On the molecular weight and subunit composition of calf thymus ribonuclease H1.
We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Rong
Y W
YW
Department of Pharmacology, University of North Carolina Medical School, Chapel Hill 27599.
Carl
P L
PL
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases; D046911:Macromolecular Substances; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H; D013950:Thymus Gland / Q000201:enzymology*
D046911:Macromolecular Substances; D011480:Protease Inhibitors; D004722:Endoribonucleases; D016914:Ribonuclease H