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

• Published in: The Journal of biological chemistry 1973-09, Vol.248 (17), p.6029-6034
• Main Authors: Vosbeck, Klaus D., Chow, Kai-Fu, Awad, William M.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43503-5

We described earlier the purification of Streptomyces griseus trypsin. This enzyme was demonstrated to bind diaminoalkanes and as a consequence was selectively retarded during CM-cellulose chromatography (Awad, W. M., Jr., Soto, A. R., Siegel, S., Skiba, W. E., Bernstrom, G. G. & Ochoa, M. S. (1972) J. Biol. Chem. 247, 4144–4154). In order to facilitate this procedure an affinity matrix, 1,6-diaminohexane-agarose, was synthesized and demonstrated to bind the trypsin. More significant was the finding that this agarose derivative also bound the two components with aminopeptidase activity present in Pronase. The trypsin was completely separated from the aminopeptidases by initial passage through a CM-cellulose column. Thereafter, the trypsin was selectively retarded by the hexanediamine-agarose; all associated components were eluted with 1 m NaCl at pH 8, whereas the trypsin could be eluted only after application of an acidic (pH 3) buffer. This enzyme appeared to be homogeneous by other studies. Bovine trypsin was not retarded by this chromatographic procedure. The aminopeptidases were purified by passage through the same column. Their complete selective retardation was achieved only after prior treatment with sodium ethylenediamine-tetraacetate. Calcium ion at low concentrations eluted the two enzymes separately. Calcium ion was required for the activity of each enzyme and also stabilized each enzyme against heat denaturation. Strontium ion could restore about two-thirds of the activities to the metal-free proteins as compared to the activities noted with calcium; other divalent cations provided much less activity. The maximum activity of each aminopeptidase lies between pH values of 7.5 and 10; each enzyme is stable between pH values of 6 and 11. These enzymes have been tentatively designated aminopeptidase 1 and aminopeptidase 2 after the order of their elution from hexanediamine-agarose; gel filtration revealed their approximate molecular weights to be 23,000 and 25,000, respectively. A single band was seen for each protein after acrylamide gel electrophoresis.