Chromatographic fractionation of multiple forms of red blood cell hexokinase (original) (raw)
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Rat red blood cell hexokinase purification, properties and age-dependence
Molecular and Cellular Biochemistry, 1986
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/rag 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.
Archives of Biochemistry and Biophysics, 1983
Pig erythrocytes, in contrast to red blood cells from other mammals (M. Biochewa. ht. 4,6'73), have been shown to contain hexokinase (EC 2.7.1.1) types II and III. Hexokinase type III is the predominant form, accounts for 98% of the total glucose phosphorylating activity, and has been purified 290,000-fold by a combination of ion-exchange chromatography and affinity chromatography on Sepharose-N-hexanoylglucosamine. The enzyme was shown to be homogeneous by polyacrylamide and sodium dodecyl sulfate-gel electrophoresis. The highest specific activity obtained was 190 units/mg protein with a yield of 60%. Because the amount of hexokinase II was small, it was only partially purified by ion-exchange chromatography.
Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase
The Journal of experimental zoology, 1981
The mature erythrocyte of the pig has been observed to possess the slowest metabolic rate of any mammalian cell type. Previous studies in this laboratory suggested that the hexokinase isolated from these cells was inhibited by glucose in concentrations in excess of 0.2 mM. In the present study, the enzyme was isolated by utilizing DEAE-Sephadex A-50, ammonium sulfate precipitation, DEAE-cellulose (DE-52), and Sephadex G-100 gel-filtration. Studies on the hexokinase isolated from the pig mature erythrocyte by the above procedures revealed two distinct isozymes of hexokinase that do not behave kinetically and electrophoretically as those previously found in other mammalian red blood cells. The isozyme isolated from the erythrocyte of the young adult pig (less than six months of age) migrated at a slower electrophoretic rate than the one isolated from the adult pig (more than six months of age). Coupled with the observed difference in electrophoretic mobilities were changes in the appa...
Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1984
The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23 + 1.90 ~mol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg • ATP 2-. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 ltM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP 4-and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94 + 0.02 l~mol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 + 0.3 ttmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.
Molecular forms of red blood cell hexokinase
Molecular and Cellular Biochemistry, 1982
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 maj or forms (Ia, 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.
Molecular and Cellular Biochemistry, 1988
Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ionexchange 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.
Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role
Archives of Biochemistry and Biophysics, 1983
The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n u = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mM). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration.
Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation
Journal of Biological …, 1986
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 hexokinase activity and the degradation of 1261-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. 1251-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 1251-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 ATPand ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system o f reticulocytes.
Hexokinase type I multiplicity in human erythrocytes
Biochemical Journal, 1988
Hexokinase I in human erythrocytes exists in multiple molecular forms that differ in isoelectric points. By means of Western blotting and immunodetection of total glucose-phosphorylating activity by using an antibody raised in rabbit against homogeneous human placenta hexokinase I, a single protein band was detected. Identical results were also obtained by immunoaffinity chromatography of the partially purified enzyme. Separation of the three major hexokinase I subtypes (Ta, Tb and Ic) by h.p.l.c. ion-exchange chromatography and immunodetection following electrophoretic blotting confirmed that each hexokinase subtype showed the same apparent Mr of 112000, which is the value obtained for the high-M, hexokinase I from human placenta. Purification of erythrocyte hexokinase by a combination of several procedures including dye-ligand and affinity chromatography that were previously successfully applied to the purification of other mammalian hexokinases type I produced a 35000-fold-purified enzyme that showed several contaminants after SDS/polyacrylamide-gel electrophoresis. Only one of these peptides was found to be recognized by anti-(hexokinase I) IgG, suggesting that proteolytic degradation does not occur and that hexokinases Ia, Tb and Ic have the same apparent Mr.