Decay pattern of rabbit erythrocyte hexokinase in cell aging (original) (raw)

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.

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.

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.

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...

Galactokinase activity and red blood cell age

AGE, 1980

Human erythrocyte galactokinase has been studied during red cell aging. The decay rate of enzyme activity is slower than glucose-6.phosphate dehydrogenase which is used as red cell age marker. The Michaelis constants for galactose and ATPMg 2 -of galactokinase of young cells are similar to the Km's of the enzyme of total cells, while the Km increases in old cells. The behaviour of galactokinase during cell aging is similar to that of other erythrocyte enzymes.

Cell age dependent decay of human erythrocytes glucose-6-phosphate isomerase

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1989

Glucose-6-phnsphate isomerase shows a biphasic decay pattern during red blood cell aging, which is very fast during the first part of cell's life span in circulation. This decay is not due to accumulation of inactive enzyme molecules, as shown by immtmolegieai studies, but is accompanied by the formation of secondary isozymes (i.e., chemically modified forms). Electrophoretic and ion-exchange chromatographic experiments show that giucose-6-phosphate isomerase (D.glucose-6-phnsphate ketul-isomerase, EC 5.3.1.9) consists of only one isnzymic form in young erythrncytes but is present in two components, with different electric charge, in mature and old cells. This secondary Jsozyme is more stable to heat treatmeut and is inactivated by lgG anti-giucose-6-phosphate isomerase with a lower affinity than the native isozyme. In vitro iaeehation of hantogeneons human giueose.6-phosphate isomcrase under conditions known to produce enzyme deamination does not reproduce the isnzymic pattern found in erythrneytes, suggesting that one or more mechanisms other than those previeusly described to explain giucose-6-phosphate Jsomerase mlcroheterogeneity occur in the human erythrocyte.

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.

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.

In vivo aging of red cell enzymes: Study of biotinylated red blood cells in rabbits

American Journal of Hematology, 1990

It is generally recognized that the activities of some of the red cell enzymes decline as the cell ages. However, there is still a controversy regarding the rate at which this aging occurs. In the present study we applied newly developed technology for the specific isolation of maturing reticulocytes/erythrocytes for a more comprehensive study of in vivo aging of red cell enzymes in rabbits. Anemia was induced by repeated phlebotomy, and reticulocyte-rich erythrocytes were labeled with N-hydroxy succinimido-biotin and then transfused into a normal rabbit. These biotinylated cells were isolated at various time points by their affinity for an avidin support, and the enzymatic activity of 19 red cell enzymes was measured. We observed a biphasic pattern of decay for the activity of six age-dependent enzymes—aldolase, glutamate-oxaloacetate transaminase, glucose 6-phosphate dehydrogenase, hexokinase, pyrimidine 5′-nucleotidase, and pyruvate kinase.