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Kinetic, Electrophoretic, and Chromatographic Studies on Glucose-ATP Phosphotransferases in Rat Hepatomas¹

The Fels Research Institute and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and the Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20014

Supernatant fractions obtained by high-speed centrifugation of homogenates of liver and chemically induced primary and transplantable hepatomas were assayed kinetically, and by starch gel electrophoresis and column chromatography on diethylaminoethyl (DEAE)-cellulose, for isoenzymes of glucose-ATP phosphotransferase activity. Well and highly differentiated hepatomas were similar to liver in having, by kinetic assay, low hexokinase and low-to-moderate glucokinase activity, whereas the poorly differentiated tumors had high hexokinase but no detectable glucokinase activity. Starch gel electrophoresis revealed similar patterns of the four phosphotransferase isozymes in liver and well-differentiated tumors. All three hexokinase isozymes were present, and glucokinase was also invariably present, although the intensity of its band varied greatly. When glucokinase activity was high, it generally appeared as a double band, whereas when low in activity, only the "slower" band appeared. Poorly differentiated hepatomas revealed strong bands of hexokinase Isozymes I, II, and III, but also displayed an extremely faint though unmistakable Isozyme IV band. Fetal liver also displayed a faint Isozyme IV band, in addition to those of the three hexokinase isozymes. Normal mammary gland and kidney, primary methylcholanthrene- and dimethylbenzanthracene-induced primary mammary tumors, and three early generation transplantable kidney tumors all had the three hexokinase isozymes. In addition, they contained traces of Isozyme IV, undetectable by kinetic assay, but unmistakably evident by starch gel electrophoresis. Chromatography of tumor extracts on DEAE-cellulose with a KCl concentration gradient gave elution patterns similar to that of liver, with peaks corresponding to the isozymes detectable by electrophoresis.

¹ This study was supported by Grants CA-07174, CA-10729 and AM-05487, from the NIH, and P-202 from the American Cancer Society.

² Present address: Howard University School of Medicine, Department of Biochemistry, Washington, D. C.

Received 9/12/68. Accepted 1/16/69.