J. Biol. Chem., Vol. 261, Issue 18, 8327-8333, Jun, 1986
Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation
M Magnani, V Stocchi, L Chiarantini, G Serafini, M Dacha and G Fornaini
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.
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