| Autor: |
Awad, William M., Soto, Aida R., Siegel, Steven, Skiba, William E., Bernstrom, Gary G., Ochoa, Maria S. |
| Zdroj: |
Journal of Biological Chemistry; July 1972, Vol. 247 Issue: 13 p4144-4154, 11p |
| Abstrakt: |
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 (A1and 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 A1was 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 A2hydrolyzed 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. |
| Databáze: |
Supplemental Index |
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