An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): primary structure, enzymatic characterization, and its interaction with SSI.

We have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J. Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-P26, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253. Complete amino acid sequence determination revealed that SAM-P26 is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-P20, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-P20. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-P26 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Da) and that of the S-pyridylethylated form (18,808.4 Da) of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SAM-P26 exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-P26 exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degreesC. The complex formation of SAM-P26 with SSI was confirmed by native-PAGE analysis.