Inhibitory Activities of Aromatic Amino Acid Esters and Peptides against Ovalbumin Permeation through Caco-2 Cell Monolayers

Trp, Phe, and Tyr ethyl esters and their dipeptides with Gly at the C-terminals inhibited ovalbumin (OVA) permeation through Caco-2 monolayers. The inhibitory activity of Trp ethyl ester was the highest at near the concentration of 10^-6 M. It was suggested that Trp ethyl ester inhibited transcellular permeation of OVA.     

An active compound against allergen absorption in hypoallergenic wheat flour produced by enzymatic modification

Hypoallergenic wheat flour produced by modification with cellulase and actinase showed inhibitory activity against ovalbumin permeation in an in vitro model by using the Caco-2 cell monolayer. The activity was found in the cellulase preparation used for producing the flour. An active compound was isolated by HPLC and identified as Trp-Ser-Asn-Ser-Gly-Asn-Phe-Val-Gly-Gly-Lys by 1H-NMR data and Edman degradation. The undecapeptide, some oligopeptides with the N-terminal sequences and Trp ethyl ester showed activity at 10(-7) M, acetyl Trp being active at 10(-2) M. These data suggest that the Trp residue without a free carboxyl group would be required for the inhibitory activity of ovalbumin absorption through the intestinal tract.     

Enzyme-modified cheese exerts inhibitory effects on allergen permeation in rats suffering from indomethacin-induced intestinal inflammation

We have found that an enzyme-modified cheese (EMC) inhibited the permeation of allergens such as ovalbumin (OVA), using Caco-2 cells as an in vitro intestinal epithelial model. In addition, NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of alphas(2)-casein) was isolated from EMC and identified as one of the responsible peptides for this inhibitory activity (Tanabe et al., J. Agric. Food Chem., (2007)). In this study, we aimed to clarify the mechanism by which NPWDQ inhibited allergen permeation in vitro, and also to evaluate the effects of EMC on allergen permeation in vivo. Intestinal permeability for both fluorescein isothiocyanate conjugated dextran and horseradish peroxidase was decreased in Caco-2 cells by the addition of NPWDQ, indicating that NPWDQ might inhibit both paracellular and transcellular transports. Next, intestinal inflammation was induced by subcutaneous injections of indomethacin to rats. When OVA was injected into the jejunal and ileac loops of indomethacin-administered rats with and without NPWDQ, it was found that the addition of NPWDQ effectively diminished OVA permeation from both loops. Although the plasma OVA concentration of indomethacin-administered rats after oral OVA challenge was markedly elevated over that of normal rats, supplemental administration of EMC to the rats effectively suppressed OVA permeation. These results suggest that EMC is useful for the prevention of food allergy by inhibiting allergen permeation probably by enforcing the intestinal barrier.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis

3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure–activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.     

Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

Non-oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5, 7 and 11 mS, respectively. The major synthase was purified 8900-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-human albumin affinity-chromatographies with an overall yield of 25%. SDS-PAGE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (Vmax = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). Km values for oleate, linoleate, arachidonate, palmitate and stearate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised or two identical, or nearly identical, subunits (Mr = 26000).     

Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.17) was found to depend greatly on pH: it was not observed at pH's above 7, but it was observed at pH's lower than 6. In addition, at pH's between 6 and 7 the NBS modification showed a delta epsilon pH profile similar to a "titration curve," giving a pK (congruent to 6.5) nearly equal to the pK (congruent to 6.2) of a catalytic residue, Glu 35. The stopped-flow chemical (NBS) modification of N-acetyl-L-tryptophan ethyl ester, a model compound of Trp 62, does not depend on pH at the pH's examined, approximately 3.5-8.5. These experimental results suggest that a change in the state of Trp 62 at Subsite C is induced by protonation-deprotonation of an ionizable residue, which could be Glu 35 (catalytic site), indicating that stopped-flow NBS modification is a good probe for detection of changes in the micorenvironment around the tryptophan residue(s) of enzymes.     

Analysis of proteases involved in phagocytic activity of macrophages through the use of various amino acid esters

Inhibitory effects of various amino acid esters on the phagocytic activity of guinea pig peritoneal macrophages were studied with sensitized 51Cr-sheep erythrocytes (51Cr-EAb) as well as 125I-alpha-amylase complexed with homologous IgG2 antibody (Ag-Ab complex). The intracellular uptake of 51Cr-EAb was markedly inhibited by N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt) and N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), but not by N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), N-alpha-acetyl-L-arginine methyl ester (Ac-Arg-OMe), N-alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) or N-alpha-acetyl-L-lysine methyl ester (Ac-Lys-OMe). When phagocytosis of the Ag-Ab complex was assayed by measuring the amount of digested products released from macrophage cells, Ac-Tyr-OEt also inhibited it as markedly as Ac-Phe-OEt, Ac-Trp-OEt, and Bz-Tyr-OEt did, whereas Bz-Arg-OEt again did not show any effect. The results of analysis of the intracellular fate of the Ag-Ab complex taken up by macrophages through the use of analytical density gradient fractionation of the homogenized cells suggest that Ac-Phe-OEt inhibits the ingestive process since the distribution of Ag-Ab complex showed a single peak, closely accompanying the plasma membrane. Ac-Tyr-OEt, on the other hand, caused a marked accumulation of Ag-Ab complex in the lysosome fraction, reflecting the inhibition of intralysosomal digestion of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semisynthetic ‘acid-esterase’: Conformational modification of ribonuclease

Bovine pancreatic ribonuclease has been modified by exposure to acidic conditions, addition of indole propionic acid and crosslinking with glutaraldehyde. The ‘acid-esterase’ generated was purified up to 100-fold by ammonium sulphate fractionation and gel filtration on Biogel P-30. The partially purified acid-esterase hydrolysed tryptophan ethyl ester (TrEE) and $\text{N-}\text{benzoyl}\text{-}\text{l}\text{-}\text{arginine}$ ethyl ester (BAEE) effectively at pH 6.0–6.3, but it had very little activity towards glycine ethyl ester and lysine ethyl ester. Hydrolysis of TrEE was competitively inhibited by tryptophan. The acid-esterase exhibited amidase activity towards benzoyl-l-arginine p-nitroanilide.     

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: A gene for alcohol-induced cardiomyopathy

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.     

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.     

The effect of calcium ions on the hydrolysis of benzoylarginine ethyl ester by porcine enteropeptidase.

Calcium ions are shown to have a marked pH-dependent effect on the kinetics of benzoyllarginine ethyl ester hydrolysis by porcine enteropeptidase (EC 3.4.21.9). Below pH 6.0, calcium ions stimulate benzoylarginine ethyl ester hydrolysis but inhibit this activity above pH 6.0. This effect is mainly on the Km for benzoylarginine ethyl ester. At pH 5.3, 2mM calcium ions reduce the Km for benzoylarginine ethyl ester from 0.31 mM to 0.26 mM while at pH 6.5 the Km increases four-fold from 0.035 mM to 0.12 mM in the presence of calcium ions. Enteropeptidase activity is not inhibited by ethylenediaminetetra-acetate indicating that calcium ions are a non-essential cofactor for benzoylarginine ethyl ester hydrolysis.     

Production of ethyl acetate from dilute ethanol solutions by Candida utilis

The conversion of ethanol to ethyl acetate has an advantage as a method of ethanol recovery since ethyl acetate is amenable to simple solvent extraction. The potential of Candida utilis in this conversion was studied. The kinetics of accumulation of ethanol and ethyl acetate in glucose-grown C. utilis showed that ester formation resulted from ethanol utilization under appropriate aeration and was inhibited by Fe(3+) supplementation. Candida utilis converted ethanol to ethyl acetate optimally at pH 5.0-7.0. The five-hour rate of ester production increased as the ethanol concentration increased to 10 g/L, and rapidly declined to zero at concentrations exceeding 35 g/L. Thus, C. utilis has potential to recover dilute ethanol in the form of ethyl acetate.     

<Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">Ex Vivo</Emphasis> Evaluation of Novel Curcumin-Loaded Excipient for Buccal Delivery

This study aimed to develop a mucoadhesive polymeric excipient comprising curcumin for buccal delivery. Curcumin encompasses broad range of benefits such as antioxidant, anti-inflammatory, and chemotherapeutic activity. Hyaluronic acid (HA) as polymeric excipient was modified by immobilization of thiol bearing ligands. L-Cysteine (SH) ethyl ester was covalently attached via amide bond formation between cysteine and the carboxylic moiety of hyaluronic acid. Succeeded synthesis was proved by H-NMR and IR spectra. The obtained thiolated polymer hyaluronic acid ethyl ester (HA-SH) was evaluated in terms of stability, safety, mucoadhesiveness, drug release, and permeation-enhancing properties. HA-SH showed 2.75-fold higher swelling capacity over time in comparison to unmodified polymer. Furthermore, mucoadhesion increased 3.4-fold in case of HA-SH and drug release was increased 1.6-fold versus HA control, respectively. Curcumin-loaded HA-SH exhibits a 4.4-fold higher permeation compared with respective HA. Taking these outcomes in consideration, novel curcumin-loaded excipient, namely thiolated hyaluronic acid ethyl ester appears as promising tool for pharyngeal diseases.     

Purification and properties of the thermostable acid protease of Penicillium duponti.

An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.     

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester.

The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: Influence of aeration and inhibition of yeast growth by ethyl acetate

The ability of the yeast Kluyveromyces marxianus to convert lactose into ethyl acetate offers good opportunities for the economical reuse of whey. The formation of ethyl acetate as a bulk product depends on aerobic conditions. Aeration of the bioreactor results in discharge of the volatile ester with the exhaust gas that allows its process‐integrated recovery. The influence of aeration (varied from 10 to 50 L/h) was investigated during batch cultivation of K. marxianus DSM 5422 in 0.6 L whey‐borne medium using a stirred reactor. With lower aeration rates, the ester accumulated in the bioreactor and reached higher concentrations in the culture medium and the off gas. A high ester concentration in the gas phase is considered beneficial for ester recovery from the gas, while a high ester concentration in the medium inhibited yeast growth and slowed down the process. To further investigate this effect, the inhibition of growth by ethyl acetate was studied in a sealed cultivation system. Here, increasing ester concentrations caused a nearly linear decrease of the growth rate with complete inhibition at concentrations greater than 17 g/L ethyl acetate. Both the cultivation process and the growth rate depending on ethyl acetate were described by mathematical models. The simulated processes agreed well with the measured data.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.