Simplified method for purification of ribonuclease H from calf thymus.
An improved purification procedure for the isolation of ribonuclease H(hybrid nuclease; RNA-DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from the thymus of 4-to 6-months-old calves yields two highly active forms of the enzyme, designated as ribonuclease H1 and H2. Their isoelectric points are 5.0 +/- 0.05 and 5.25 +/- 0.05, respectively; their molecular weight, estimated from gel filtration, is in both cases 64,000 +/- 2000. On sodium dodecyl sulfate gel electrophoresis, two principal bands were identified, with molecular weights of 32,000 and 21,000. The nature of the nucleotides at the 3'-OH terminals, produced initially by the enzymic hydrolysis of hybridized RNA, was examined and shown to be a function of the divalent metal ion employed as activator.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007526:Isoelectric Point; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013950:Thymus Gland / Q000201:enzymology
D002413:Cations, Divalent; D013439:Sulfhydryl Reagents; D012260:Ribonucleases