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Biochem J. 1998 Apr 15;331 ( Pt 2):539-45.

Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin.

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  • 1Department of Microbiology, College of Natural Science and Research Centre for Molecular Microbiology, Seoul National University, Seoul 151-742, Korea.

Abstract

Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatus SMF13 by ammonium sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 degrees C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation.

PMID:
9531495
[PubMed - indexed for MEDLINE]
PMCID:
PMC1219386
Free PMC Article
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