Characterization of two cysteine proteinases secreted by Fasciola hepatica and demonstration of their kininogenase activity.
We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.
Cordova
M
M
Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Urbanización Ingeniería-San Martin de Porras, Lima, Peru.
Jara
J
J
Del Nery
E
E
Hirata
I Y
IY
Araújo
M S
MS
Carmona
A K
AK
Juliano
M A
MA
Juliano
L
L
D000577:Amides; D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005210:Fasciola hepatica / Q000201:enzymology*; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism*; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism; D007705:Kinins / Q000378:metabolism; D008969:Molecular Sequence Data; D013194:Staining and Labeling; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates
D000577:Amides; D019679:Kininogen, High-Molecular-Weight; D007705:Kinins; D062367:ortho-Aminobenzoates; D007610:Kallikreins; D003546:Cysteine Endopeptidases