The Proteolytic Enzymes of the K-1 Strain of Streptomyces griseus Obtained from a Commercial Preparation (Pronase)

Abstract

Abstract The commercial protease mixture, Pronase, which is obtained from the K-1 strain of Streptomyces griseus, contains many exo- and endopeptidases. Among the latter enzymes are several which are inhibited by diisopropyl fluorophosphate (DFP). Three of these DFP-reacting components, being of low molecular weight, were partially separated by filtration through Sephadex G-75. Chromatography through CM-cellulose of the filtration fractions with the smaller proteins yielded two well separated peaks (A and B) after the start of a sodium chloride gradient. Peak B consisted of a single enzyme which hydrolyzed N-α-acetyll-tyrosine ethyl ester (Ac-Tyr-OEt). It was homogeneous by gel electrophoresis and its approximate molecular weight was 17,500 as determined by gel filtration. The material in Peak A was heterogeneous with activities against both AcTyr-OEt and N-α-benzoyl-l-arginine ethyl ester (Bz-Arg-OEt). The component which hydrolyzes Bz-Arg-OEt was previously shown to be homologous with mammalian trypsin. Because of reports of the binding of alkylamines by trypsin, rechromatography of components in the earlier peak was carried out in the presence of n-butylamine. Slight retardation of the trypsin-like component was achieved. When a diamine, cadaverine, was substituted for the monoamine, further selective retardation was noted with almost complete resolution of the components in Peak A into two peaks (A1 and A2). These resolutions were thought to be due to the formation of an amine-enzyme complex at the active site; for, if the protein corresponding to Peak A was first reacted with DFP, no retardation was noted. Material from Peak A1 was homogeneous by gel electrophoresis and had an approximate molecular weight of 15,500. It hydrolyzed Ac-Tyr-OEt selectively. As expected the protein corresponding to Peak A2 hydrolyzed Bz-Arg-OEt selectively; its approximate molecular weight was 20,500. This enzyme demonstrated two bands by gel electrophoresis; this heterogeneity was tentatively attributed to the presence of both native enzyme and a partially autolyzed product. A larger DFP-reacting protein which hydrolyzed Ac-Tyr-OEt was partially purified by chromatography through CM-cellulose. Its approximate molecular weight was 27,000. An analysis was made of the partially hydrolyzed [32P]diisopropylphosphoryl (DIP) derivative of this protein. Electrophoretic studies of the migration of the radioactive peptides revealed a significant difference from the pattern of the radioactive peptides of [32P]DIP-α-chymotrypsin. This was in marked contrast to the results obtained with the three smaller enzymes. When a comparative electrophoretic analysis was made with the radioactive peptides of both the new enzyme and the [32P]DIP subtilisins excellent correspondence of patterns was noted. Therefore, the larger enzyme probably contains the sequence Thr-Ser-Met around the reactive seryl residue in contrast to the Asp-Ser-Gly sequences of the smaller enzymes.