Leishmania (Leishmania) amazonensis: purification and enzymatic characterization of a soluble serine oligopeptidase from promastigotes.
A soluble proteinase was purified 90-fold from extracts of promastigotes of Leishmania (Leishmania) amazonensis using a combination of ion-exchange chromatography in Q-Sepharose Fast Flow, gel filtration chromatography in Sephacryl HR S-200, and chromatofocusing. The enzyme appeared as a single band with an apparent molecular weight of 101 kDa by silver staining following SDS-PAGE, under both reducing and nonreducing conditions. The proteinase has a pH optimum between 8.0 and 8.5 and an isoelectric point between 5.12 and 5.23, belongs to the serine proteinase class, and is inhibited by Mg2+, Ca2+, and K+. The primary specificity determined using synthetic substrates is for basic amino acids. The kinetic parameters for the Bz-L-Arg-Nam substrate are Km = 26 microM, kcat = 32 min(-1), and Ksi = 1270 microM (the proteinase showed inhibition by excess substrate). The enzyme does not hydrolyze casein, albumin, and gelatin or large peptides like the oxidized insulin B chain, but hydrolyzes small peptides like bradykinin and fragment 4-10 of the adrenocorticotropic hormone, at the carboxyl side of basic residues and aromatic residues preceding basic residues. The enzyme appears, thus, to be restricted in its action, cleaving only small peptide substrates, which characterizes the proteinase as an oligopeptidase. This is the first report of purification of a serine peptidase from Leishmania species and it increases the short list of known oligopeptidases.
de Andrade
A S
AS
Radiobiology Laboratory, AT4, Nuclear Technology Development Center (CDTN), Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
Santoro
M M
MM
de Melo
M N
MN
Mares-Guia
M
M
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D008274:Magnesium; D011188:Potassium; D002118:Calcium