Decay mechanisms of rabbit hexokinase during reticulocyte maturation.
In rabbit erythrocytes hexokinase (EC 2.7.1.1) specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by an in vitro made policlonal antibody shows that this maturation dependent hexokinase decay is not due to the accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system made from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinase 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. 125I-hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mecaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugate 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 involves both the hexokinase molecular forms (hexokinase Ia and Ib) present in reticulocytes. "In vivo", hexokinase Ia is mitochondrial bound while hexokinase Ib is soluble. The energy dependent degradation system of reticulocytes is active only on the soluble enzyme, namely hexokinase Ib. As the cell mature mitochondria are degradated, hexokinase Ia becomes soluble but there is a concomitant decay also of the proteolytic system resulting in a mature erythrocyte that contains only hexokinase Ia in a soluble form.
D000255:Adenosine Triphosphate / Q000097:blood; D000818:Animals; D002454:Cell Differentiation; D002474:Cell-Free System; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000378:metabolism; D006019:Glycolysis; D006593:Hexokinase / Q000097:blood*; D066298:In Vitro Techniques; D007700:Kinetics; D011817:Rabbits; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D000255:Adenosine Triphosphate; D006593:Hexokinase