The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: l>hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70-80% of total activity), the remaining 20-30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from iymphocytes shows a slightly decreased K m apparent for glucose (P < 0.05).

No abstract

Abstract-The activity, isoenzyme distribution and compartmentation of hexokinase (ADP: D-hexose-6_phosphotransferase, EC 2.7.1.1) were compared in slowly growing, well-differentiated medullary thyroid carcinoma (DMTC) and rapidly proliferating anaplastic thyroid carcinoma (AMTC) in the rat. Individual isoenzymes from either soluble or particulate fractions after solubilization were obtained by fast protein liquid chromatography and were kinetically analyzed either in soluble form or after (re)binding to rat liver mitochondria. These studies were undertaken to test the hypothesis that the growth rate of tumors is correlated with the activity of mitochondrial-bound hexokinase in our tumor system. In contradiction to this hypothesis, wefound no difference in either enzyme activity or compartmentation of both kinds of tumors. The major part of enzyme activity was soluble (73 and 78% in DMTC and AMTC respectively). In addition, no major differences were observed in the kinetic properties of the individual isoenzymes of both tumors. Only soluble type II hexokinase from AMTC had a slightly decreased apparent K, for glucose. There appeared to be some differences in isoenzyme composition: both tumors contained type I and type II hexokinase in the solubleas well as in the particulate fractions. However, the proportion was shifted in favor of type II hexokinase in the soluble fraction of AMTC. Additional findings of this study were the following: the affinity of type II hexokinase to both substrates glucose and MgATP'-was significantly less compared to type I hexokinase. However, the inhibition constant for glucose-1,6-diphosphate of both isoenzymes was exactly the same. The bound form of both isoenzymes had the same substrate affinities as the soluble form but was considerably less inhibited by glucose-l,6 diphosphate. In the latter respect, type I and type II hexokinase behaved in the same way.

SummaryIn order to study the three regulator enzymes of glycolysis, hexokinase (HK). phosphofructokinase (PFK) and pyruvate kinase (PK), in relation to lymphocyte nlaturatjon, lymphocytes of different origin were investigated. Lymphocytes from bone marrow, thymus, cord blood, adult peripheral blood and mitogen-stimulated lymphocytes were investigated. The enzyme activities were determined and the isozyme patterns were studied by means of electrophoresis, kinetic measurements and immunoprecipitation.The young lymphocytes from bone marrow and the mitogen-stimulated lymphocytes could be distinguished from the other lymphocytes by a higher residual HK activity in the presence of the inhibitor glucose-1,Gdiphosphate. Peripheral blood T lymphocytes differed from non-T lymphocytes in the PK isozymes distribution.All the cells contained PK type K, and the hybrid K,M. In T cells a smaller amount of the K isozyme was seen than in non-T cells. The PK residual activity in the presence of alanine was significantly higher in peripheral blood T cells than in non-T cells.Thymocytes are characterised by a larger amount of PFK M-subunits than peripheral blood T and non-T lymphocytes.The stimulation of PFK by the positive effector glucose-1,Gdiphosphate was higher in thymocytes than in the peripheral blood lymphocytes.

SummaryThe enzyme activities and isozyme distribution of the three glycolytic regulator enzymes hexokinase, phosphofructokinase and pyruvate kinase were studied in lymphocytes of patients with chronic lymphocytic leukemia. Isozyme distribution patterns were determined by kinetic measurements, electrophoresis and immunoprecipitation.The CLL lymphocytes were different from normal non-T lymphocytes with respect to hexokinase residual activity in the presence of glucose-l ,6-P,, pyruvate kinase residual activity in the presence of alanine, and phosphofructokinase activity after stimulation by glucose-1,6-P,.No differences could be discerned in enzyme activities between the CLL and the normal T and non-T lymphocytes.