Purification and characterization of an estrogen-regulated Xenopus liver polysomal nuclease involved in the selective destabilization of albumin mRNA.
A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had properties one might expect for a messenger ribonuclease involved in the regulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This report describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa and a small amount of a 40-kDa peptide. In situ analysis on both denaturing and nondenaturing gels using an albumin transcript as substrate showed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa peptides to be isoforms, and the 40-kDa peptide to be a degradation product of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, with isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect on either a full-length albumin antisense transcript or full-length ferritin 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 nuclease with micrococcal nuclease had no effect, indicating that this enzyme does not require an RNA cofactor for activity. Finally, primer extension mapped the major cleavage site to an overlapping repeated sequence APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.
Dompenciel
R E
RE
Department of Pharmacology, Uniformed Services University of the Health Sciences, School of Medicine, Bethesda, Maryland 20814-4799.
Garnepudi
V R
VR
Schoenberg
D R
DR
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004967:Estrogens / Q000494:pharmacology*; D005293:Ferritins / Q000096:biosynthesis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011132:Polyribosomes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012709:Serum Albumin / Q000096:biosynthesis*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014981:Xenopus
D017931:DNA Primers; D004967:Estrogens; D007527:Isoenzymes; D012333:RNA, Messenger; D012709:Serum Albumin; D005293:Ferritins; D012260:Ribonucleases