Theoretical and experimental approach of fuel gas and carbon black production from coal tar pitch by thermal plasma process.

The processing of coal tar pitch (CTP) to produce clean fuel gas and carbon black (CB) is studied in a plasma reactor equipped with a direct-current plasma torch. The composition of the gas produced and energy costs were estimated theoretically for the CTP pyrolysis and gasification processes by two oxidants, namely oxygen and water vapor. We have found that the main gaseous compounds obtained in the pyrolysis and gasification processes are hydrogen (H2), carbon monoxide (CO), and very often carbon dioxide (CO2). For the pyrolysis case, the mean value of the synthesis gas concentration reaches a major value of 98 vol.% (H2 - 81 vol.%, CO - 17. vol.%). However, only 23% of the initial CTP is transformed into gas phase at 1100 K and its content increases up to 37.4% at a temperature of 3000 K. For oxygen gasification, the syngas quantity is little less compared to the pyrolysis case and attains 96.6 vol.% (H2 - 26.5 vol.%, CO - 70.1 vol.%) for T > 1100 K. An intermediate syngas content for the water steam gasification is 97.8 vol.% (with H2 - 55.8 vol.% and CO - 42.0 vol.%). The CB produced was composed of well-defined spherical particles of 30-nm size. Furthermore, it is composed of carbon (98.2%), and followed by oxygen (1.8%) with a surface area of 97 m2 g-1. The thermal plasma system shows high efficiency in conversion of CTP into high-value-added products.

Keto analogues and amino acids supplementation induces a decrease of white blood cell counts and a reduction of muscle damage during intense exercise under thermoneutral conditions.

This study evaluated the acute effect of keto analogue and amino acid (AA-KAAA) supplementation on both white blood cell counts and the established biomarkers of muscle damage during exercise under thermoneutral conditions. Sixteen male cyclists received a ketogenic diet for two days and were divided into two equal groups: a group taking AA-KAAA (KA) or a control group (PL). The athletes performed a two hour cycling session followed by a maximum incremental test until voluntary exhaustion (VExh). Blood samples were obtained at rest and during exercise for further hematological and biochemical analyses. Exercise-induced ammonemia increased in the PL group at VExh (75%) but remained unchanged in the KA group. Both groups exhibited a significant increase in leukocyte and neutrophil counts of ∼85% (∼13 × 109 L-1), but the shape of the lymphocytes and the eosinophil counts suggest that AA-KAAA supplementation helps prevent lymphocytosis. AA-KAAA supplementation induced a decrease in creatine kinase and aspartate aminotransferase levels at VExh while showing a significant decrease in lactate dehydrogenase at 120 min. We found that AA-KAAA supplementation decreases both the lymphocyte count response in blood and the established biomarkers of muscle damage after intense exercise under a low heat stress environment.

Comparison of human and porcine tissue kallikrein substrate specificities.

Little is known about the species specificity of tissue kallikrein-kininogen interaction since the kinetic parameters for Lys-bradykinin release from kininogen by tissue kallikreins from different animal species have not been reported. We have now determined the kinetic parameters for hydrolysis by human and porcine tissue kallikrein, hK1 and pK1, respectively (Berg et al., 1992) of two series of intramolecularly quenched fluorogenic peptides having the sequences that flank the scissile Arg-Ser or Met-Lys bond in human and bovine kininogen. Results have shown that peptides having sequences from human kininogen are better substrates for hK1 and peptides derived from bovine kininogen are better substrates for pK1. Kinetic data for hydrolysis of the Arg-Ser bond showed that differences in the interaction of residue(s) in positions P2'-P10' contribute to the efficiency of the cleavage and may be responsible for differences in their susceptibilities to the two kallikreins. Significant variations in the kinetic data were observed for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension at sites P3-P9. The highest k(cat)/Km value in the hydrolysis of Abz-[Gln370-Gln381]-bkng-EDDnp by pk1 demonstrates an important interaction of subsites S5-S4 with Gln and Thr residues in the bovine kininogen segment. A Gln370-Gln391 bovine kininogen fragment used to study the cleavage of both Met-Lys and Arg-Ser bonds in the same molecule confirmed the importance of an extended interaction site for species specificity among tissue kallikreins.

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 reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin, Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/Km of 12400 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 hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments with Abz-TSVIRRPQ-EDDnp, which is resistant to most kallikreins, and Abz-TSVIRRVQ-EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki values similar to the Km values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/Km = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semipurified sample of submandibular 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-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.

Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites.

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in Nalpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (Ki 0.4 microM) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The Ki value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine], was 0.08 microM for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo.

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3-200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 microM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 microM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 microM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 microM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 microM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 microM; ESP1), Bz-F-F-pNa (Ki = 0.4 microM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 microM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 microM; ESP10) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.

Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments.

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P'4 to P'11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P'11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.

Proteolytic specificity of two hemorrhagic factors, LHF-I and LHF-II, isolated from the venom of the bushmaster snake (Lachesis muta muta).

Two hemorrhagic metalloproteinases (LHF-I and LHF-II) were previously isolated from Lachesis muta muta (bushmaster snake) venom. The proteolytic activities of these hemorrhagic factors and of the crude venom were investigated using as substrate the oxidized B-chain of bovine insulin. LHF-II cleaves the Ala14-Leu15 bond of insulin B-chain very rapidly and the Phe24-Phe25, His10-Leu11 and His5-Leu6 more slowly, whereas LHF-I hydrolyzed only the Ala14-Leu15 bond. Both hemorrhagic factors cleaved the Leu-Leu bond in the fluorogenic peptide Abz-Pro-Leu-Gly-Leu-Leu-Gly-Arg-EDDnp. When the insulin B-chain was incubated with crude venom previously treated with 2.5 mM PMSF, the Ala14-Leu15 bond was also rapidly cleaved. In addition, the hemorrhagic activity and the digestion of casein remained unaltered. Both hemorrhagic and proteolytic activities were inhibited when the crude venom was treated with EDTA, confirming that only metalloproteinases are responsible for these activities. The hydrolysis of insulin B-chain and the fluorogenic heptapeptide by these proteinases was found to be in inverse relationship to their hemorrhagic activities.

Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins.

Kinetic data for the hydrolysis by human tissue kallikrein of fluorogenic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group and different leaving groups demonstrate that interactions with the S'1, S'2 and S'3 subsites are important for cleavage efficiency. In addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond [Abz-M-I-S-L-M-K-R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'1, P'2 and P'3 showed that (a) the presence of a proline residue at P'3 and the interactions with the tissue kallikrein-binding sites S2 to S'2 are determinants of Met-Lys bond cleavage and (b) residues P3, P4 and/or P5 arc important for cleavage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstrated that lysyl-bradykinin-releasing tissue kallikreins also have a primary specificity for phenylalanine. The replacement of arginine by phenylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykinin-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.

Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.

The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.

Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins.

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

Identification of receptors in the liver that mediate endocytosis of circulating tissue kallikreins.

The liver plays an important role in the clearance, by receptor-mediated endocytosis, of circulating glycoproteins. It has been demonstrated that tissue kallikreins, which are acid glycoproteins, circulate in plasma, where they are poorly inhibited by plasma proteins. We have shown that the liver is the main organ that clears tissue kallikreins from the circulation. We now report the identification of receptors involved in this clearance. Using a perfused rat-liver system, and as models, pig pancreatic (PPK) and horse urinary (HoUK) kallikreins, we have found that: (a) the binding of PPK to the perfused liver was inhibited by 50 mM methyl alpha-D-mannoside and 20 microM mannan, was partially inhibited by 50 mM mannose and was unaffected by 1.5 microM asialofetuin; (b) binding of HoUK to the perfused liver was inhibited by 1.5 microM asialofetuin, 50 mM galactose and 50 mM lactose and was unaffected by 50 mM mannose; (c) the clearance rate of both kallikreins followed the equation y = a.xb; (d) their binding was Ca2+-dependent and their clearance was inhibited by 3 mM chloroquine and 10 mM methylamine. Using isolated liver cells and tritiated HoUK, we calculated that 500,000 receptors/cell were present and the Scatchard plot showed that there were two apparent affinity constants: 0.24.10(9) l/M) (high-affinity) and 0.3.10(8) l/M (low-affinity). These results show that PPK is recognized by a liver mannose receptor and HoUK by the galactose receptor. The liver uptake of native and circulating tissue kallikreins thus emerges as a mechanism by which their levels in plasma are regulated.

Horse urinary kallikrein, I. Complete purification and characterization.

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.

Horse urinary kallikrein, II. Effect of subsite interactions on its catalytic activity.

The effect of secondary-subsite interactions on the catalytic efficiency of horse urinary kallikrein was studied using as substrates oligopeptides and peptidyl-4-nitroanilides with L-Arg at P1. The known secondary specificity of tissue kallikreins for hydrophobic residues at P2 was also demonstrated for horse urinary kallikrein and a higher preference of this enzyme for L-Phe over L-Leu at P2 was evident. Interaction of subsites S3 with D-Pro and D-Phe enhanced the catalytic efficiency but tripeptidyl-4-nitroanilides with acetyl-D-Pro, L-Pro and acetyl-L-Pro at P3 were no better substrates than acetyl-dipeptidyl-4-nitroanilides. The importance of the leaving group for the catalysis was proved by higher kcat/Km values for the peptides in relation to peptidyl-4-nitroanilides containing a common acyl-chain. The low kcat value for the peptide with L-Pro at P'2 stresses the importance of a hydrogen bond between P'2 amide and the carbonyl group at S'2. One L-arginine residue at the leaving group, specially at the P'2 position, decreases the value of the apparent Km. This effect resulting of side-chain interactions with S'2, is impaired by a second L-Arg at P'1.

Substrate activation of porcine pancreatic kallikrein by N alpha derivatives of arginine 4-nitroanilides.

Hydrolysis of several N alpha-substituted L-arginine 4-nitroanilides with porcine pancreatic kallikrein was studied under different conditions of pH, temperature, and salt concentration. At high substrate concentrations a deviation from Michaelis-Menten kinetics was observed with a significant increase in the hydrolysis rates of almost all substrates. Kinetic data were analyzed on the assumption that porcine pancreatic kallikrein presents an additional binding site with lower affinity for the substrate. Binding to this auxiliary site gives rise to a modulated enzyme species which can hydrolyze an additional molecule of the substrate through a second catalytic pathway. The values of both Michaelis-Menten and catalytic rate constants were higher for the modulated species than for the free enzyme, suggesting a mechanism of enzyme activation by substrate. Kinetic data indicated similar substrate requirements for binding at the primary and auxiliary sites of the enzyme. Tris(hydroxymethyl)aminomethane hydrochloride and NaCl were shown to alter the kinetic parameters of the hydrolysis of N alpha-acetyl-L-Phe-L-Arg 4-nitroanilide by porcine pancreatic kallikrein but not the enzyme activation pattern (ratio of the catalytic constants for the activated and the free enzyme forms). Similar observations were made when the hydrolysis of D-Val-L-Leu-L-Arg 4-nitroanilide was studied under different pH and temperature conditions.

Tetrapeptide substrates for the discrimination among kallikreins and other trypsin-like serine proteinases.

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.

Receptor-mediated clearance of tissue kallikreins by rat liver.

The exsanguinated, isolated and perfused rat liver clears from the perfusate, at comparable rates, some native tissue kallikreins: human and horse urinary as well as hog pancreatic; the clearance rates were dependent on the initial enzyme concentration in the perfusing fluid. Contrary-wise, rat urinary kallikrein was cleared at negligible rates. Neuraminidase pretreatment of these four kallikreins did not alter their clearance rates. Horse urinary kallikrein binding to isolated prefixed hepatocytes was calcium-dependent and inhibited by asialofetuin (but not by fetuin) and some sugars; these characteristics are compatible with the interpretation that this native tissue kallikrein is recognized by the hepatocyte asialoglycoprotein-receptor. It was calculated that there are about 300,000 receptor sites per cell either using perfusion experiments at 4 degrees C or isolated hepatocytes.

Tissue kallikreins and related enzymes: characterization by model oligopeptides.

The first purpose of this work was to obtain direct evidence that tissue kallikreins cleave arginyl bonds when the leaving group is Arg-Val, and on the contrary, do not split them when it is Arg-Pro; the second aim was to ascertain whether this specificity could be used as a criterion, for characterizing tissue kallikreins. Two tetrapeptides Ac-Phe-Arg-Arg-Val-NH2 and Ac-Phe-Arg-Arg-Pro-NH2 were synthesized by the solid phase method and purified to homogeneity. They were used as substrates for homogeneous preparations of tissue and plasma kallikreins, as well as for some related serine proteases. Products identification and kinetic analyses were made by HPLC.

Active-site titration of horse urinary kallikrein.

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.