LEISHMANIA (LEISHMANIA) AMAZONENSIS - PURIFICATION AND ENZYMATIC CHARACTERIZATION OF A SOLUBLE SERINE OLIGOPEPTIDASE FROM PROMASTIGOTES

A soluble proteinase was purified 90-fold from extracts of promastigot es of Leishmania (Leishmania) amazonensis using a combination of ion-e xchange chromatography in Q-Sepharose Fast Flow, gel filtration chroma tography in Sephacryl HR S-200. and chromatofocusing. The enzyme appea red as a single band with an apparent molecular weight of 101 kDa by s ilver staining following SDS-PAGE, under both reducing and nonreducing conditions. The proteinase has a pH optimum between 8.0 and 8.5 and a n isoelectric point between 5.12 and 5.23, belongs to the serine prote inase class, and is inhibited by Mg2+, Ca2+, and K+. The primary speci ficity determined using synthetic substrates is for basic amino acids. The kinetic parameters for the Bz-L-Arg-Nam substrate are K-m = 26 mu M, k(cat) = 32 min(-1), and K-si = 1270 mu M (the proteinase showed i nhibition by excess substrate). The enzyme does not hydrolyze casein, albumin, and gelatin or large peptides like the oxidized insulin B cha in, but hydrolyzes small peptides like bradykinin and fragment 4-10 of the adrenocorticotropic hormone, at the carboxyl side of basic residu es and aromatic residues preceding basic residues. The enzyme appears, thus, to be restricted in its action, cleaving only small peptide sub strates, which characterizes the proteinase as an oligopeptidase. This is the first report of purification of a serine peptidase from Leishm ania species and it increases the short list of known oligopeptidases. (C) 1998 Academic Press.