Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity.
The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)
Freeman-Wittig
M J
MJ
Department of Biochemistry, University of Nevada-Reno 89557.
Lewis
R A
RA
D000818:Animals; D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000187:drug effects / Q000201:enzymology*; D002215:Captan / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D004722:Endoribonucleases / Q000378:metabolism*; D005990:Glycerol; D009693:Nucleic Acid Hybridization; D016914:Ribonuclease H; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D004722:Endoribonucleases; D016914:Ribonuclease H; D002215:Captan; D005990:Glycerol