Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase (original) (raw)
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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.
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.
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.
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.
Chromatographic fractionation of multiple forms of red blood cell hexokinase
Journal of Chromatography A, 1982
It is well established that four hexokinase (E-C. 2-7.1-l) isozymes (named I, II, III and IV in order of increasing electrophoretic mobility towards the anode) are present in mammalian tissues 1*2 Conflicting results have been reported for the hexo-_ kinase pattern in the erythrocytes~'5. The discrepancies are mainly due to the presence of sub-types or multipIe forms of hexokinase I and to the dilTiculties related to their separation. Electrophoresis on starch gel, agarose gel, cellulose acetate membrane, polyacrylamide ge1, and isoeIectric focusing or ion-exchange chromatography have been employed as separation techniques of the red blood cells isozymic pattern IJnfortunately starch gel, a,oarse gel and cellulose acetate have been found unsuitable as media for hexokinase isozymes separation owing to the lack of resolution_ Polyacrylamide disc gel electrophoresis or electrofocusing, on the other hand, inactivate some isozymes.
Improved metabolic properties of hexokinase-overloaded human erythrocytes
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1988
Human erythrocytes were loaded with homogeneous hexokinase purified from lamm i~aeenta (an enzyme species apparently idenlical to the erythrocyte enzyme), using a precedmm of eneaps.~la~on based on hYlmtonk hemolysis, isotonic resealing and reannealing. The hexol~,~-~.overllo~led er~lmm'oeyt~ eontalned 4.77 :k. 0.75 IU ol hexokinase activity per ~ of lmcked erythroeytes, a value 15-times higher than that of enrm~onding mlonded or native red cells. The bexoklnase-loaded erytbrocytes were fmmd to metabolize twice the memt oi glucose consumed by the unloaded ce!Is through a nearly doubled glyeo~jtie activity, while the aetivit~ of the hexese ~te slmat pathway was ummdifl~ Estimates el glyeelytie intermedhtes showed increased levels of most metabolites with respect to the tmi(mded erytbrocytes, while the inUacelhdar concentrations el adenine nudeotldes and Z,3-blsphespbo~cerate were unaffected by entnqmaent el hexokinase. The new steady-state condition characterized by imla~ed glycolyti¢ flaicti~ was denmmtmted to be ~]reetly related to enhanced levels of bexokinase activity and not to the use el a rejuvenation solution dining the procedure of entrapment. These results are consistent with saggestiom by several investigators that glucose metabolism in human eqOm)eytes is regulated by hexokimse, and they open new perspeeflves for manipulating erythrocytes with the ultimate aim el imlxoving tlmir survival under different storage comations.
Hexokinase in developing rabbit erythroid cells
Biochimica et Biophysica Acta (BBA) - General Subjects, 1984
The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits separated 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. Most of the falls in hexokinase activity take place when 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.
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.