BINDING OF ACTIVATED CYCLOSOME TO P13(SUC1) - USE FOR AFFINITY PURIFICATION

Previous studies have indicated that a similar to 1,500-kDa complex, d esignated the cyclosome or anaphase-promoting complex, has a regulated cyclin-ubiquitin ligase activity that targets cyclin B for degradatio n at the end of mitosis. The cyclosome is inactive in the interphase o f the embryonic cell cycle and is converted to the active form in late mitosis in a phosphorylation-dependent process initiated by protein k inase Cdc2-cyclin B. We show here that the active, phosphorylated form of the cyclosome from clam oocytes binds to p13(suc1), a protein know n to associate with Cdc2, The following evidence indicates that the bi nding of the cyclosome to p13(suc1) is not mediated via the Cdc2-cycli n B complex: (a) activated cyclosome binds to p13(suc1)-Sepharose foll owing its separation from Cdc2-cyclin B by gel filtration chromatograp hy; (b) cyclosome from interphase extracts, activated by a kinase in w hich cyclin B has been replaced by an N-terminally truncated derivativ e fused to glutathione S-transferase, binds well to p13(suc1) Sepharos e but not to glutathione-agarose. An alternative possibility, that the phosphorylated cyclosome binds directly to a phosphate-binding site o f p13(suc1), is supported by the observation that the cyclosome is eff iciently eluted from p13(suc1)-Sepharose by phosphate-containing compo unds. This information was utilized to develop a procedure for the aff inity purification of the cyclosome. A factor abundant in the fraction not adsorbed to p13(suc1)-Sepharose stimulates the activity of purifi ed cyclosome. It is suggested that binding of Suc1 may have a role in the regulation of cyclosome activity.