Multiple forms of hexokinase. Activities associated with subcellular particulate and soluble fractions of normal and streptozotocin diabetic rat tissues.

The multiple molecular forms of hexokinase associated with the soluble and several subcellular particulate fractions of various tissues of normal and streptozotocin diabetic rats were studied. Hexokinase activities extracted from the particulate fractions, and those in the soluble fractions, were determined according to spectrophotometric assays and staining intensities of the hexokinase types separated by DEAEcellulose chromatography and gel electrophoresis. According to these criteria, the amounts of type Il hexokmase associated with the 13,000 x g “Pg)’ (mitochondrial-rich), 105,000 x g “P;’ and “Pd” (sarcoplasmic vesicle-rich), and 105,000 x g supernatant “&” (soluble) fractions of heart, diaphragm, and gastrocnemius muscles from diabetic rats were deficient when compared with those from normal rats. However, type II increased in the soluble fraction of the diabetic small intestine. While diabetes had no discernible effect on type I hexohmase content in any of the fractions of these tissues, brain, or kidney, it resulted in an increase in type III hexokmase in the soluble fraction of cardiac and skeletal muscles. The loss of type II activity in diabetic muscle extracts appeared to be due to a loss in amount of enzyme rather than to inhibition by glucose-6-P or other metabolites. Within 2 hours after insulin administration to diabetic rats net hexokinase activity in the soluble fraction of gastrocnemius muscle was restored to normal values, while in this fraction from cardiac muscle more than 48 hours were required for complete restoration. The only forms of hexokinase associated with all of the particulate fractions of all tissues studied were found to be indistinguishable from the hexokinase types I and II that are also present in the soluble fractions. Particle-bound type III hexokinase could not be shown. Latency of hexokinase activity, previously discovered in brain mitochondria in other laboratories, is found to be a general phenomenon characteristic of other particulate fractions of brain as well as skeletal and heart muscles. Such latency is attributable to only type I hexokinase and can be rendered assayable as the “overtbound” activity or the solubilized activity by treatment of the particulate fractions with various membrane-disruptive agents. Examination of the hexokinase patterns associated with the P2, PL Pq, and soluble fractions of skeletal and heart muscles revealed that, with each tissue, the proportion of type I to II hexokinases varied from fraction to fraction. It is suggested that the observed differences in the association of the three hexokinases with different subcellular fractions, as well as their different responses to lack of insulin, may reflect some type of compartmentalization of the molecular forms of hexokinase in the cell.