Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

Molecular forms of red blood cell hexokinase

Summary Mammalian red blood cell hexokinase has been shown to exist in two or more distinct molecular forms, which are separable by ion-exchange chromatography. Of these forms just one corresponds to hexokinase type I from other tissues, while the others differ from any previously reported hexokinase isozyme. Analysis of several molecular properties of the three major forms (la, Ib and Ic in the order of their elution from DE-52 columns) of hexokinase prepared from human red cells and of the two forms purified from rabbit reticulocytes, shows significant differences in the isoelectric point. The kinetic and regulatory characteristics, the molecular weight, the temperature and pH-dependence of the various isozymes were similar.The hexokinase isozymic pattern is largely dependent upon red blood cell age. Among all, hexokinase Ib is the predominant form in rabbit reticulocytes and becomes the minor component in the older cells; a similar situation has also been found in the human erythrocyte. At present the molecular basis of hexokinase heterogeneity remains unknown, however preliminary experimental findings indicate a post-translational modification as a possible mechanism.     

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase la can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during ‘in vivo’ reticulocytes maturation. This new hexokinase micro-heterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.     

Effects of Ca2+ and lipoxygenase inhibitors on hexokinase degradation in rabbit reticulocytes

In rabbit reticulocytes more than half of the total hexokinase activity is mitochondrial bound and shows a fast decay during reticulocyte maturation. During in vitro incubation of rabbit reticulocytes, Ca²⁺ increases the decay of hexokinase while salicylhydroxamate (SHAM), an inhibitor of lipoxygenase, reduces the decay. Swelling of mitochondria, by incubation of the cells in hypotonic solutions, greatly enhances hexokinase decay, but both the Ca²⁺ and SHAM are still appreciable suggesting that Ca²⁺ and the swelling act by additive mechanisms, both able to influence hexokinase decay. This was confirmed by incubation of rabbit brain mitochondria in hypotonic solutions which does not promote any hexokinase decay, while the presence of Ca²⁺ does. Analyses of hexokinase isozymic pattern after incubation of reticulocytes in hypotonic solution both with and without Ca²⁺ and SHAM showed that the decay of hexokinase mainly involves the mitochrondrial bound isozymic forms.Abbreviations SHAM Salicylhydroxamate - HPLC High-Performance Liquid Chromatography     

Rabbit red blood cell hexokinase

Summary Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in reticulocytes as two distinct molecular forms, designated hexokinase Ia and Ib, but only one of these was consistently present in mature red cells. In vivo, hexokinase la and Ib show a decay rate of 3 and 8% a day, respectively, while in vitro they show a similar stability.The possibility that the proteolytic activities of the reticulocyte could be responsible for the fast decay of hexokinase was investigated. No differences were found in the decay rates of hexokinase la and Ib during in vitro reticulocyte maturation in presence or absence of proteolytic inhibitors. Contrariwise, many findings indicate the ATP-dependent proteolytic system of the reticulocyte as a possible mechanism. In fact, the decay of hexokinase and the degradation of 3H-globins are both stimulated by ATP and ubiquitin; they show similar kinetic properties and both disappear during reticulocyte maturation.The cellular localization of hexokinase la and Ib was shown to be responsible for the differences found between their decay rates.Abbreviations PMSF phenylmethylsulfonyl fluoride - TPCK 1-1-tosylamide-2-phenylethyl-chloromethyl ketone - TLCK N -p-tosyl-L-lysine chloromethyl ketone     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

The Genetics of Hexokinase in a Mosquito, CULEX PIPIENS

The genetics of hexokinase (HK) variants in a mosquito, Culex pipiens L., was studied using starch gel electrophoresis. Three isozymic forms of HK, all migrating anodally, were present in all three body regions, but in differing proportions. No obvious differences in specificity for three hexose sugars was detected among the three isozymic bands. However, qualitative differences in staining intensity indicate the following order of substrate affinity: glucose greater than fructose greater than mannose. The inheritance of the HK variants is controlled by a pair of co-dominant alleles at a single genetic locus.     

A study of hexokinase isoenzymes in rat sarcoma M-1

Using a wide spectrum of criteria, the isozyme composition of hexokinase from sarcoma M-1 reinoculated to rat m. gastrocnemius was studied. The structural, physicochemical and functional properties of the homogeneous enzyme which is represented in sarcoma M-1 by one molecular form, were investigated. Some properties of the enzyme (amino acid composition, resistance to mild proteolysis, Mr, pH-dependence of enzyme activity, electrophoretic mobility, kinetic behaviour) indicate that sarcoma M-1 hexokinase is a specific form of the enzyme which differs markedly from other known isozymes of mammalian hexokinase. The observed peculiarities of sarcoma M-1 hexokinase are discussed in terms of present-day concepts on the structure of isozymic spectra of enzymes in neoplastic tissues.     

Rat red blood cell hexokinase purification,properties and age-dependence

Summary Rat erythrocytes, in contrast to red blood cells from other mammals, have been shown to contain only one hexokinase isozymic form identified as type I by chromatographic and kinetic properties. Rat reticulocytes contain 3.6-times the hexokinase activity found in mature erythrocytes but exactly the same isozyme. By a combination of ion-exchange chromatography, dye-ligand chromatography and high-pressure liquid chromatography the rat erythrocyte hexokinase was purified more than 84 000-fold to a specific activity of 143 units/mg protein and shown to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native protein showed a molecular weight of 100 000 by gel-filtration and an apparent molecular weight of 98 000 under denaturating conditions in sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was shown to be 6.3 pH units. This data provides evidence of only one form of hexokinase in the erythrocytes of a mammal.     

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin.Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition.In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates.     

Utilization of metabolic energy under saline conditions: changes in properties of ATP dependent enzymes in plant cells grown under saline conditions

The effect of growth in saline medium on the activity of two ATP utilizing enzymes was studied. Hexokinase in carrot (Daucus carota L.) cells grown in suspension culture either in the absence or presence of 150 ml NaCl, and tonoplast H⁺-ATPase in tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells grown in suspension culture either in the absence of presence of 428 mM NaCl. There was no difference in the pH profiles, NaCl sensitivity and kinetic parameters towards glucose of hexokinase activities from carrot cells grown in the presence or the absence of NaCl, but the activity from cells grown in the presence of NaCl was more resistant to inhibition by N-ethylmaleimide and to inactivation by heat. Two separate apparent Km values toward ATP were delineated in the extract from cells grown in presence of NaCl while extracts from cells grown in the absence of NaCl had only one apparent Km value. The tonoplast H⁺-ATPase from NaCl grown tobacco cells showed changed kinetic compared to this activity from cells grown in the absence of NaCl. These data may indicate that growth in NaCl results in the appearance of isozymic activity that enhances the ability of plant cells to utilize metabolic energy more efficiently.     

Distribution of hexokinase isoenzymes depending on a carbon source in Saccharomyces cerevisiae.

DEAE cellulose chromatography and agar gel electrophoresis of glucose-phosphorylating enzymes in Saccharomyces cerevisiae showed the existence of glucokinase and two hexokinase isoenzymes ( designated as hexokinase I and II ). The distribution of hexokinase isoenzymes was dependent on a carbon source in the medium, while that of glucokinase was not dependent. The cells grown on 3 % ethanol as carbon source showed the isoenzyme pattern with predominant hexokinase I and a little hexokinase II. The isoenzyme pattern of the cells grown on 6 % glucose, which was differnt from that of the cells grown on ethanol, showed that hexokinase I and II were minor and major parts respectively. When the cells grown on 3 % ethanol were incubated on the medium containing 6 % glucose, hexokinase I was repressed and hexokinase II inducted. These facts suggest that two hexokinase isoenzymes, but not glucokinase, are adaptive enzyme.     

Studies on Myogenesis in vitro: Changes of Creatine Kinase, Phosphorylase and Phosphofructokinase Isozymes

During myogenesis in vitro , a rat muscle cell subclone has an isozymic type modification of the 3 enzymes studied: creatine kinase, phosphorylase and phosphofructokinase.
When fusion occurs in myoblasts, the resulting myotubes take on a new muscle isozymic pattern which is different from the one found in non-differentiated cells. The isozymic modification is demonstrated by electrophoretic and immunological techniques and permits the following of muscle cell differentiation in vitro: , while the isozymic pattern of the differentiated cells is the same as the adult muscle type, the presumptive myoblasts have either a foetal type for the creatine kinase or another isozyme type. We conclude that the studies in vitro , may give a model for the understanding of the differentiation in vivo.     

Aluminum detoxification mechanism in Pseudomonas fluorescens is dependent on iron

Abstract Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant ( frA ). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans , while the fr A1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the fr A1 mutation. The absence of hexokinase activity in the fr A1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism. This suggests that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.     

Role of hexokinase in the regulation of glucose metabolism in human erythrocytes

Red blood cell glucose metabolism was studied in erythrocytes from a patient with trisomy 10 p which resulted in + 50% hexokinase specific activity, in normal controls and in cases of heterozygous hexokinase deficiency. The results obtained show that the hexokinase activity level is an important factor in the control of the erythrocyte's glycolytic rate while having no appreciable effect on the hexose monophosphate pathway under resting conditions. No clear conclusion could be drawn when an oxidative stress was present.     

Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.     

Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice.

The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.     

Effects of kainate on glucose metabolising enzymes in the brain

Hexokinase and glucose-6-phosphate dehydrogenase activities were studied in brain regions after intraventricular injection of kainic acid. Hexokinase activity was decreased by 10-15% in various regions while glucose-6-phosphate dehydrogenase activity remained unaltered. Soluble hexokinase activity, which remained the smaller fraction of total hexokinase activity, showed slightly more dramatic decreases of 15-35% compared to normal activities in brain regions. This decrease of hexokinase activity in the cytosolic compartment could partly account for the kainate-induced decreases seen in glucose metabolism.     

Altered distribution of hexokinase and glucokinase between parenchymal and nonparenchymal cells of rat liver after methapyrilene intoxication

The cell number as well as the hexokinase and glucokinase activity of liver parenchymal and nonparenchymal cells were studied in methapyrilene treated rats. The number of nonparenchymal cells was doubled after treatment with methapyrilene for two weeks while that of hepatocytes remained constant. The hexokinase activity was increased fourfold in the nonparenchymal cell fraction while it was unchanged in the parenchymal cells. The glucokinase activity was decreased in the hepatocytes to one third. Hence, the increased hexokinase activity was due to a proliferation of nonparenchymal cells rather than to a toxic dedifferentiation of hepatocytes.     

Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture.

Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca²⁺ from the growth medium or when protein synthesis was inhibited by cycloheximide.