Figure 4.
The degradation of endogenous cyclin B in mitotic clam oocyte extracts is inhibited by C→S mutants of human UbcH10 and clam E2-C. Incubations contained in a volume of 12 μl: 10 μl extract of early M-phase clam oocytes (75 min postfertilization, see ref. 7), 2 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 50 μg/ml creatine kinase, 0.5 mM DTT, 250 μM emetine, and recombinant E2-C derivatives as specified. After incubation at 16°C for the time periods indicated, samples of 2 μl were withdrawn into SDS electrophoresis sample buffer. The samples were separated on a 10% polyacrylamide gel, blotted on nitrocellulose, probed with an antiserum directed against clam cyclin B (25), and detected by ECL. (A) Effects of human UbcH10 and clam E2-C C→S mutants on the degradation of clam cyclin B. (B) Reversal of the effects of the C→S mutant by wild-type human UbcH10. The various recombinant Ubc derivatives were added at the concentrations indicated. The position of cyclin B is indicated. The identity of the crossreactive protein that migrates ahead of cyclin B is not known. WT, wild type.