Inhibition of Serine Proteinases by P-Carbethoxyphenyl Esters of ϵ-Guanidino- And ϵ-Amino Caproic Acid: Thermodynamic and Molecular Modeling Study

Abstract

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of ?-guanidino-caproic acid metha-nesulphonate (?-GCA-CEP) on the catalytic properties of human LYS⁷⁷-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine α-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine β-trypsin (EC 3.4.21.4), porcine pancreatic β-kallikrein-B (EC 3.4.21.39, human urinary kallikrein (EC 3.4.21.35) and the M_r 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M;T = 21 ? 0.5?C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl ?-amino-caproate hydro chloride (?-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (K_i) for ?-GCA. CEP and ?-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) ?-GCA-CEP interacts with bovine factor Xa and bovine α-thrombin with an higher affinity than that observed for ?-ACA-CEP binding; (ii) both inhibitors associate to bovine β-trypsin with the same affinity; and (iii) ?-ACA-CEP inhibits human Lys⁷⁷-plasmin and the M_r 54,000 species of human urokinase with an higher affinity than that reported for ?-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of ?-ACA-CEP for ancrod, crotalase, porcine pancreatic β-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by ?-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of ?-GCA-CEP and ?-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.4), alpha-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and beta-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for beta-trypsin; at variance with the affinity of TAPP for alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B which is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, values of Ki for TAPP binding to beta-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases: TAPP and :benzamidine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study.

Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: Ka = 4.9 x 10(4) M-1, delta G degree = -6.3 kcal/mol, and delta S degree = -37 entropy units (all at 21.0 degrees C); and delta H degree = +4.6 kcal/mol (temperature independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

Primary specificity of ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom

Values of steady-state and pre-steady-state parameters for the hydrolysis of ZArgONp and ZLysONp catalysed by ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom, have been determined, between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C, and analysed in parallel with those of bovine alpha-thrombin and porcine pancreatic beta-kallikrein-B. In addition to the well-known coagulating behaviour, ancrod also shows catalytic properties, in the hydrolysis of ZArgONp and ZLysONp, reminiscent of those of porcine pancreatic beta-kallikrein-B.     

Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin > human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine alpha-chymotrypsin and bovine beta-trypsin. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

Catalytic properties of bovine alpha-thrombin: a comparative steady-state and pre-steady-state study

Values of steady-state and pre-steady-state parameters for the bovine alpha-thrombin-catalyzed hydrolysis of ZArgONp and ZLysONp have been determined between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degree C. Kinetic properties of bovine alpha-thrombin have been analyzed in parallel with those of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin, all acting on cationic substrates. The different primary specificity and catalytic behaviour of these three serine proteinases reflect subtle structural differences at their S1 subsite, especially at residue positions 190, 221 and 226 as well as in the 217-219 segment.     

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.5 degrees C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine beta-trypsin/Kazal inhibitor and bovine beta-trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzyme/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine beta-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine beta-trypsin. The existence of different discrete states suggests that the zymogen-to-active enzyme transition should not be considered as a two-state process but as a multistep event.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

Inhibition of proteases in coagulation, kinin-forming and complement systems by alpha2-plasmin inhibitor.

Inhibitory activities of alpha2-plasmin inhibitor against various proteases were investigated. The inhibitor promptly inhibited the esterolytic activity of alpha-chymotrypsin and progressively inhibited the esterolytic or amidolytic activities of bovine plasma kallikrein, bovine thrombin and bovine activated factor X. Heparin had no effect on the reaction of the inhibitor with thrombin or activated factor X. However, the inhibitor had no effect on the activities of human C-1-esterase, papain and snake venom kininogenase. On the basis of its rapid inhibition of kallikrein, alpha2-plasmin inhibitor is considered to exert some regulating effect on kallikrein activity in plasma.     

Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator

A low Mr form (Mr 32,000) of single-chain urokinase-type plasminogen activator (scu-PA) was isolated from conditioned culture medium of a human lung adenocarcinoma cell line, CALU-3 (ATCC, HTB-55). The purified material (scu-PA-32k) consists of a single polypeptide chain and is immunologically similar to Mr 33,000 urokinase. Its NH2-terminal sequence is identical to that beginning at Leu-144 of Mr 54,000 urokinase. Whereas low Mr urokinase is derived from mature Mr 54,000 scu-PA by limited hydrolysis by plasmin first of the Lys-158-Ile-159 peptide bond and then of the Lys-136-Lys-137, scu-PA-32k is generated by specific hydrolysis of the Glu-143-Leu-144 peptide bond by an unidentified protease. scu-PA-32k resembles its Mr 54,000 scu-PA counterpart by its very low activity on chromogenic substrates for urokinase, by plasminogen-dependent fibrinolytic activity on fibrin plates, and by the lack of specific binding to fibrin. It activates plasminogen directly with high affinity, Km = 0.9 microM, but low turnover number, kcat = 0.0028 s-1. It is converted to fully active two-chain urokinase by plasmin with Km = 12 microM and kcat = 0.3 s-1. Like Mr 54,000 scu-PA, it causes significant lysis of a 125I-labeled fibrin clot in human plasma with relatively less fibrinogen breakdown as compared to urokinase. scu-PA-32k, which also has conserved fibrin specificity, represents a molecular variant which may be more suitable for large scale production as a fibrin-specific thrombolytic agent by recombinant DNA technology.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Glu1-, Lys77-, Val442-, and Val561-plasmin: a comparative study

Thermodynamic and kinetic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human Glu1-, Lys77-, Val442- and Val561-plasmin (EC 3.4.21.7) have been determined between pH 3.0 and 9.5, and from 5.0 to 45.0 degrees C. The inhibitor-binding properties to human Glu1-, Lys77-, Val442- and Val561-plasmin suggest a possible role of BPTI in modulating plasmin activity when the inhibitor is used therapeutically.     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.     

Trypsin-like serine proteinase action: determination of the catalytic parameters KS, k+2 and k/3 under conditions where the substrate exceeds the enzyme concentration

A method for the determination of the catalytic parameters Ks, k+2 and k+3 describing trypsin-like serine proteinase action has been developed from the quantitative analysis of the kinetics of hydrolysis of two specific chromogenic substrates, N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester, catalyzed by porcine pancreatic betta-kallikrein B, bovine betta-trypsin and human urokinase (Mr 54,000 species), under conditions where the concentration of the substrate exceeds that of the enzyme. Value of Ks, k+2 and k+3 have been estimated from the effect of substrate concentration on the apparent first-order rate constant of the time-course of the burst phase of p-nitrophenol release preceding the steady-state reaction.     

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some physico-chemical and enzymatic properties of tissue kallikrein from human urine

A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.