Abstract
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 hexokinae activity and the degradation of 125I-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. 125I-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 125I-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 ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.
Highlights
In rabbitreticulocytes, the hexokinase (EC 2.7.1.1)- more of the following: the actionof specific proteases
By a polyclonal antibody made in vitro shows that this we report detailed studies on the mechanism of hexokinase maturation-dependent hexokinase decay is not due to decay during rabbitreticulocyte maturation
Hexokinase in Rabbit Erythrocytes and Reticulocytes-In rabbit reticulocytes, the hexokinase-specific activity is 3.8 f 0.2 units/g of hemoglobin, a value 4-5 times that found in the corresponding mature erythrocytes (0.9 k 0.05 unit/g of hemoglobin)
Summary
A cell-free system derived becoming a rate-limiting step of glycolysis [24,25,26,27] It from rabbit reticulocytes, but not mature erythrocytes, could be a useful model providing insight into the mechawas found to catalyze the decay of hexokinase activity and the degradation of 1261-labeled enzyme.This degradation is ATP-dependent and requirebs oth ubiquitin and a proteolytic fraction retainedby DEAE-cellulose. The hybridoma selected produced antibodies that in the enzyme-linked immunosorbent assay test recognized active hexokinase, boiled hexokinase, inactive hexokinase obtained by dialysis in 10 mM sodium phosphate buffer, pH 7.0, without glucose and mercaptoethanol, and hexokinase that had lost activity upon storage for 2 months at -20 "Cin the absence of glucose.the enzyme was inactivated when incubated with these antibodies. It is worth noting that the antibody used was a polyclonal capable of detecting both active and inactive enzyme molecules, as described under “Experimental Proce-
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