Abstract
The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs). In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated. The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site. IC is able to form the complex with diisopropyl fluorophosphate-modified CPY. Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY. These findings demonstrate the multiple site binding of IC with CPY. Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs.
Highlights
The serine carboxypeptidase inhibitor in the cyto- acetyl group in its inhibitory activity were recently reported (3, plasm of Saccharomyces cerevisiae, IC, in- 5, 10), precise information on the mechanism involved in its hibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs)
Dissociation and Activity of the CPY-IC Complex in the Presence of GdnHCl—Gel filtration of the CPY-IC complex in the presence of various concentrations of GdnHCl revealed a single peak in 1 M GdnHCl or below, but a complex elution profile was obtained in 1.5 M GdnHCl or above (Fig. 1A)
When the CPY-IC complex was mixed with glutaraldehyde, a cross-linked product with a molecular mass in excess of 97.4 kDa was detected in both the absence of GdnHCl and the presence of 1 M GdnHCl, whereas free CPY and free IC failed to cross-link in a similar experiment (Fig. 2)
Summary
Materials—Guanidine hydrochloride (GdnHCl), PCMB, and PMSF (purity of more than 99%) were from Nacalai Tesque, Kyoto, Japan. The CPY-IC complex (140 g) was applied and eluted with buffer A containing various concentrations of GdnHCl at a flow rate of 0.5 ml/min. Reaction with PMSF, PCMB, and DFP—The CPY-IC complex (2 M) was incubated with a 20-fold molar excess of PMSF or PCMB in buffer A containing either 1 M GdnHCl or none at 25 °C for 0 –15 min. Residual peptidase activities after the reaction were determined in buffer A containing 1 mM CBZ-Ala-Phe-OH and 1.5 M GdnHCl. PMSF and PCMB were extensively diluted under such conditions. To assay for peptidase activity, the CPY-IC complex (32.8 nM) was added to buffer A containing 1 mM CBZ-Ala-Phe-OH and various concentrations of GdnHCl. The initial rates of hydrolysis were determined at 25 °C as described previously [17]. Native PAGE was performed on an 8% polyacrylamide gel, and the sample was prepared without either SDS or 2-mercaptoethanol
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