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Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae (BmTI) were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris-HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins (BmTI-B and BmTI-A, respectively). Both purified proteins inhibited trypsin with Ki of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase (Ki 1.4 nM) and plasma kallikrein (Ki 120 nM). BmTI-A did not change prothrombin time (PT) and thrombin time (TT), but it increased activated partial thromboplastin time (APTT) was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor (BPTI)-Kunitz type inhibitor family. These inhibitors (by their properties) play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable.

The action of two Bowman-Birk and several plant Kunitz-type inhibitors were studied on trypsin, chymotrypsin, plasma kallikrein and factor XII. The primary structure of some of them was completely defined. The results showed that the Bowman-Birk type inhibitors, although potent inhibitors for trypsin (Ki in the range of 1-2 nM), are not able to inhibit plasma kallikrein. Factor XII (Ki = 1.4 microM) and chymotrypsin (Ki = 5.0 nM) are inhibited by Torresea cearensis trypsin inhibitor (TcTI) but not by Dioclea glabra trypsin inhibitor (DgTI). Both inhibitors reactive site regions are highly homologous, and the amino acid residues in P1 position are the same, Lys and His; major differences are in the charge of the C-terminal portion of the molecules. The studied Kunitz-type inhibitors were all able to inhibit plasma kallikrein (Ki between 4 and 80 nM), with the exception of Schizolobium parahyba chymotrypsin inhibitor (SpCI), that is specific for chymotrypsin. All Kunitz-type inhibitors inactivate chymotrypsin, but with a dissociation constant in the range of 0.1 to 0.6 microM. Factor XIIf is inhibited with Ki in the range of 0.1 microM. Bauhinia bauhinioides trypsin inhibitor (BbTI) did not promote factor XIIf inhibition. The Kunitz-type inhibitors are a highly homologous, sharing 60% identity in the N-terminal portion of the loop containing the reactive site, and 28.6% identity in the C-terminal portion of the same loop.

Human plasma kallikrein (HuPK) is a serine protease found in mammalian plasma. Following limited proteolysis, the enzyme is activated and forms two chains. The light chain occurs with molecular weight 36,000 or 33,000 and contains the active site. The heavy chain occurs with molecular weight of 45,000 and contains the binding site for high-molecular-weight kininogen. Both chains were prepared from active HuPK, following mild reduction with dithiothreitol and carboxymethylation with iodoacetamide. The light chain was isolated in SBTI-Sepharose and its kinetic properties were determined with synthetic derivatives of arginine-p-nitroanilides, to investigate any possible alteration of the active site. These studies showed that substrate modifications affected the hydrolytic activity more than the binding capacity. The ability to cleave high-molecular-weight kininogen, as observed for intact kallikrein, was reduced in beta-kallikrein and absent in the light chain, even when equimolar amounts of both light and heavy chains were tested together. Anti-kallikrein antiserum formed immunoprecipitates not only with kallikrein itself, but also with the separated chains. The immunoreactivity of the light-chain was not identical with that of kallikrein. Immunoselected specific antibodies for both chains, depending on the selectivity, reacted with the heavy chain and kallikrein, or with the light chain and kallikrein. These antibodies were shown to be effective in binding radio-iodinated HuPK in the radioimmunoassay developed for intact kallikrein.

Human plasma kallikrein (HuPK) was detected in normal non-activated and dilution-activated plasma by immunoblotting, using polyclonal antibodies. In non-activated plasma, the predominatly detected protein corresponds to prokallikrein (Mr 80,000-90,000). Activated plasma, besides kallikrein, contains larger proteins (Mr > 130,000) that possibly represent complexes between kallikrein and proteinase inhibitors. Plasma also contains species (Mr 43,000) which corresponds to kallikrein heavy chain. In activated plasma, monoclonal antibodies against kallikrein heavy chain detected, besides these same bands described above, two additional bands (Mr 30,000 and 20,000) possibly correspondent to fragments of kallikrein heavy chain.

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.

Ala-Phe-Lys-CH2-Cl is a chloromethylketone derivative which is able to promote the inhibition of several proteolytic enzymes. In this paper the inhibition of horse urinary and plasmatic kallikreins is described and this inhibition is compared to that produced in human plasma kallikrein. This compound was designed based upon the structure of bradykinin. This enzyme substrate system can provide a model for the study of the interactions between bradykinin and its receptor. The inhibition of the enzymes was achieved both for its esterase and kinin-releasing activities.

Acid-activated human plasma kallikrein (HuPK) was purified from human Cohn fraction IV by affinity chromatography, using a ligand soybean trypsin inhibitor and aminobenzamidine. The purified enzyme does not inactivate bradykinin and lysyl-bradykinin by cleavage of their peptide bonds. Methionyl-lysyl-bradykinin is converted to the more potent peptide, bradykinin, by incubation with plasma kallikrein. The enzyme does not show aminopeptidase activity when assayed with amino-acyl-naphthylamides. Arginine-rich polypeptides and proteins, such as polyarginine, salmine, and histones were cleaved by the enzyme. HuPK does not show any detectable caseinolytic activity. A kinin is released from a non-homologous plasma (horse) by this kallikrein. The enzyme is not affected by calcium or EDTA, and it is strongly inhibited by copper ion.

The isolation procedure for horse urinary kallikrein was considerably improved by the introduction of two new purification steps: a) removal of mucoproteins and concentration of the urine by ultrafiltration and b) affinity chromatography on benzamidine-Sepharose conjugate. The homogeneity of the enzyme preparations, regarding their protein moiety, was demonstrated by: 1) a single symmetric peak on DEAE-Sephadex chromatography, with constant values for A280/A260 ratios, esterolytic and amidolytic specific activities; 2) a single band, although dispersed, on gel-electrophoresis at pH 8.3, also in the presence of sodium dodecyl sulfate, and 3) a unique sequence for the six amino-terminal residues. The isolated enzyme was shown to be a single chain glycoprotein (alpha-kallikrein), similar to human urinary and porcine-pancreatic kallikreins regarding the protein moiety molecular mass, amino-acid composition, and partial amino-terminal sequence; differences were found in their total sugar content and even more conspicuously in their carbohydrate composition. In contrast to porcine pancreatic beta-kallikrein, horse urinary kallikrein was not substrate-activated and unlike other alpha-kallikreins, did not present the biphasic time-course in benzoyl-L-arginine ethyl ester hydrolysis. The specificity constants (kcat/Km) for ester and 4-nitroanilide substrates were lower for horse urinary than for pancreatic beta-kallikrein and as observed with the latter enzyme, were affected by NaCl.

A four-step procedure was used to purify rat plasma kallikrein (RPK) with a relative molecular mass (Mr) of 87 kD (obtained both by gel filtration and SDS-PAGE), which indicates the purification of an alpha (intact) kallikrein, in contrast to previously described RPK preparations which had lower Mr (beta or degraded form). RPK is a neutral (pI 6.7) serine proteinase glycoprotein (15% carbohydrates) and contains (residues/mol): galactose (27), N-acetylglucosamine (24), mannose (13), glucose (13) and fucose (7). This purified alpha form of RPK has properties very similar to those of pure human and bovine kallikreins.

Active plasma kallikrein (Mr = 90,000) can be separated by reduction of the disulfide bridges into a heavy-chain (Mr = 45,000) and a light-chain (Mr = 36,000). A partially active fragment, corresponding to the light-chain, was isolated during the purification of active human plasma kallikrein. Bovine trypsin can form a fragment corresponding to the heavy-chain when incubated with plasma kallikrein; plasmin also causes the formation of the heavy-chain but at a slower rate. High molecular weight kininogen decreases the rate of cleavage of kallikrein by both enzymes. Light-chain does not accumulate during incubation with these proteases and the heavy-chain, after prolonged incubation is also digested. Incubation of labelled active kallikrein with plasma causes formation of fragments corresponding to the heavy- and the light-chain.

Horse urinary kallikrein was titrated with the reagent 4-nitrophenyl 4-guanidino-benzoate. The titration was shown to be dependent upon the concentration of the titrant. This finding, which distinguishes horse urinary kallikrein from other enzymes, is explained by the relatively small ratio between its rate of acylation and deacylation (k2/k3 = 16.8) and by a low affinity of the reagent (Km = 1.16 microM). By an appropriate kinetic treatment, it was possible to establish the relationship between the hydrolysis of 4-nitrophenyl 4-guanidinobenzoate and the actual concentration of the active enzyme.

The three tetrapeptides Ac-Phe-Arg-Arg-Val-NH2 (I), Ac-Phe-Arg-Arg-Pro-NH2 (II) and Ac-Phe-Lys-Arg-Val-NH2 (III) were shown to form a most convenient substrate system for the discrimination of the serine proteinases listed below. Tissue kallikreins (porcine pancreatic, horse and human urinary) have the unique feature of cleaving well the Arg-Arg bond in peptide I (P'2 = Val), hardly splitting it in peptide II (P'2 = Pro). The kcat/Km for the hydrolysis of peptide II by horse urinary kallikrein was 600-fold lower than that for peptide I. Trypsin, plasma kallikreins (human and rat), tonin and rat urinary kallikrein were distinguished from each other by the sequence of the N-terminal fragments formed in the hydrolysis of peptides I and/or II. Differences in the cleavage sites in these peptides are explained by differences in the specificities of the proteinase subsite S2 and/or in their preference for Arg or Lys residues. The three tetrapeptides were not substrates for plasmin.

We report observations regarding the in vivo distribution of labelled kallikreins in plasma, liver and some other organs, twenty minutes following their intravenous injection in the rat. The kallikreins used were: tritiated homogeneous human plasma (HuPK) and horse urinary (HoUK) as well as highly purified iodinated rat plasma kallikrein (RPK). The main findings were: the liver cleared 15% of HuPK, 38% of RPK and 69% HoUK; with both types (plasma and tissue) of native kallikreins the liver was the main clearing organ.

Platelet aggregation induced by ADP, collagen and adrenaline is increased by low concentrations of human plasma kallikrein, which does not cause aggregation by itself and inhibits the aggregation induced by arachidonic acid and Thrombofax in platelet rich plasma, and the aggregation induced by thrombin in washed platelets. At higher concentrations however, plasma kallikrein inhibits the aggregation induced by all the agents tested with platelet rich plasma and also causes a decrease in the aggregation induced in whole blood.