Kinetic, electrophoretic, and chromatographic studies on glucose-ATP phosphotransferases in rat hepatomas.

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 di ethylaminoethyl (DEAE)-cellulose, for isoenzymes of glucose ATP phosphotransferase activity. Well and highly differenti ated hepatomas were similar to liver in having, by kinetic assay, low hexokinase and low-to-moderate glucokinase activi ty, whereas the poorly differentiated tumors had high hexo kinase but no detectable glucokinase activity. Starch gel elec trophoresis revealed similar patterns of the four phosphotrans ferase 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 methyicholan threneand dimethylbenzanthracene-induced primary mam mary tumors, and three early generation transplantable kidney tumors all had the three hexokinase isozymes. lit 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 KC1 concentration gradient gave elution patterns similar to that of liver, with peaks corresponding to the isozymes detect able by electrophoresis. Recent biochemical investigations of the Morris hepatomas (22) havemadeit clear that the neoplastictransformationof the liver cell may occur without complete disruption of cell function, and have created renewed interest in the initial en zymatic alterations associated with neoplasia. As part of an investigation of the loss or retention of enzyme activities as sociated with normal tissue function that occur with liver neo plasia (43), we have studied alterations in rat liver ATP-glucose phosphotransferase activities in liver and liver tumors (34, 35). Normal liver of the rat and other species contains two distinct types of glucose-ATP phosphotransferase. One of these (EC.2.7.1.2), termed glucokinase, is relatively specific for glu cose, is dependent for its activity on dietary glucose in normal animals, requires insulin for full activity, and has a high Km for glucose, thus making it highly responsive to ambient glu cose concentrations (3, 11, 32, 34, 39, 40). This enzyme is considered to play an important regulatory role in hepatic giucose utilization. The other, relatively less specific type (EC.2.7.1.1) is similar to other nonhepatic hexokinases and is now known to consist of three isozymes separable from each other and from glucokinase by starch gel electrophoresis (13, 14, 18, 21) and by chromatography on DEAE-cellulose (11, 13, 18).@@4 These are generally present in low activity, which is not affected by diet or insulin; they have low K@ucose and appear to be electrophoretically identical with hexokinases found in nonhepatic tissues. In previous work on the Morris hepatomas (43) and various chemically induced, primary rat hepatomas (34, 35), we explored some of the relationships between the proportions of hexokinase and glucokinase iso zymes and the degree of tumor differentiation. However, limi tations of the kinetic procedure used in these studies left un