Puridication and properties of an intracellular ribonuclease from Candida lipolytica.
1. A ribonuclease (RNAase CL) (EC 3.1.4.23, ribonucleate 3'-oligonucleotide hydrolase) was extracted by EDTA/acetate buffer, pH 5.6 from acetonedried cells of Candida lipolytica and purified 1350-fold by acetone and (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography. 2. RNAase CL is an acidic protein having an isoelectric point of 4.2, and an approximate molecular weight of 32 000. 3. Optimal pH and temperature for the enzyme were 6.0 and 60 degrees C, respectively. It is stable at neutral pH up to 50 degrees C. At 64 degrees C for 30 min, 95, 49 and 64% inactivation of the enzyme occurred at pH values 4.2, 6.6 and 10.0, respectively. 4. RNAase CL inhibited by Zn2+ and Cu2+, sulfhydryl reactants and by high concentration of salts, but not by chelating agents. 5. RNAase CL degraded ribosomal RNA, transfer RNA, polyadenylic acid, polycytidylic acid and polyuridylic acid into acid-soluble nucleotides. Among the synthetic homopolymers, polycytidylic acid was most rapidly degraded. Polyguanylic acid and duplexes of synthetic homopolymers were less sensitive. DNA was not attacked. Specificity studies showed that RNAase CL preferentially cleaves pC-purine bonds. 6. Digestion of poly (C) by RNAase CL resulted in the liberation of cyclic 2',3'-CPM from the start of the reaction with no observable formation of intermediate oligonucleotides. This suggests that the enzyme depolymerizes by an exonucleolytic mechanism.
D000096:Acetone; D002175:Candida / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology
D002614:Chelating Agents; D013439:Sulfhydryl Reagents; D000096:Acetone; D003300:Copper; D004492:Edetic Acid; D004720:Endonucleases; D005092:Exonucleases; D012260:Ribonucleases; D015032:Zinc