Purification and properties of a novel pyrimidine-specific endoribonuclease termed endoribonuclease VII from calf thymus that is modulated by polyadenylate.
Endoribonuclease VII, a novel endoribonuclease from calf thymus, was identified and purified by us. The purified enzyme has Mr = 74,000; its homogeneity was checked by analysis in polyacrylamide gels (both in the presence and in the absence of sodium dodecyl sulfate). The nuclease cleaves poly(U) and poly(C) while other single-stranded homopolyribo- as well as polydeoxyribonucleotides are not degraded; poly(A,C) is hydrolyzed to a smaller extent, while poly(U) X poly(A) is not degraded at all. Poly(A) modulates the poly(U)-degrading activity; at a molar ratio of approximately 1 [poly(A)]:10 [poly(U)], a more than 100% stimulation of the enzyme activity was achieved, while at lower ratios an almost complete inhibition of the enzyme activity resulted. Binding studies revealed that endoribonuclease VII has a marked affinity for poly(A) and poly(U). During hydrolysis, oligo(U)12 fragments with 3'-OH and 5'-P termini are formed. The basic enzyme (pI = 8.5) has its activity optimum at pH 7.2, requiring neither monovalent nor divalent cations; the enzyme is not inhibited by thiol group reagents. Several lines of evidence suggesting a role of endoribonuclease VII in mRNA processing are presented.
D000818:Animals; D002417:Cattle; D004722:Endoribonucleases / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D011061:Poly A / Q000494:pharmacology*; D011072:Poly U; D011089:Polydeoxyribonucleotides; D011131:Polyribonucleotides; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D011089:Polydeoxyribonucleotides; D011131:Polyribonucleotides; D011061:Poly A; D011072:Poly U; D004722:Endoribonucleases; C038251:endoribonuclease VII