Purification, subunit structure, and serologicai analysis of calf thymus ribonuclease H I.

作者：BÜsen · 发表于：Journal of Biological Chemistry · 年份：1980 · DOI：10.1016/s0021-9258(19)70581-x · 被引用次数：74 · 研究领域：Transgenic Plants and Applications、Toxin Mechanisms and Immunotoxins、Trypanosoma species research and implications

Calf thymus ribonuclease H I (for nomenclature, see Büsen, W., and Hausen, P. (1975) Eur. J. Biochem. 52, 179-190) has been purified to near homogeneity. The large scale purification procedure results in a 1,000-fold enrichment of enzyme protein over the crude extract. The enzyme has a molecular weight of about 80,000, and S value of about 5, and an isoelectric point of about 4.9 under nondenaturing conditions. The purified enzyme sample contains two forms of ribonuclease H I, possibly isozymes, named ribonuclease H I 1 and ribonuclease H I 2. They can be activated by Mn2+ or Mg2+ ions. The most highly purified fraction is composed of four polypeptides named A, B, C, and D with molecular weights of 31,6000, 26,6000, 24,800, and 24,300, respectively. Polypeptides A, C, and D are acidic, whereas Polypeptide B is basic. Each form consists of three polypeptides. Ribonuclease H I 1 and ribonuclease H I 2 have Polypeptides A and B in common and differ from each other in the third. The data are consistent with a trimeric (A, B, C/D) or tetrameric (A, B2, C/D) structure for calf thymus ribonuclease H I. When alkalisensitive supercoiled DNA molecules containing ribonucleotides covalently inserted in one of the DNA strands are used as substrate, the products of the reaction are relaxed circles; thus, ribonuclease H I has an endonucleolytic mode of action. The final preparation is free of ribonuclease, and also of endodeoxyribonuclease activity single- and double-stranded DNA. Rabbit an...