Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological.
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 degrees C (pH 7.2) was 10.23 +/- 1.90 mumol/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 X ATP2-. 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 microM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP4- 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 mumol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 +/- 0.3 mumol/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.
D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate / Q000097:blood; D000818:Animals; D004163:Diphosphoglyceric Acids / Q000097:blood; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase; D006019:Glycolysis; D006593:Hexokinase / Q000097:blood*; D007700:Kinetics; D008274:Magnesium / Q000097:blood; D011817:Rabbits; D013696:Temperature
D004163:Diphosphoglyceric Acids; D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D008274:Magnesium