Protein inhibitor of acid deoxyribonucleases. Improved purification procedure and properties.
A method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate versus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme.
D000818:Animals; D002417:Cattle; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006372:Helix, Snails / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors
D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; D007461:Iodoacetates; D010726:Phosphodiesterase Inhibitors; D003851:Deoxyribonucleases