Compartmentation of hexokinase in human blood cells. Characterization of soluble and particulate enzymes.

The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP:D-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 lymphocytes 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 lymphocytes shows a slightly decreased Km apparent for glucose (P less than 0.05).

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung.

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.

Isozyme distribution of hexokinase, phosphofructokinase and pyruvate kinase in lymphocytes from patients with chronic lymphocytic leukemia.

The 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-1,6-P2, pyruvate kinase residual activity in the presence of alanine, and phosphofructokinase activity after stimulation by glucose-1,6-P2. No differences could be discerned in enzyme activities between the CLL and the normal T and non-T lymphocytes.

Hexokinase, phosphofructokinase and pyruvate kinase isoenzymes in lymphocyte subpopulations.

In order to study the three regulator enzymes of glycolysis, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), in relation to lymphocyte maturation, 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-1,6-diphosphate. Peripheral blood T lymphocytes differed from non-T lymphocytes in the PK isozymes distribution. All the cells contained PK type K4 and the hybrid K3M. 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-1,6-diphosphate was higher in thymocytes than in the peripheral blood lymphocytes.

Divergence between the occurrence of antibody and cellular immune reactivity to cervical carcinoma cell lines in preinvasive and macroinvasive stages of cervical carcinoma.

A lymphocyte stimulation assay is described which detects immune reactivity to antigens derived from the CaSki cervical carcinoma cell line. Taking a stimulation index of greater than 4.1 as positive, the peripheral blood lymphocytes of 14/20 patients (70%) with untreated dysplasia or carcinoma-in-situ, 8/19 patients (42%) with untreated macroinvasive squamous cell carcinoma of the uterine cervix and 8/38 controls (21%) showed positive reactions. Statistical analysis revealed a significant difference between the group of patients with dysplasia or carcinoma-in-situ and the controls. The sera of patients and controls were simultaneously tested for the presence of tumour-directed antibody. There was no correlation between the occurrence of cellular immune reactivity and of serum antibody, both directed to cervical carcinoma antigens. Cellular immune reactivity tended to occur more frequently in patients with preinvasive stages of cervical carcinoma, and serum antibody in patients with macroinvasive carcinoma.

Hexokinase isoenzymes from anaplastic and differentiated medullary thyroid carcinoma in the rat.

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, we found 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 Km for glucose. There appeared to be some differences in isoenzyme composition: both tumors contained type I and type II hexokinase in the soluble as 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 MgATP2- 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-1,6-diphosphate. In the latter respect, type I and type II hexokinase behaved in the same way.