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Experimentally induced diabetic rats were treated separately with insulin and vanadate. The activities of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6PDH) were increased in reticulocyte hemolysate isolated from the diabetic rats and were restored to normal levels by insulin. The restoration was not detected in vanadate treated diabetic animals. The enzymes of glutathione metabolism namely glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) exhibited increases in their activities with diabetes and were restored to almost control values by insulin treatment. Vanadate given to diabetic animals further increased GPx, and GST. The level of superoxide dismutase(SOD) decreased in the reticulocytes of diabetic rats and catalase (CAT) was unchanged. Both CAT and SOD had normal values when the diabetic rats were treated with insulin and vanadate. It is proposed that vanadate may cause an increase in the activity of GR which may stimulate glucose transporters and glucose metabolism.

Rabbit red blood cells (RBC) were exposed in vitro to an oxygen-radical-generating system represented by iron and ascorbic acid. Under these experimental conditions we have investigated the effect of this system on some intracellular rabbit reticulocyte and erythrocyte enzymes. The results obtained have shown a pronounced decay of hexokinase activity both in the erythrocytes and reticulocytes when exposed to these radical species. We have found that the amount of hexokinase inactivated is at least three times higher in a blood sample with a percentage of reticulocytes of 50-60%. This different behaviour of the hexokinase decay in the erythrocytes and reticulocytes could be due to its different intracellular distribution related to the two distinct cells. In addition we have evaluated some important intracellular compounds involved in maintaining the redox and the energetic state of the cell such as the reduced glutathione and the adenine nucleotides and their degradation products, in order to understand if there is any correlation between the hexokinase decay and a change concerning the metabolic conditions of the rabbit reticulocytes and erythrocytes exposed to free radicals.

Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in the soluble fraction of reticulocytes as two distinct molecular forms, designated hexokinase Ia and hexokinase Ib, which are separable by ion exchange chromatography and polyacrylamide gel electrophoresis. Hexokinase Ia was found to be similar to the brain enzyme, while hexokinase Ib differs from every other previously reported hexokinase isozyme. Reticulocyte hexokinase Ia and Ib have been purified 55,000-and 50,000-fold, respectively, by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis, as proteins homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native proteins have the same molecular weight of 105,000 by gel filtration and sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels have a molecular weight of 104,000, indicating that the two forms are monomers. Hexokinase Ia had a pI of 6.2 to 6.3 pH units while hexokinase Ib had a pI of 5.7 to 5.8 pH units by isoelectric focusing. The two enzymes were specific for Mg.ATP and Mg.ITP as the nucleotide substrates. Several hexoses could be phosphorylated by hexokinase Ia and Ib with different affinities.

Mitochondria from rabbit reticulocytes contain about 50% of the total reticulocyte hexokinases. The proportion of mitochondrial hexokinases may be changed under different metabolic conditions. Mitochondrial bound and soluble hexokinases exhibit different kinetic properties (KMATP and glucose-6-phosphate inhibition). The respiratory rate of isolated reticulocyte mitochondria in the presence of glucose depends on the glucose-6-phosphate concentration, as the ADP generation by the endogenous hexokinases is strongly inhibited by glucose-6-phosphate. In the experimental system all intermediary states of mitochondrial respiration can be adjusted between the state of maximal activity (state 3 or active state) and the controlled or resting state (state 4) by different glucose-6-phosphate levels. The stationary levels of the extramitochondrial adenine nucleotides in this experimental system have been measured. The rate of mitochondrial respiration and ATP formation depends on the extramitochondrial ATP/ADP ratio. At ratios of about 10 and lower the mitochondria are in their maximum phosphorylation state, at higher ratios the mitochondrial ATP formation is controlled by the extramitochondrial ATP/ADP ratio. It is postulated that the close intercounnection between the mitochondrial hexokinase and the mitochondrial ATP forming system in reticulocytes is of funcitonal significance for mitochondrial-cytosolic interactions in rabbit reticulocytes and probably in other types of cells with mitochondrial hexokinases, too.

Respiration and phosphorylation in functionally intact isolated reticulocyte mitochondria are controlled by the extramitochondrial ATP/ADP ratio. Mitochondrially bound hexokinase exhibits an apparent preference against mitochondrially formed ATP, as indicated by the lower apparent Km values under phosphorylating conditions. Thereby the control characteristics are shifted to higher ATP/ADP ratios compared to hexokinase free mitochondria. Respiration of intact reticulocytes was estimated to be smaller than had to be expected from the ATP/ADP ratios of total cells roughly corresponding to the cytosolic values. Inhibition of respiration leads to small drop of total adenine nucleotides and lowers the phosphorylation potential directly determined or calculated from metabolite indicators. Compartmentation of ADP by protein binding is discussed as a possible reason for the difference of determined and calculated phosphorylation potentials and the apparent discrepancy between the regulation of mitochondrial ATP-formation in isolated mitochondria and intact reticulocytes.

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 Ia 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 microheterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.

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.

The rate at which the activities of red cell enzymes decay during maturation of the reticulocyte and ageing of the erythrocyte is poorly understood. It has recently been suggested that loss of enzyme activity may be rapid during reticulocyte maturation and occur slowly thereafter. We have now devised a rabbit model in which reticulocytosis is combined with transfusional polycythaemia. This makes it possible to follow the enzymatic activity of a cohort of reticulocytes without the interfering effect of newly formed cells. In this model the reticulocyte count of the experimental animals falls from about 60% to subnormal levels within 4 d. Red cell hexokinase activity declined rapidly with more than half of the activity being lost within 7 d, little change occurring thereafter. Glucose-6-phosphate dehydrogenase activity declined more gradually, but also followed a biphasic course. The fall of pyrimidine 5' nucleotidase activity was the slowest of the three age-dependent enzymes studied. This is consistent with our observations in children with transient erythroblastopenia of childhood, in which this enzyme, alone of the age-dependent enzymes, was found to be decreased in a senescent red cell population. In contrast to the three age-dependent enzymes studied, the activity of lactate dehydrogenase remained unchanged during the course of the experiment. These studies provide direct verification of the suggestion that the decline of red cell enzyme activities may be biphasic. They show, moreover, that enzyme decay is not only rapid in reticulocytes, but also in young erythrocytes.

The isoenzyme pattern of hexokinase in rabbit red cells (erythrocytes, fetal erythrocytes and reticulocytes) were determined by means of agarose gel and disc electrophoresis. One duplicated hexokinase (4a and 4b according to the IUPAC-nomenclature) was detected in rabbit erythrocytes as also described for human erythrocytes. Besides the isoenzymes 4a and 4b reticulocytes also contain hexokinase 2 and 3 like rabbit and rat liver. The high KM glucose phosphorylating enzyme, hexokinase 1 could be demonstrated only under specific conditions in the reticulocytes during the initial stage of the anemia. After the fractionation of reticulocyte homogenates the total hexokinase activity was recovered in the mitochondria and cytosol to nearly equal amounts as revealed by the distribution of markers. Hexokinase 2 and 3 were detectable in reticulocytes and in isolated mitochondria only after the addition of certain dissociating agents. In contrast to the tightly bound mitochondrial hexokinases 2 and 3 the type 4a and 4b are more loosely bound and exhibit a bilocal distribution between mitochondria and cytosol of reticulocytes.

Proteolysis of hexokinase in cell-free systems prepared from rabbit reticulocytes has been shown previously to be ATP-dependent and apparently mediated by the ubiquitin system (Magnani et al. J. Biol.Chem.261, 8327-8333). We have investigated this phenomenon, but found no substantial loss of hexokinase in cell-free systems prepared from fresh lysates. Storage of lysates at -20 degrees C or addition of a free radical generating system was required to demonstrate rapid ATP-dependent decay. It appears that initial oxidative damage to hexokinase does not abolish its activity but allows it to be recognized by an ATP-dependent proteolytic system. The relevance of this mechanism to in vivo degradation of hexokinase is discussed.

Rabbit red blood cells contain hexokinase type I whereas in the reticulocyte two distinct molecular forms (HK Ia and Ib) are present. One (HK Ia) corresponds to hexokinase type I from other tissues, while the other differs from any previously reported isozyme. Rabbit bone marrow cells contain hexokinase type I and II. However, when the erythroid precursor cells become predominant over the non-erythroid cells (during phenylhydrazine anemia) a great increase of HK Ia can be observed concomitant with the appearance of HK Ib. Fractionation of the bone marrow cells on density gradients provides evidence that basophil erythroblasts and proerythroblasts contain only HK Ia while HK Ib appears at the reticulocyte stage. Maturation and ageing of circulating reticulocytes are associated with the decrease of hexokinase activity. Since the decay rate of HK Ib is about three times higher than the decay rate of HK Ia, the mature erythrocytes do not contain appreciable amounts of HK Ib. Furthermore, in vitro, HK Ia and Ib possess similar stabilities so that a cellular mechanism must be responsible of their in vivo different decay rates. This mechanism, as reported in this paper, is ATP-dependent, could be found in the soluble fraction, and is active only at the reticulocyte stage. These properties are similar to those of the ATP-dependent proteolytic system. Pure ubiquitin, an essential polypeptide of the ATP-dependent proteolytic system, is also able to catalyze the decay of hexokinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 Ia 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 Ia 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 Ia and Ib was shown to be responsible for the differences found between their decay rates.

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 submitochondrial distribution of hexokinase was studied by repeated specific solubilizations and by tryptic digestion of isolated rabbit reticulocyte mitochondria. Whereas most of the enzyme is dissociably bound to the outer side of outer mitochondrial membrane, a small tightly bound portion is localized more internally. Electrophoretic separations did not reveal a specific isoenzyme pattern of the internal mitochondrial enzyme. Relationships between mitochondrial hexokinases and the intramitochondrial ATP pool, generated by oxidative phosphorylation, were studied by measuring 32P-fluxes following gamma-32P-ATP pulses on phosphorylating and non-phosphorylating mitochondria. Under both conditions, the specific activities in deoxyglucose-6-phosphate correspond closely to that of total gamma-ATP, thus not supporting a preferential use of intramitochondrial generated ATP by part of the mitochondrial hexokinases.

Erythrocytes from adults and newborn infants (at term and premature) were separated by Percoll density gradient centrifugation into four fractions of increasing density. Glycolytic enzymes, especially the age-dependent ones, hexokinase (EC 2.7.1.1, HK), pyruvate kinase (EC 2.7.1.40, PK), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) were studied during reticulocyte maturation and further red cell senescence. Analysis of the fraction with lowest density showed an almost linear and steep decline of HK, PK, and G6PD activity with a decreasing number of reticulocytes. In the next three fractions of increasing density, the activity decline was far less. These data are therefore illustrative for a biphasic activity decay pattern of HK, PK, and G6PD during both adult and neonatal red cell aging. The strong decline in HK activity could not be ascribed to the disappearance of a particulate (mitochondrial) bound fraction of the enzyme during reticulocyte maturation. All hexokinase activity in human reticulocytes was found to be cytosolic in contrast with rabbit reticulocytes in which 70% of HK activity was particulate.

In this paper we have used a new method which allows the simultaneous extraction and HPLC determination of ATP, ADP, AMP, NADP, NADPH, NAD and NADH to evaluate the changes in concentration of these compounds during maturation of rabbit reticulocytes and cell aging. The results show a significant increase of ATP concentration, higher ATP/ADP, ATP/AMP ratios and lower NADP+/NADPH, NAD+/NADH ratios in rabbit reticulocytes when compared to mature cells. Similar results were also obtained when the whole red blood cell population was separated into fractions of increasing mean age on discontinuous gradients of Percoll-BSA. These metabolic modifications are discussed in relation to the age-dependent metabolic decline of the erythrocyte.

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.

In rabbit erythrocytes hexokinase (EC 2.7.1.1) specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by an in vitro made policlonal antibody shows that this maturation dependent hexokinase decay is not due to the accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system made from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinase activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. 125I-hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mecaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugate to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP and ubiquitin dependent and involves both the hexokinase molecular forms (hexokinase Ia and Ib) present in reticulocytes. "In vivo", hexokinase Ia is mitochondrial bound while hexokinase Ib is soluble. The energy dependent degradation system of reticulocytes is active only on the soluble enzyme, namely hexokinase Ib. As the cell mature mitochondria are degradated, hexokinase Ia becomes soluble but there is a concomitant decay also of the proteolytic system resulting in a mature erythrocyte that contains only hexokinase Ia in a soluble form.