PURIFICATION AND CHARACTERIZATION OF AN ESTROGEN-REGULATED XENOPUS LIVER POLYSOMAL NUCLEASE INVOLVED IN THE SELECTIVE DESTABILIZATION OF ALBUMIN MESSENGER-RNA

A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had proper ties one might expect for a messenger ribonuclease involved in the reg ulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This r eport describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa an d a small amount of a 40-kDa peptide, In situ analysis on both denatur ing and nondenaturing gels using an albumin transcript as substrate sh owed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa pe ptides to be isoforms, and the 40-kDa peptide to be a degradation prod uct of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, wi th isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect o n either a full-length albumin antisense transcript or full-length fer ritin transcript, A number of properties of the purified nuclease were characterized, including the effects of salt, divalent cations, EDTA, sulfhydryl reagents, and temperature. Treatment of the polysomal nucl ease with micrococcal nuclease had no effect, indicating that this enz yme does not require an RNA cofactor for activity. Finally, primer ext ension mapped the major cleavage site to an overlapping repeated seque nce APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.