Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role (original) (raw)
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Comparative studies of glucose metabolism on mammals' red blood cells
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1980
1. Glucose utilization was measured in human, pig, cow, rabbit, mouse and rat red blood cells. Mean values are variable from species to species and range from 0.27 #mol/hr/ml RBC for pig erythrocytes to 2.85 #mol/hr/ml RBC in mouse red cells.
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...
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.
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.
Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.