Details

Autor(en) / Beteiligte

• BUTTON, ELIZABETH ELLEN

Titel

PURIFICATION AND CHARACTERIZATION OF PORCINE THYROID, CYTOSOLIC, ALKALINE RIBONUCLEASE FROM THE ALKALINE RIBONUCLEASE: RIBONUCLEASE INHIBITOR COMPLEX

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1984

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Porcine thyroid, cytosolic, alkaline RNase (ptAR) was purified from the alkaline RNase:RNase inhibitor complex (ptAR:RI) by a procedure which excludes heat and acid. Purified ptAR shows a single area of activity on polyacrylamide gel electrophoresis which corresponds with the major stainable protein area. The enzyme has a molecular weight of 51,000 on Sephadex G-75 and from 55,000 to 58,000 on Sephacryl S-300. Gel electrophoresis of ptAR in the presence of sodium dodecyl sulfate revealed a group of bands with molecular weights from 50,000 to 60,000 and a band at 37,000 was seen. Purified ptAR also displayed apparent heterogeneity when chromatographed on CM-cellulose; up to three interconvertable forms of the enzyme were detected. The pH optimum was 8.1 in both Tris and HEPES buffer. The enzyme was stimulated by monovalent chloride and potassium salts at ionic strengths between 10 and 70 mM; these salts were inhibitory above 100 mM with the exception of ammonium chloride. Both MgCl(,2) and CaCl(,2) were stimulatory at 0.5 mM whereas CuCl(,2) and ZnCl(,2) (1.0 mM), EDTA (1.0 mM) and p-chloromercuriphenylsulfonate (pCMPS) (1.0 mM) were inhibitory. Both native and denatured DNA slightly stimulated enzyme activity. The hydrolytic products of natural and synthetic substrates indicates that ptAR is a pyrimidine specific endoribonuclease which hydrolyzes singled-stranded RNA to mono- and oligonucleotides terminating in pyrimidine 2'3'-cyclic phosphate. The Km for tRNA was 1.06 x 10('-5) M. Poly(A) and poly(G) inhibited ptAR and this inhibition could be reversed by low concentrations of spermine and to some extent by increases in ionic strength. Spermine alone inhibited ptAR at 0.5 mM and stimulated activity at 0.05 mM. A comparison between ptAR purified using an acid treatment and ptAR purified by a procedure which excludes acid indicated that the use of acid in the purification may alter the properties of ptAR. Initial investigations into the characteristics of the ptAR:RI complex indicated that the two proteins combine in a 1:1 stoichiometry. Treatment of the complex with pCMPS results in a dissociation of the components which appears to be reversible. An attempt to find a physiologically significant activator of the complex was conducted. Of the compounds tested, (CuCl(,2), CaCl(,2), glutathione, calmodulin, 3'5'-cyclic AMP, bacterial alkaline phosphatase, protein kinase, and spermine) only CuCl(,2) was able to effect the release of RNase activity but the significance of this response is questionable. Comparisons between ptAR and pancreatic RNase A indicate that they are distinct enzymes.