Ca²⁺-Stimulated Ribonucleases from Rat Mammary Gland and R3230ac Mammary Adenocarcinoma Nuclei

Ribonuclease activity in the nuclei of lactating rat mammary gland and R3230AC mammary tumor is several times higher with Ca²⁺ (5 mM optimum) in the assay than with Mg²⁺ or no added divalent cations. The Ca²⁺-stimulated RNase (Ca²⁺ RNase) activities from the nuclei of both mammary gland and R3230AC tumor are found primarily in the nucleoli-free nuclear sonicate. High-speed centrifugation of the sonicate yields Ca²⁺ RNase activity in both supernatant (Sap I) and pellet fractions. DEAE-cellulose chromatography of Sap I revealed a single peak of activity (Ca²⁺ RNase I) that was eluted at 0.15 M NaCl. Similar chromatography of the 1 M NaCl extract of the pellet (Sap II) yielded a peak (Ca²⁺ RNase Ha) that appeared prior to the salt gradient and a major peak (Ca²⁺ RNase IIb) that appeared at 0.08 M NaCl. The molecular weights of Ca²⁺ RNases I and IIb are approximately 52 500 and 32 400, respectively, as determined by Sephadex G-100 gel filtration. Ca²⁺ RNases I and IIb are both endonucleases as judged by sucrose density gradient analysis of ³H-labeled 45S RNA reaction products. Ca²⁺ RNases I and IIb catalyze the degradation of ³H-labeled poly(U), but are less active against poly(C). The mammary gland enzymes are inactive against poly(G) and poly(A); tumor Ca²⁺ RNase I is inactive against poly(G) but is slightly active against poly(A). The potential substrates, poly(dT), DNA-RNA hybrids, ribosomal RNA, and DNA, were not degraded into ethanol-soluble products by tumor Ca²⁺ RNase I. The enzymes are inhibited by polyamines, heparin, polydextran sulfate, NaF, and sodium pyrophosphate. PEI-cellulose thin-layer chromatography of labeled poly(U) and poly(C) degradation products catalyzed by Ca²⁺ RNases I and IIb revealed the presence of 2′,3′-cyclic mononucleotides and oligonucleotides with 3′-phosphoryl and 5′-hydroxyl termini. In summary, nuclei from lactating rat mammary gland and R3230AC mammary tumor contain Ca²⁺-dependent, pyrimidine-specific, 3′-phosphate-forming RNA endonucleases.