KININOGEN-DERIVED FLUOROGENIC SUBSTRATES FOR INVESTIGATING THE VASOACTIVE PROPERTIES OF RAT-TISSUE KALLIKREINS - IDENTIFICATION OF A T-KININ-RELEASING RAT KALLIKREIN

Peptide substrates with intramolecularly quenched fluorescence that re produce the rat kininogen sequences at both ends of the bradykinin moi ety were synthesized and used to investigate the kinin-releasing prope rties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Subst rates derived from rat H- and L-kininogen were cleaved best by rK1, es pecially that including the N-terminal insertion site of bradykinin; A bz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-di nitrophenyl), which was cleaved at the R-R bond with a k(cat)/k(m) of 12 400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz- TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hyd rolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl -N-methyl coumarylamide substrates, which lack prime residues, also ha d low specificities. The importance of the P2' residue for rK1 specifi city was further demonstrated using a human-kininogen-derived substrat e that included the N-terminal insertion site of bradykinin (Abz-LMKRP -EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing s ite), but at the K-R bond (bradykinin-releasing site) by rK1. Competit ion experiments, with Abz-TSVIRRPQ-EDDnp. which is resistant to most k allikreins. and Abz-TSVIRRVQ-EDDnp. a general kallikrein substrate. de monstrated that the former competitively inhibited hydrolysis by rK9 a nd hK1, with K-i values similar to the K-m, values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme act ive site, but impairs cleavage of the scissile bond. The T-kininogen-d erived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp ) was cleaved by rK10 (k(cat)/K-m = 2310 mM(-1) s(-1)) and less rapidl y by rK1, rK7 and hK1, at the R-L bond, while that corresponding to th e N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all fiv e kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semi-purified sample of submand ibular gland extract. Another kallikrein, identified as kallikrein rK3 , was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP -EDDnp: rK3 also specifically released T-kinin from purified T1/T2-kin inogen after HPLC fractionation. Injection of purified rK3 and of Abz- ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesize d rats caused transient falls in blood pressure, as did purified rK1 b ut none of the other purified rat or human kallikreins. This effect oc curred via activation of the kinin system since it was blocked by Hoe1 40, a kinin receptor antagonist.