Reference: Hrycyna CA and Clarke S (1993) Purification and characterization of a novel metalloendopeptidase from Saccharomyces cerevisiae. Biochemistry 32(42):11293-301

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Abstract


We previously identified an activity in the soluble fraction of the yeast Saccharomyces cerevisiae that is a candidate for catalyzing the proteolytic maturation of farnesylated-CXXX precursor polypeptides. We describe here a 1259-fold purification of this activity by chromatography on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose, and Sephacryl S-200. Sodium dodecyl sulfate gel electrophoresis of this preparation demonstrated a single 68-kDa polypeptide chain. The experimentally determined N-terminal amino acid sequence was identical at all 20 positions with residues 28-47 of the deduced sequence of the S. cerevisiae YCL57w gene product. This analysis suggests that the YCL57w gene encodes this enzyme and that the initial translation product may contain a leader peptide. Its complete deduced amino acid sequence shows significant homology to a number of zinc metallopeptidases and is most closely related to rat metalloendopeptidase 24.15 (E.C. 3.4.24.15), an enzyme that preferentially cleaves after hydrophobic residues. Using the purified yeast enzyme, we show a unique cleavage site in the peptides bradykinin and beta-neoendorphin four residues from the C-terminus on the carboxyl side of a hydrophobic amino acid. The cleavage pattern for neurotensin revealed a major site three residues from the C-terminus also on the carboxyl side of a hydrophobic residue and a minor site four residues from the C-terminus of the peptide. This specificity is similar to that of rat endopeptidase 24.15 and may explain why the farnesylated peptide employed in our studies is a good substrate for the yeast enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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Comparative Study | Journal Article | Research Support, U.S. Gov't, P.H.S.
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Hrycyna CA, Clarke S
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