these tissues (81, 96, 97, 233, 336). The predominating comparisons (see preceding paragraph). Both aspartate aminotransferase enzymes have been purified from phosphate-independent glutaminase enzymes showed sigmoid the cytoplasm and mitochondria from most of these tissues kinetics in the absence of activator and normal linear kinetics and were found to be distinguishable by their electrophoretic, in the presence of activator (malÃ©ate). However, rat liver Chromatographie, kinetic, immunochemical, and solubility phosphate-independent glutaminase increased in molecular properties (31-33, 233, 252); but they appear to have similar weight in the presence of substrate, rat kidney primary sequences at their active sites (51, 253, 381). The phosphate-independent glutaminase did not (168, 170). Thus, cytoplasmic enzyme migrated as a single anionic component glutaminase activity is found in a phosphate-dependent and a on paper or agar gel electrophoresis, however, it was observed phosphate-independent form, the latter also existing as 2 as 3 components in starch gel electrophoresis (48, 50, 103). distinct enzymes which are tissue specific. The mitochondrial aspartate aminotransferase migrated as a Glutaminase activity is high in kidney and brain, moderate single cationic component on paper or agar electrophoresis, in liver and heart, and low in hepatomas (139, 211, 301) and but it was separated into 2 components by other tumors (193). Studies with synthetic glutamine carboxymethylcellulose column chromatography (33, 48). derivatives on the inhibition of glutaminase in rat liver and These 2 mitochondrial forms were found to have different several oncogenic tissues (Sarcoma 180, reticulum cell Chromatographie, solubility, and mitochondrial membrane sarcoma, and a diazo dye-induced hepatoma) indicate that the properties (33, 244). Thus, there appear to be distinct liver and tumor glutaminase are different enzymes (361). cytoplasmic and mitochondrial aspartate aminotransferases in Katunuma et al. (170, 173), using kidney mammalian tissues; either may exist in multiple forms. phosphate-independent glutaminase antiserum, found Aspartate aminotransferase activity has been shown to be complete cross-reactivity with kidney phosphate-independent elevated in the Morris Hepatoma 5123 and decreased in the glutaminase and fetal liver phosphate-independent Morris Hepatoma 3683 and Dunning and Novikoff hepatomas glutaminase, partial cross-reactivity with Hepatoma AH-130 (87). An increased supernatant activity was observed in a series phosphate-independent glutaminase, and no cross-reactivity of fast-growing hepatomas when compared to adult, with regenerating or normal adult liver phosphate-independent embryonic, or regenerating liver (335, 337). Otani and Morris glutaminases. (274) observed at least 2 forms of aspartate aminotransferase Similarly, the major glutaminase activity found in a series of in rat hepatomas. Both cationic and anionic enzymes were hepatomas was of the kidney type (217). It would appear that qualitatively similar to the normal liver forms, but there was the glutaminase activity of tumors is caused by an enzyme an increased amount of the cytoplasmic isozyme in Hepatoma

1530 CANCER RESEARCH VOL. 31

5123B (273, 274). A study of hormone responsiveness in Adenylate kinase has been separated into several Morris Hepatoma 5123B revealed that adrenalectomy caused a electrophoretic forms (36, 76, 101, 189) and has been decrease in the total specific activity of aspartate observed to occur in different subcellular compartments of aminotransferase in the tumor without isozyme changes, while mammalian tissues (2, 66, 74, 75, 181, 187, 210, 228, 300). hydrocortisone treatment caused an increase in the total Two major isozymic forms have been distinguished in rat specific activity and an alteration of the isozyme distribution tissues (75). Adenylate kinase II was the major form in skeletal favoring the cationic (mitochondria!) enzyme. The enzymatic muscle, heart, diaphragm, brain, lungs, and uterus. It was activity from the liver of normal rats remained unaltered under found in the cytoplasm of these tissues. The predominant similar chronic conditions (274). Immunochemical studies isozyme in kidney, liver, and testis was adenylate kinase III with the antiserum of the anionic (cytoplasmic) enzyme which was located in the mitochondria in these tissues. The revealed an increase in the anionic form of aspartate liver mitochondrial adenylate kinase was observed to respond aminotransferase in the Dunning hepatoma and Morris to dietary changes in the host (76). Hepatoma 5123B and a slight decrease of this enzyme in Adenylate kinase activity has been studied in a series of Novikoff Hepatoma (262). However, fluctuations of the Morris hepatomas (76). Although the mitochondrial isozyme various subspecies of the cytoplasmic and mitochondrial was high in a group of highly differentiated hepatomas, it was aspartate aminotransferases in tumors have not been reported. decreased almost 10-fold in a group of poorly differentiated Therefore, it would appear that aspartate aminotransferase hepatomas. The cytoplasmic adenylate kinase was relatively remains responsive to hormones in at least 1 tumor, but there unchanged in the latter experiments. Thus, it would appear is a shift in the isozyme pattern which results in increased that the rat liver adenylate kinase, which was under hormonal levels of the cytoplasmic enzyme in tumors. or dietary control, was the form which in hepatomas decreased Aspartate aminotransferase activity has been implicated in with decreasing differentiation. the synthesis of glucose from noncarbohydrate precursors Since adenylate kinase is an integral part of the adenylate (343). Glyceraldehyde-3-P, a glycolytic and gluconeogenic energy storage system, which has been defined and elucidated intermediate, inhibited both aspartate aminotransferase by Atkinson (6â€”9) and his colleagues as being capable of isozymes (195). The mitochondrial enzyme was much more regulating entire metabolic pathways, thus providing the cell susceptible to inhibition than the cytoplasmic enzyme. If, in with a very sensitive regulatory control device, decreases in the vivo, the intermediate, glyceraldehyde-3-P, has a negative mitochondrial form of this enzyme could decrease the ability feedback effect upon the gluconeogenic enzyme, aspartate of the mitochondria to maintain or reestablish a proper aminotransferase, we would anticipate that in tumors, where "adenylate charge" in the tumor cell or possibly even hamper the cytoplasmic to mitochondrial isozyme ratio was increased, tumor mitochondrial transphosphorylation. this controlling effect would be diminished. Carbamyl Phosphate Synthetase Adenylate Kinase Nature has provided that the synthesis of carbamyl Adenylate kinase activity (ATPrAMP phosphotransferase, phosphate, an important precursor in urea and pyrimidine EC 2.7.4.3) has been found in mammalian (143, 159, 263, synthesis, could be derived from citrulline (134, 301), 265, 349) and nonmammalian tissues (66, 359). The major creatinine (364), or glutamine (70, 127). However, in animal forms of the enzyme were highly purified from bakers' yeast tissues, there appear to be 2 major forms of carbamyl (66), calf lens (188, 189), bovine liver mitochondria (228, phosphate synthetase (EC 2.7.2.5) which are specific for either 229), pig liver (64), rat liver (77, 321), and rabbit muscle ammonium or glutamine (57, 71, 130, 132, 133, 232, 266). (263). Numerous similarities and differences can be observed Carbamyl phosphate synthetase I formed carbamyl phosphate from the reported kinetic and structural data. from ammonia, carbon dioxide, and ATP; required The molecular weight for adenylate kinases from calf lens, ./V-acetylglutamate as a catalytic cofactor; was not inhibited by bovine liver mitochondria, rabbit muscle, and yeast were 6-diazo-5-oxo-L-norleucine; and was located predominantly in determined to be 21,000, 21,500, 21,000, and 40,000, liver mitochondria. Carbamyl phosphate synthetase II used respectively (188, 189, 223, 228, 229, 263, 264). Adenylate glutamine as substrate, did not require A'-acetylglutamate for kinase III, from rat liver mitochondria (74), was observed at 3 activity; was inhibited by 6-diazo-5-oxo-L-norleucine; and was different molecular weight values, 23,000, 46,000, and 68,000 located in the supernatant of muscle, spleen, testis, heart, and (77). Adenylate kinase II from rat liver cytosol (74, 75) had lungs. Examination of carbamyl phosphate synthetase activity only 1 molecular weight near 46,000 (321). Considerable in Novikoff ascites tumor (235), Ehrlich ascites tumor (131), variation was observed in substrate specificity and apparent Walker carcinosarcoma and several mammary carcinomas Michaelis constants between the purified adenylate kinases. (420), fetal rat liver (132), and hematopoietic spleen (155, KJJJ values for all the adenine nucleotides with bovine liver 369) has revealed that increased amounts of Enzyme II were mitochondrial enzyme were above 1 mM ; the the K^ values correlated with increased tissue growth rates. Very little, if for the rabbit muscle and both rat liver enzymes were from any, measurable amounts of carbamyl phosphate synthetase I 0.12 to 0.73 mM; K^ values for the yeast enzyme were were detected in the tissues with higher metabolic rates. The reported from 0.05 to 0.27 mM. Substrate feedback inhibition cytoplasmic synthetase II may be a prerequisite for accelerated at levels of 1 mM AMP was observed with both of the rat liver rates of growth in a variety of tissues (420). Further studies adenylate kinases (321). concerning the possible regulation of carbamyl phosphate

NOVEMBER 1971 1531

Table 1 A summary ofneoplastic changes This is a tabulation of the normal isozymic system, tumor isozymic system, and the theoretical changes which could result in the intermediary metabolism of tumors because of the observed alterations in the isozymic system.

Isozyme from corresponding Enzyme Isozymes from normal tissue malignant tissues Isozymic change would result in:

DNA polymerase I. Use denatured DNA Increased II; decreased III Preference for denatured vs. native II. Use denatured DNA DNA as "primer" substrale III. Use native DNA

Hexokinase I. 1 Low Km (glucose); Increased hexokinases; de- Decreased response lo hormones; in II. > most lissues; inhibited decreased glucokinase creased product inhibition (glucose- III. J by glucose-6-P and ADP 6-P) IV. High Km (glucose); responsive to hor mones; liver; inhibiled by ADP

Fructose 1,6-di- L. Liver and kidney M. muscle enzyme Increased sensitivity to AMP inhibition; phosphatase M. Skelelal muscle; inhibited by AMP loss of response to hormones

Aldolase A. Skeletal muscle; splils only fruclose- Increased aldolase A Increased breakdown of fructose-1,6- 1,6-di-P inhibited by ATP di-P over thai of splilting fruclose- B. Liver; splits fructose-1,6-di-P and fruc- 1-P; increased sensitivity to inhibilion lose-l-P;no/ inhibited by ATP by ATP C. Brain; intermediate

Pyruvate kinase L. Liver; aclivaled by fruclose-1,6-di-P; in Increased total activily; pos Increased aclivity may be due lo de hibited by ATP sible new "type" isozyme creased level of inhibitor (ATP) M. Skelelal muscle; inhibited by ATP

LÃ¡clatedehydro- L. Liver; inhibited by high pyruvate Increased total activity; in Isozyme form which allows high rates of genase M. Skeletal muscle; not inhibited by high creased muscle enzyme glycolysis pyruvale

Adenylate kinase Mitochondria!: liver and kidney Decreased lolal activity, de Loss of response to hormones, shift in Cytoplasmic: skeletal muscle, heart, brain, creased mitochondrial iso ability of subcellular compartmenls lungs zyme to regulale levels of adenine nucleo- lide pool

Isocitrate dehydro- NAD-linked; mitochondria! Decreased cyloplasmic NADP- Increased sensilivily lo control by ade genase NADP-linked; mitochondria! and cyloplas- linked form; predominanl nine and pyridine nucleotide levels mic form is mitochondrial NAD form

Malate dehydro- Mosl lissues: Decreased tolal aclivily; de Increased sensitivily lo environmenlal genase Milochondrial; sensilive lo environmenl creased cytoplasmic isozyme parameters; decreased malale-aspar- and subslrale inhibilion tate shuttle activity Cyloplasmic; Â«orsensitive

Glycerol-P dehy- Cytoplasmic: NAD-linked, NADP-linked Decreased tolal activily, de Decreased glycerol-P shuttle aclivily drogenase Mitochondria): FAD-linked creased cyloplasmic NAD- linked enzyme

Glu lamina se Phosphate dependent Decreased lotal activily; in Loss of NH4*-> urea mechanism; pos Phosphate independent creased kidney-specific sibly a decreased reducing power for 1. Liver-specific phosphale-independent glu- Ihe milochondrion 2. Kidney-specific taminase

Asparla te amino- Most lissues: Increased cyloplasmic isozyme Decreased inlracellular control Iransferase Milochondrial; more sensilive lo inhibi lion by glyceraldehyde-3-P Cyloplasmic; less sensitive to inhibition of glyceraldehyde-3-P

Carbamyl phos- I. Ammonia + CO2 + ATP; mitochondria! Increased II Isozyme form which correlates with ac phale synlhelase II. Glutamine as subslrale; cytoplasmic celerated growth rates

LaclÃ³sesynlhetase A. Galactosyllransferase Both present Respond lo hormones similar lo aduli B- a-Laclalbumin tissue and dissimilar lo immature tis sue

1532 CANCER RESEARCH VOL. 31

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1971 American Association for Cancer Research. Isozymes in Cancer synthetase II are necessary before we can postulate a role for it in the review, the enzyme systems which are directly or in the neoplastic processes. indirectly engaged in intermediary metabolism would rate a very high priority. Upon reflection, there is excellent rationale Lactose Synthetase why key pathway enzymes of intermediary metabolism exist as isozymic systems. It is therefore not unreasonable to find Lactose synthetase (UDP-galactose: D-glucose-1-galactosyl- that many of these fundamental isozymic systems have transferase EC 2.4.1.22) is composed of 2 individual proteins undergone some form of alteration or complement shift in the and is found only in mammary tissue (12-14, 28, 53-55, neoplastic state. 237, 238, 396). Protein A was found to be a UDP Considerable data are available which support the idea that galactose:jV-acetylglucosamine galactosyltransferase and was neoplastic tissues have lost many of their differentiated located in the microsomes of mammary glands from the cow, functions. Loss of function can probably be attributed to loss rat, goat, sheep, human, and guinea pig (12-14, 53, 55, 158, of the key enzyme or isozymic complement within the 280, 376, 396). Protein B has been crystallized and was pathway which performs that function. Since the function identical to a-lactalbumin by substitution in enzymatic rate may not be present in the corresponding tissue in the fetal assays, spectra, immunological titration, amino acid animal, it is not surprising to find the enzymatic and isozymic composition, mobility on starch gel electrophoresis, molecular complement patterns of the fetal tissue resembling the weight, and cochromatography on diethylaminoethyl cellulose neoplastic tissue. and Sephadex G-100 (13, 53, 88, 118). The B protein Similarly, fetal tissues are not subject to the same modified the substrate specificity of the A protein from extracellular and intracellular controls as the corresponding ./V-acetylglucosamine to glucose, resulting in lactose synthesis. normal adult tissues (121 â€”123, 151, 265). Hence, one must Proteins A and B are both necessary for lactose synthesis (53, anticipate a change in the controlling mechanisms in tissues 55,237,238,377). which have become neoplastic. The new modes of control In immature female rats, the activity of lactose synthetase which are established in tumor cells need not be unique to all was very low. In adult female rats, the specific activity of the neoplastic tissues or identical to the corresponding fetal tissue, A protein increased 30-fold and the specific activity of the B but need only provide an environment which is conducive to protein increased 100-fold upon lactation or in response to increased cell growth and increased cell division. One has but prolactin. A transplantable rat mammary carcinoma to look at the many cell types in all of nature to realize that (R3230AC) apparently synthesized significant quantities of numerous combinations of enzymatic and isozymic systems lactose synthetase (237). However, perphenazine, which is a can produce in or respond to such an environment with the potent stimulator of prolactin release, did not increase the result being a potentially neoplastic state. Therefore, we activity of lactose synthetase in the immature rat mammary should not necessarily expect all neoplastic tissues to be gland but did increase the activity 2- to 4-fold in the mammary biochemically similar, nor should we expect them to respond carcinoma (237). McGuire (238) also observed increases in the in concerted fashion with concerted control mechanisms. A and B proteins of lactose synthetase in the rat mammary Numerous experimental approaches on a variety of neoplastic carcinoma upon 4 days of in vivo treatment with perphenazine tissues are necessary to establish any possible biochemical or ovine prolactin. It would appear that the immature control correlates within and between tumor species. mammary glands were not sufficiently differentiated to respond to prolactin, whereas the mature glands and the mammary tumor were differentiated enough to respond to the ACKNOWLEDGMENTS hormone. I sincerely thank Dr. Sidney Weinhouse for his continued encouragement and critical suggestions concerning the manuscript.

DISCUSSION REFERENCES I have attempted to survey several intracellular isozymic systems which show alterations during differentiation from the 1. Mulinimi!km, A. K., and Nikolaev, A. J. Separation and Purification of Glutaminase and Desamidase of Isoglutamine from normal to the neoplastic state (Table 1). I have purposely Yeast. Biokhimiya, 32: 859-866, 1967. omitted the isozymic changes found in the blood plasma, such 2. Adelman, R. C., Lo, C., and Weinhouse, S. Dietary and Hormonal as acid and alkaline phosphatases, lÃ¡clatedehydrogenase,etc., Effects on Adenosine Triphosphate: Adenosine Monophosphate Phosphotransferase Activity in Rat Liver. J. Biol. Chem., 243: because these systems have been reviewed many times and lead 2538-2544, 1968. to an entirely different type of interpretation. The latter 3. Adelman, R. C., Morris, H. P., and Weinhouse, S. Fructokinase, Irii)k iii.-i-.i-and Aldolases in Liver Tumors of the Rat. Cancer Res., interpretation can be of diagnostic significance, but it reveals 27. 2408-2413, 1967. only a very small portion of the multiple changes in a tissue 4. Anstall, H. B., Lapp, C., and Trujillo, J. M. Isozymes of Aldolase. which occur during the neoplastic transformation. Science, 154: 657-658, 1966. It was not the original intent to center the review around 5. App, A. A., and Jagendorf, A. T. Purification of Alkaline Fructose Diphosphatase from Euglena gracilis. Biochim. Biophys. any specific isozymic systems, but simply to review those Acta, 85: 427-434, 1964. isozymic systems which have been reported to undergo change 6. Atkinson, D. E. Biological Feedback Control at the Molecular Level. Science, ISO: 851-857, 1965. in cancers, especially in liver cancers. It would appear that, if 7. Atkinson, D. E. Regulation of Enzyme Activity. Ann. Rev. there is a mutual area of involvement for most of the enzymes Biochem., 35: 85-118, 1966.

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8. Atkinson, D. E. The Energy Charge of the Adenylate Pool as a 36. BÃ¶ckelmann,W., Wolf, U., and Ritter, H. Polymorphism of the Regulatory Parameterâ€”Interaction with Feedback Modifiers. Phosphotransferases Adenylate Kinase and Pyruvate Kinase. Biochemistry, 7: 4030-4034, 1968. Humangenetik, 6: 78-83, 1968. 9. Atkinson, D. E. Regulation of Enzyme Function. Ann. Rev. 37. Bollum, F. J. Thermal Conversion of Nonprinting Microbio!., 23: 47-68, 1969. Deoxyribonucleic Acid to Primer. J. Biol. Chem., 234: 10. Atkinson, D. E., Hathaway, J. A., and Smith, E. C. Kinetics of 2733-2734, 1959. Regulatory Enzymes. J. Biol. Chem., 240: 2682-2690, 1965. 38. Bollum, F. J. Oligodeoxyribonucleotide Primers for Calf Thymus 11. Auerbach, V. H., and Waisman, H. A. Tryptophan Polymerase. J. Biol. Chem., 235: PC18-PC20, 1960. Peroxidase-Oxidase, Histidase, and Transaminase Activity in the 39. Bollum, F. J. Calf Thymus Polymerase. J. Biol. Chem., 2.?5: Liver of the Developing Rat. J. Biol. 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Wayne E. Criss

Cancer Res 1971;31:1523-1542.

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