Conversion of Met-enkephalin-Arg6-Phe7 by a purified brain carboxypeptidase (cathepsin A).

A carboxypeptidase A-like enzyme known as cathepsin A was purified from rat brain by extraction with Triton X-100, followed by chromatography on DEAE-Sephadex A-50 and gel-filtration. Purified enzyme was devoid of contamination of tryptic-like enzymes, by dipeptidyl carboxypeptidase (angiotensin converting enzyme) and of enkephalinnases cleaving the Tyr-Gly and Gly-Phe bonds of Met-enkephalin. Incubation of purified enzyme with Met-enkephalin-Arg6-Phe7, a naturally occurring enkephalin surrogate, was accompanied by the release of three products as detected by reverse phase HPLC. Subsequent amino acid analysis identified these as Phe, Met-enkephalin-Arg6, and Met-enkephalin, indicating cleavage at the Arg6-Phe7 and Met5-Phe6 bonds. Breakdown followed a precursor-product-relationship with the hexapeptide appearing as an intermediate and the pentapeptide as the final product. The Km for cleavage of the Arg-Phe site was 0.09 mM. Rates of cleavage of hexa- and heptapeptide accord with those found for synthetic N-protected dipeptide substrates. Cathepsin A does not act as an enkephalinase in the accepted sense, since no breakdown of Met-enkephalin was observed.