Hexokinase Specificity of Some Tumor Tissue Extracts*

CHARLES F. LARGE, JR.,t AND PAUL KOHN

(Department o] Biological Ckemistry, University of Illinois College of Medicine, Chicago, Illinois)

SUMMARY The substrate specificity of hexokinase preparations from Ehrlich ascites cells, a spontaneous adenocarcinoma, Jensen sarcoma, and Walker carcinoma was studied with a number of sugars which differed in structure from glucose at one or more carbon atoms used as substrates. A marked variation in specificity was found. The preparations from Jensen sarcoma and Ehrlich ascites cells were relatively specific for glucose and some C-~ analogs. The preparations from the adenocarcinoma and Walker carcinoma were nonspecific and brought about phosphorylation of a number of sugars differing from glucose in many positions. Inhibition of the hexokinase preparation from Ehrlich ascites tumor cells by altrose and talose was observed.

In 1957 Krebs (7) referred to two "pacemakers" aldohexoses and that, therefore, hexoses other of anaerobic glycolysis, the hexokinase reaction than glucose might act as inhibitors of glycolysis. and the triosephosphate dehydrogenase reaction, More specifically, since glucose serves as a sub- and discussed the fundamental importance of strate for a hexokinase, the specificity of these en- these reactions to the entire metabolic economy of zymes was of particular interest. the organism. Many hexose analogs substituted in The present investigation entailed a study of the C-s position have been shown to have a par- the specificity of hexokinases derived from a num- ticular capacity to serve as glycolytic inhibitors ber of tumor tissues. For this purpose, hexokinase (~, 8, 5, 9, ~8, 24). The importance of the hexo- preparations were obtained from Ehrlich ascites kinase reaction can thus be clearly seen. It ap- cells and a spontaneous adenocarcinoma of mice peared, therefore, that a study of the hexokinase and from Jensen sarcoma and Walker carcinoma activity in various tumor tissues should prove of of rats. Studies were made of the ability of the interest, especially in light of the difference in rare aldohexoses, D-allose, altrose, gulose, idose, glycolytic rates of these tissues as compared with and talose, and of glucosamine, N-acetylglucos- that of normal tissues. The pronounced require- amine, and ~teoxyglucose to serve as substrates ment of tumor cells for glucose, whatever the un- for, or inhibitors of, these enzyme preparations. derlying metabolic demand, may offer a possible The findings indicate distinct differences in the avenue of attack for cancer chemotherapy. specificities of the hexokinases in the enzyme Because of the structural relation of known preparations. In studies of a similar nature (8), competitive inhibitors to normal substrates, it differences in the specificities of the hexokina.ses seemed reasonable to assume that a similar rela- in preparations derived from normal rat tissues tionship might exist between D-glucose and other were observed. * Supported in part by research grant P-161 from the Ameri- can Cancer Society and by research grant A-4~5 from the Na- MATERIALS AND METHODS tional Institutes ~f Arthritis and Metabolic Diseases of the The sugars used were obtained commercially National Institutes of Health, United States Public Health or prepared synthetically as follows: glucosamine Service. hydrochloride was obtained from PfanstieM Labo- This work is taken from a thesis by C.F.L. submitted to the University of Illinois Graduate College in partial fulfillment of ratories, N-acetylgtucosamine from Chas. Pfizer the requirements for the degree of Doctor of Philosophy. and Company, Inc., ~-deoxygluoose from Aldrich t Present address: The Hektoen Institute for Medical Ile- Chemical Company, Inc. The sugars were research, Chicago, Illinois. crystallized from suitable solvents before use. The Received for publication April 7, 1961. rare aldohexoses allose, altrose, gulose, and talose 1055

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1961 American Association for Cancer Research. 1056 Cancer Research Vol. 21, September 1961 were prepared as described previously (8). Idose and the cells were separated from the fluid by cen- was prepared from xylose by the procedure of trifugation at 400 X g for 5 minutes. The layer lsbell (6). Adenosine triphosphate (ATP) was ob- of red blood cells was carefully removed by aspira- tained from Pabst Laboratories as the disodium tion, and the precipitate was repeatedly washed salt. Ethylenediaminetetraacetic acid (EDTA) with Krebs-Ringer bicarbonate buffer solution at was obtained from the Eastman Organic Chemi- pH 7.4 (20) until the supernatant fluid was clear cals Division of the Eastman Kodak Company. and the tumor cells almost entirely free of red Assay procedure.--The method used for assay- blood cells. One hundred ml. of washed, packed ing the enzyme preparations was the constant pH cells was obtained from an original volume of 500 microtitrimetric procedure of Schwartz and Myers ml. of aseites fluid. (17), carried out as previously described (8). In- The cells were homogenized for 5 minutes in a cubations were carried out in 4-ml. volumes at VirTis "45" blender with 2 volumes of a buffer 30 ~ C. and pH 7.4 with 0.02 ~ MgC12, 0.002 M consisting of 0.115 M KC1, 0.02 M K2HPO4, 0.01 M ATP, 0.002 M EDTA, enzyme, and varying con- EDTA at pH 7.4. The homogenate was then cen- centrations of the substrate. A unit of enzyme ac- trifuged at 2000 X g for 30 minutes. The floating tivity was defined as the amount of enzyme re- lipide layer and the precipitate were discarded, quired to produce 1 X 10-s acid equivalents per and the supernatant fluid was centrifuged at

TABLE 1 DISTRIBUTION OF HEX0KINASE ACTIVITY IN TISSUE HOMOGENATES

CENTRIFUGATION* HEXOKINASE RECOVERED AMOUNT IN TISSUE SUPEItNA TANT ttomogenate Supernatant Speed Time (units/gin (units/gm (rER Cr~NT) (rain.) (xg) tissue) tissue)

Ehrlich ascites cells 100,000 60 ]47t 10St 71 Spontaneous adenocarcinoma 100,000 60 5O8 105 51 Jensen sarcoma 50,000 45 6O4 327 54 Walker carcinoma 50,000 45 551 365 70

* Following an initial centrifugation at 600 X g for ~ minutes to remove unbroken cells and other debris. Expressed as units per 100 ml. of washed, packed cells. minute at 30 ~ C. For the determination of each 100,000 X g for 1 hour. The clear supernatant Km value, the enzyme concentration was adjusted liquid containing soluble hexokinase activity was to produce about 25 X 10-s acid equivalents per removed and used in the assays (Table 1). This minute. Phosphatase and ATPase activities were fluid had a protein concentration of 14.5 mg/ml inhibited during assay by the use of fluoride with a specific activity of 6.5 units/nag. The prepa- (15, 21) and n-octanol (13). The activity of these ration was stable for several months at --15 ~ C. interfering enzymes was decreased further as a Hexokinase from a spontaneous adenocarcinoma. 1 result of the freezing and thawing (13) entailed in --A mammary adenocarcinoma which had de- carrying out the assays. veloped spontaneously in 21 mice was collected Enzyme preparation.--All enzyme isolation and found to weigh 50 gm. The tissue was homoge- work was performed at 00-5 ~ C. Tumors were nized and treated by the procedure described grown in female Sprague-Dawley strain rats or above. The supernatant fluid had a protein con- Strong A mice. Protein determinations were car- centration of 20 mg/ml with a specific activity of ried out by the method of Lowry et al. (11), or by 3 units/rag (Table 1). The preparation was found digestion and nesslerization as described in Hawk, to be stable for several months at --15 ~ C. Oser, and Summerson (3), after dialysis against Jensen tumor hexokinase.2--Whole tumors were running water for 15 hours. excised from 26 rats, and most of the connective Ehrlich ascites tumor hexokinase.l--Ascites fluid tissue was removed. The tumor tissue was washed from tumor-bearing mice was harvested in Luster- in cold phosphate buffer at pH 7.4 to remove sur- old centrifuge tubes 10 days after inoculation, face blood. After being washed, the tissue was 1 Tissue kindly donated by Dr. Harry Monsen, Department Tumor transplants were kindly donated by Dr. Kathryn of Anatomy, University of Illinois. Sydnor, Ben May Laboratories, University of Chicago.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1961 American Association for Cancer Research. LANGE AND KorIN--Hexokinase Specificity of Tumor Extracts 1057 placed in petri dishes on ice, sliced longitudinally, are shown in Tables r and 3. The behavior of the and necrotic material was scraped away. The test sugars as substrates is compared to glucopy- clean, soft, translucent tumor tissue was then care- ranose, on the assumption that all the aldohexoses fully scraped away from any remaining connective exist primarily in the pyranose form in aqueous tissue. The tissue, which weighed 115 gin., was solution (16). Michaelis constants (Kin) were cal- homogenized in a Waring Blendor for 3 minutes culated by the method of Lineweaver and Burk in ~ volumes of a buffer consisting of 0.115 M/(C1, (10). Comparison of these values provides infor- 0.0~ M K2ttP04, 0.01 M EDTA at pH 7.4. An ini- mation pertaining to the identity of enzymes iso- tial centrifugation at 800 X g for 30 minutes related from different sources. Relative maximal moved all the heavy material. The supernatant rates, obtained from the expression fluid was centrifuged at r X g for 45 minutes. Vm~x (substrate) The clear supernatant fluid so obtained was used Relative maximal rate = for the assays (Table 1). Fluid having a protein Vmax (glucose) concentration of 4r mg/ml with a specific activity of 4 units/rag was obtained. This preparation was are of value in comparing the behavior of an en- stable for several months at --15 ~ C. zyme toward different substrates. Walker tumor hexokinase.2--From 95 gin. of tu- The phosphorylation coefficient (18), calculated mor tissue, obtained from ~5 rats, a soluble hexo- from the expression kinase (Table 1) was prepared by the procedure Phosphorylation coefficient described above. A clear supernatant fluid having a protein concentration of 60 mg/ml with a specific = Vmax (substrate) X Km (glucose) activity of 3 units/mg was obtained. This prepa- Vm~x (glucose) Km (substrate) ration was stable for several months at -15 ~ C. indicates the suitability of a substance as a sub- RESULTS AND DISCUSSION strate for hexokinase and serves to indicate the Since the assay procedure was dependent on the sensitivity necessary to assay enzyme activity. liberation of H +, control experiments were carried Values indicating the extent of phosphorylation out to determine the extent of interference by of a substrate in a given time are shown in Table 3. ATPase and other acid-producing systems which To obtain these values, assays were carried out may have been present in the enzyme preparations under conditions of optimal concentration of all and the effectiveness of the inhibitors used to re- constituents except the substrate, the concentra- press their activity. At pH 7.4 glucose-6-phosphate tion of which was limiting. Comparison of these is a stronger acid than phosphoric acid. Therefore, values gives information about the activity of an the enzymatic hydrolysis of glucose 6-phosphate enzyme toward a variety of substrates and can brings about an increase in pH, rather than a lib- also be used to ascertain conditions necessary for eration of I-I+. In previous work (8) with prepara- the determination of Michaelis constants. tions containing more active phosphatases than Ehrlich ascites tumor ceUs.--The results of the was present in these preparations, the increase was substrate specificity studies are shown in Tables found to be less than 0.1 pH units. For this reason, and 3. Only glucose and deoxyglucose were good and because octanol, fluoride, freezing and thaw- substrates in the ascites cell system. Of the other ing, and short periods at room temperature de- substances tested, gulose and allose were found to stroy the activity of glucose-6-phosphatase (13, be poor substrates. Considering that a large excess 15, 21), interference by this enzyme was neglected. of enzyme was required to demonstrate phos- The results of these experiments showed that the phorylation of these two rare aldohexoses, it is activity of ATPase and other acid-producing proc- apparent that this enzyme preparation had a pro- esses which may have been occurring was very nounced specificity and was, in fact, more highly low or absent from the preparations. Even in those specific than the other systems studied. From instances where the test substrate proved to be these experiments with relatively crude prepara- inert, or a very poor substrate, necessitating the tions it is not possible to state whether the ac- use of large amounts of the enzyme preparation, tivity observed was due to a mixture of kinases or acid production due to interfering processes was a single nonspecific kinase. On the basis of specific- less than 5 per cent of the total. This was consid- ity, however, it appears that the preparation from ered to be within the limits of experimental error Ehrlich ascites cells was more likely to have con- under these circumstances, and the blank values sisted of a single kinase than the other prepara- were neglected. tions studied. It should be noted, however, that in The results of the substrate specificity studies similar studies (14) galactose did not serve as a

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substrate, but phosphorylation of fructose was zymes of the other preparations in which they also demonstrated, suggesting that a specific fructo- were substrates. In spite of the lack of specificity kinase may also have been present. This is known and the high degree of enzyme-substrate affinity, to be the case in rat liver (4, 22). idose was found to be inert in this enzyme prepa- Spontaneous adenocarcinoma.--Owing to the re- ration, as well as in the other systems studied. stricted amount of this enzyme preparation it was In previous studies with hexokinase prepara- not possible to include studies with idose, glucos- tions from normal tissues (8), a fairly regular pat- amine, N-acetylglucosamine, or deoxyglucose. tern of specificity was observed in all tissues, Glucose, allose, altrose, gulose, and talose all which permitted certain conclusions to be drawn served as substrates, indicating considerably less associating carbohydrate structure with enzyme specificity than in the previous case. Of the four specificity. However, each of the preparations ob- preparations described here, the hexokinase de- tained from abnormal tissue, described in this re- rived from this tumor showed the lowest affinity port, had its own specificity characteristics, and for glucose, having a Km value of 1.5 X 10-4 M, a generalizations relating structure to specificity are fourfold increase over the other Km values re- difficult to make because many inconsistencies ported. ~Thereas the high degree of specificity of were found in these studies. Studies with the nor- the ascites cell preparation suggests the presence mal tissues indicated that the structure of the car- of a single hexokinase, the findings in this case sug- bohydrate at carbon s had little influence on gest that more than one hexokinase with different hexokinase activity, but a change from the glucose specificities may be present rather than a single configuration at carbon 3 (allose) or carbon 4 nonspecific enzyme. However, no evidence is (galactose) resulted in decreased activity. Changes available which would enable resolution of this at carbons 2 and 4 (talose) decreased activity still point at this time. further, and changes at ~ and 8 (altrose) or 3 and Jensen sarcoma.--Only glucose and those deriv- 4 (gulose) resulted in a loss of all activity. With atives of glucose modified at carbon 2 served as the exception that gulose was phosphorylated in a substrates in this enzyme preparation. The rare a system prepared from rat liver, the order of aldohexoses were completely inert. These results suitability of the rare hexoses as substrates for the may be interpreted to indicate the presence of a normal tissues studied was allose, talose, altrose, rather specific kinase in this tissue. In an earlier in- and gulose. vestigation, mannose and fructose were found to In the present studies, the lack of influence on be substrates for a Jensen sarcoma, but galactose enzyme activity due to changes in structure at car- was not (1). If it is assumed that the preparations bon 2 applied consistently only to 2-deoxyglu- were similar, these findings might also be interpret- cose. Glucosamine was not phosphorylated by the ed to indicate the presence of a fructokinase, as preparation from Ehrlich ascites cells, although it well as some lack of specificity in the glucokinase. was found to be a good substrate in all other prepa- The results support the conclusion that modifica- rations studied except that from rat kidney (8). tions of glucose at carbon 2 are relatively unimpor- Conversely, N-acetylglucosamine was actively tant in relation to the specificity of the enzyme, phosphorylated by the preparations from Jensen but modifications elsewhere in the molecule may sarcoma and Walker carcinoma, although it was markedly decrease or eliminate enzyme action (8). inert in the other systems in which it was tested. Walker carcinoma.--The results show that all This was an unexpected finding in the case of the the sugars tested except idose served as substrates, Jensen tumor, which otherwise showed a high de- The :Kin value for glucose was found to be com- gree of specificity, because it would appear that parable to that in the other systems studied. A the bulk of the acetyl group would be likely to considerable lack of specificity of the enzyme prep- prevent activity, t)hosphorylation of N-acetyl- aration is indicated and illustrates the marked glucosamine by hexokinases derived from a num- variation of specificities of the hexokinases derived ber of sources has, however, been reported by from different sources. The lack of specificity of others (12, 18, 19). this preparation can again be interpreted to indi- Almost no further correlations can be drawn cate the presence of either one nonspecific enzyme from the data, either by comparing the tumor tis, or more than one enzyme with different specific- sues with the results obtained from normal rat ities. tissues (8), or by comparing the tumor tissues The data in Table 2 show that, in almost every only to each other. Considering only the prepara- instance, sugars which served as substrates in this tions from ascites cells and Jensen sarcoma, both preparation had a greater affinity (lower K~ of which showed a high degree of specificity in values) for the enzyme than they did for the en- their action toward the rare sugars, differences in

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1961 American Association for Cancer Research. 1060 Cancer Research Vol. ~1. September 1961 behavior nevertheless were found, the principal were carried out by determining the reaction veloc- one being that gulose was not a substrate in the ity in assays in which the concentration of in- Jensen sarcoma preparation, but was a substrate, hibitor was kept constant and the concentration of though a poor one, in the ascites-cell system. The glucose was varied. In those experiments in which preparations from Walker carcinoma and the no inhibition occurred, calculations as described adenocarcinoma, which both exhibited a low de- by Lineweaver and Burk (10) led to a result iden- gree of specificity, also showed unique character- tical to the Kin. When inhibition occurred, an in- istics in their behavior toward the rare sugars hibitor constant (K~) was calculated (10). other than idose. In the preparation from the ade- The only instances in which inhibition was ob- nocarcinoma the order of suitability of the rare served was the inhibition of the hexokinase of sugars as substrates was altrose, gulose, allose, and Ehrlich ascites cells by altrose and talose (Table talose, whereas in the preparation from Walker 4). In similar experiments with the preparations carcinoma the order of suitability was altrose, from Walker and Jensen tumors and the adeno- talose, allose, and gulose. In both these prepara- carcinoma, no inhibitory effect of any of the rare tions, greater hexokinase activity was found toward sugars differing in configuration from glu- TABLE 4 cose at two positions than toward allose, which INHIBITION OF GLUCOSE PHOSPHORYLATION BY differs from glucose only at carbon 3. In the studies ALDOHEXOSES IN EHRLICH ASCITES CELL with hexokinases from normal rat tissues (8), HEXOKINASE PREPARATION* allose was found to be a better substrate than any Incubation mixtures contained 0.02 M MgC12, 0.002 M ATP, 0.002 M EDTA, 2 drops of n-octanol, and 25 units of enzyme of the other rare hexoses. Thus, the principal clear- and were carried out at 30 ~ C. and pH 7.4. Glucose concen- cut conclusions that can be drawn from these ob- tration was varied from 0.00014 M to 0.014 M at each concen- servations are that loss of the hydroxyl group of tration of inhibitor. glucose at carbon ~ affected these hexokinases only slightly, but changes from the glucose con- Inhibitor Inhibitor Modified at concentration Ki figuration at carbons ~, 3, and 4 (idose) caused a hexose carbont (/~moles/ml) (moles/liter) loss of all activity. Changes from the glucose configuration between these extremes, involving any Allose 3 13.9 Altrose 2, 3 13.9 6 X 10 -3 one or any two of the carbon atoms, resulted in a Talose 2, 4 13.9 3Xt0-3 pattern of behavior which was more or less unique 22.8 3.5X10 -3 for each of the enzyme systems studied. It is ap- Gulose 3, 4 25.4 J; Idose 2, 3, 4 21.8 parent that the hexokinases of these systems differ considerably in their capacity to act on sugars * In similar studies with preparations from the spontaneous other than glucose. In view of these differences it adenocarcinoma and Walker and ffensen tumors, no instances is remarkable that they are all similar in their ac- of inhibition were found. tion on glucose and ~-deoxyglucose and that, Jf Compared with D-glucose. furthermore, this activity, as judged by Km An undetectable inhibition. values, is similar to that of hexokinases derived from a wide variety of other biological sources. hexoses was detected at concentrations of the rare Nature of the reaction product.--In each assay sugars ranging from 7.8 to ~7.8/~moles/ml. in which hexokinase activity was found, the incu- Altrose and talose, which inhibited the Ehrlich bation mixture was examined to determine the preparation, were not substrates in this system, nature of the reaction product. According to the but gulose and allose, which were substrates, failed procedures previously described (8), it was found to exert a detectable inhibitory action. Further- that hexose phosphate esters were formed. Be- more, those sugars which were substrates in the cause these esters also had reducing properties, it other enzyme systems studied also failed to act as is reasonable to assume that they were the 6-phos- inhibitors of those systems. phate esters. The appearance of these esters in the To determine whether the rare sugars were incubation mixtures indicated that hexokinase ac- phosphorylated during these experiments, each in- tivity was indeed present. cubation mixture was examined by paper chroma- Inhibition of hexokinases by the rare sugars.- tography. In no case was any sugar phosphate Since a number of reports (~, 5, 9, s s have in- other than glucose 6-phosphate detected, regard- dicated that hexokinase can be inhibited by ana- less of whether or not the rare hexose had pre- logs of glucose, experiments were conducted to de- viously been found to be a substrate or inert, or termine whether the rare aldohexoses would act whether it had an inhibitory action in these as competitive inhibitors of glucose. These studies studies.

If it is assumed that two substances which hibition of Yeast Metabolism, Yeast Hexoklnase Activity, serve as substrates for the same enzyme occupy and Tissue Glycolysis, Biochim. et Biophys. Acta, 28: 127-33, 1958. the same active site on the enzyme surface, it is 6. ISBELL, H. S. ; KARABINOS, ft. V. ; I~USH, H. L.; HOLT, N. difficult to understand why inhibition did not oc- B. ; SCHWEBEL, A.; and GALKOWSKI, T. T. Synthesis of cur in those instances in which the rare sugars were D-Glucose-l-C 14 and D-Mannose-l-C 14. J. Research Nat. found to be substrates. A possible explanation for Bur. Standards, 48:163-71, 1952. this apparent contradiction in behavior may lie in 7. KREBS, H. A. Control of Metabolic Processes. Endeavour, 16:125-32, 1957. the conditions under which the experiments were 8. LAI~IGE, C. F., Jr., and KOHN, P. Substrate Specificity of carried out. In those assays in which the rare Hexokinases. J. Biol. Chem., 236:1-5, 1961. sugars were found to be substrates, the concen- 9. LASZLO, J.; LANDAU, B.; WIGHT, K.; and BURK, D. tration of the sugar and of the enzyme was much Effect of Glucose Analogs on the Metabolism of Human Leukemic Cells. ft. Nat. Cancer Inst., 21:475-83, 1958. higher than in the inhibition studies, and, as can 10. LINEWEAVER,H., and BURK, D. Determination of Enzyme be seen from the phosphorylation coefficients and Dissociation Constants. J. Am. Chem. Soc., 56:658-66, I/:m values in Table ~, the rare sugars were greatly 1934. inferior to glucose as substrates for these enzymes. 11. LOWRY, O. H.; ROSEBROUGII, N. J.; FAIR, A. L.; and RANDALL, R. J. Protein Measurement with the Folin Because glucose has such a high affinity for the en- Phenol Reagent. J. Biol. Chem., 193: 265-75, 1951. zyme and because low enzyme concentrations were 19. MCCOMB, R. B., and YUSI-IOK,W. D. Properties of Partic- used in the inhibition studies, the inhibitory effect ulate Hexokinase of the Krebs-2 Ascites Tumor. Biochim. of the rare sugars may have been decreased be- et Biophys. Acta, 34:515-26, 1959. yond the point of detection by the methods used. 1S. MEYERROF, D., and WILSON, J. Studies on the Enzymatic System of Tumor Glycolysis. I. Glycolysis of Free Sugar The fact that altrose and talose, which were in- in Homogenates and Extracts of Transplanted Rat Sar- ert as substrates, could nevertheless exert an in- coma. Arch. Biochem., 21:1-34, 1949. hibitory effect, suggests that these substances 14. NIRENBERG, M. W., and HOGG, J. F. Hexose Transport could be bound to the active site of the enzyme, in Ascites Tumor Cells. J. Am. Chem. Soc., 80:4407-12, 1958. but activation and phosphorylation could not oc- 15. OCHOA, S. Glycolysis and Phosphorylation in Brain Ex- cur. tracts. J. Biol. Chem., 141:245-51, 1941. The results obtained in both the substrate spec- 16. PIGMAN, W. The Carbohydrates. Chemistry, Biochemistry ificity and inhibition studies clearly indicate that and Physiology, p. 49. New York: Academic Press, Inc., there are significant differences in the enzyme 1957. 17. SCHWARTZ, M., and MYERS, T. C. A Simple Microtitri- systems investigated, but these differences would metric Constant pH Method for Accurate Enzyme Assays. not be readily apparent if only glucose were stud- Anal. Chem., 30:1150-51, 1958. ied as a substrate. 18. SOLS, A., and CRANE, R. K. Substrate Specificity of Brain Hexokinase. g. Biol. Chem., 210:581-95, 1954. 19. SOLB, A.; DE LA FUENTE, G.; VILLAR-PALASI, C.; and ACKNOWLEDGMENTS ASENSIO, C. Substrate Specificity and Some Other Prop- The authors are indebted to Mr. Bobby H. Biggers for his erties of Bakers Yeast Hexokinase. Biochim. et Biophys. assistance during the course of this work. Acta, 30:92-101, 1958. 20 SWANSON, M A. Phosphatases of Liver I. Glueose-6- Phosphatase. J. Biol. Chem., 184:647-59, 1950. REFERENCES 21. UMBREIT, W. W.; BURRIS, R. B.; and STAUFFER, J. F. 1. BOYLAND, E.; Goss, G. C. L.; and WILLIAMS-ASHMAN, Manometric Techniques and Tissue Metabolism, p. 149. H. G. Hexokinase Activity of Animal Tumors. Biochem. 3d ed. Minneapolis: Burgess Publishing Co., 1957. J., 49:321-25, 1951. 22. VESTIX[NG, C. S.; MYLROIE, A. K.; IRISH, U.; and GRANT, 2. EEG-LARSEN, N., and LALAND, S. G. The Effect of D- N. H. Rat Liver Fructokinase. J. Biol. Chem., 185:789- Glucose on Brain Hexokinase and Liver Glucose Dehy- 801, 1950. drogenase. Acta Physiol. Scandinav., 30: 295-302, 1954. 28. WOODWARD, G. E.; CRAMER, F. B.; and HUDSON, M. T. 3. HAWK, P. B. ; OSER, B. L.; and SUMMERSON, W. H. Prac- Carbohydrate Analogs as Antagonists of Glucose in Car- tical Physiological Chemistry, p. 602. 13th ed. New York: bohydrate Metabolism of Yeast. J. Franklin Inst., 256: Blakiston Co., Inc., 1954, 577-87, 1953. 4. HERS, H. G. La Fructokinase du foie. Biochim. et Biophys. 24. WOODWARD, G. E., and HVDSON, M. T. The Effect of 2- Acta, 8:416-23, 1952. Deoxy-D-Glucose on Glycolysis and Respiration of Tumor 5. HUDSON, M. T., and WOODWARD, G. E. Glucosone II. In- and Normal Tissues. Cancer Research, 14: 599-605, 1954.

Charles F. Lange, Jr. and Paul Kohn

Cancer Res 1961;21:1055-1061.

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