A highly specific aminotripeptidase of rat brain cytosol: Substrate specificity and effects of inhibitors

Abstract

An aminopeptidase preferentially hydrolyzing Leu- or Ala-Gly-Gly was purified from rat brain cytosol and detailed studies have been performed on its substrate specificity and the effects of inhibitors. The enzyme was devoid of di- and oligopeptidase contamination. Biologically active tripeptides such as Met-Leu-Tyr (chemotactic factor), Gly-His-Lys (liver growth factor) and Thr-Val-Leu (central nervous system tripeptide) were hydrolyzed at rates 0.05–0.15-times that of Leu-Gly-Gly. Melanostatin (Pro-Leu-GlyNH 2) did not serve as a substrate. Substrates bearing N-terminal charged groups, or ones with proline in positions 2 or 3, or those with d-amino acid in positions 1 or 2, or with C-terminal CONH 2, were poorly hydrolyzed or did not act as substrates, providing information on subsites involved in enzyme catalysis. The enzyme was inhibited competitively by bestatin ( K i 10 −7M) and by Captopril (2.5 · 10 −7 M, d-3-thio-2-methylpropanyi proline) and by low concentrations of Zn 2+ or PCMB, and at higher concentration by TPCK and PMSF. Inhibition was observed for the chemotactic factor ( I 50 13 μM) and for the central nervous system tripeptide (195 μM). The enhanced action of Captopril was attributed to the presence of the -SH and -CH 3 groups, since inhibition was shared by di- and tripeptides with proline in positions 2 and 3. The specificity pattern of brain enzyme was different from that reported for kidney and intestine.