benzoyl-L-arginine Ethyl Ester Research Articles

Kinetics of human peptidylarginine deiminase 2 (hPAD2) — Reduction of Ca2+ dependence by phospholipids and assessment of proposed inhibition by paclitaxel side chains

Multiple sclerosis is a complex human neurodegenerative disease, characterized by the active destruction of the insulating myelin sheath around the axons in the central nervous system. The physical deterioration of myelin is mediated by hyperdeimination of myelin basic and other proteins, catalysed by the Ca2+ -dependent enzyme peptidylarginine deiminase 2 (PAD2). Thus, inhibition of PAD2 may be of value in treatment of this disease. Here, we have first characterized the in vitro kinetic properties of the human peptidylarginine deiminase isoform 2 (hPAD2). Phosphatidylserine and phosphatidylcholine reduced its Ca2+ dependence by almost twofold. Second, we have explored the putative inhibitory action of the methyl ester side chain of paclitaxel (TSME), which shares structural features with a synthetic PAD substrate, viz., the benzoyl-L-arginine ethyl ester (BAEE). Using the known crystallographic structure of the homologous enzyme hPAD4 and in silico molecular docking, we have shown that TSME interacted strongly with the catalytic site, albeit with a 100-fold lower affinity than BAEE. Despite paclitaxel having previously been shown to inhibit hPAD2 in vitro, the side chain of paclitaxel alone did not inhibit this enzyme's activity.

A heat-stable trypsin from the hepatopancreas of the cuttlefish ( Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine- p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins. The trypsin kinetic constants, K m and k cat for BAPNA, were 0.064 mM and 2.32 s −1, respectively, while the catalytic efficiency k cat/ K m was 36.3 s −1 mM −1.

Enhanced Proteolytic Activity of Covalently Bound Enzymes in Photopolymerized Sol Gel

Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of N(alpha)-benzoyl-l-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N alpha-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20 degrees C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20 degrees C after 16 h of incubation time.

Nitric oxide and cyclic GMP-mediated protein secretion from cultured lacrimal gland acinar cells.

Nitric oxide (NO) donors and NO synthase (NOS) substrates were tested for their use to stimulate protein secretion from cultured lacrimal gland acinar cells, through activation of guanylate cyclase. Rabbit lacrimal gland epithelial cells (RLG cells) were incubated with NO donors and/or NOS substrates and the protein released into culture medium was determined with bicinchoninic acid assay. Guanylate cyclase activation by NO precursors was determined by measurement of c-GMP produced. Both NO donors and NOS substrates were able to stimulate protein release from RLG cells. Among 6 compounds studied, sodium nitroprusside, isosorbide dinitrate and N(a)-benzoyl L-arginine ethyl ester (BAEE) were most potent to release protein over 100% of the basal release. The guanylate cyclase activity was stimulated by these NO precursors and was inhibited by guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). NO donors and NOS substrates were able to stimulate protein release from RLG cells via activation of guanylate cyclase and c-GMP release, which was blocked by guanylate cyclase inhibitor, ODQ. It indicates that NO donors and NOS substrates could be used for the treatment of dry eye syndrome if the same holds true in dry eye animal models.

Surface Acoustic Wave (SAW)-Impedance Sensor for Kinetic Assay of Trypsin

The trypsin-catalyzed hydrolysis of benzoyl-l-arginine ethyl ester (BAEE) has been studied systematically at pH 7.8 and 32°C with a nonlinear regression model. The Michaelis constants covering the range from pH 7.0 to 8.6 were determined. The concentrations of enzyme isolated from pig pancreas were determined using the SAW-impedance sensor and the spectrophotometry, respectively. The experimental detection limit of trypsin was 0.3 mU/ml22mU/ml expresses milliunits per milliliter. One unit of enzyme is defined as the amount of trypsin required to hydrolyze 1 μmol of substrate per minute at 32°C and pH 7.8.and the recovery of the sensor system ranged from 94.7 to 105% (n= 6). The effects of temperature, pH, and some metal ions on the trypsin activity were investigated.

Proteinases Are Involved in Both DNA Fragmentation and Membrane Damage during CTL-Mediated Target Cell Killing

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N -benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsinlike and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.

γ-Carboxyglutamic Acid (Gla)-Domainless Blood Coagulation Factc IXa Species: Preparation and Properties1

To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)

β-Fibrinogenase from the venom of Vipera lebetina

An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and p I ∼ 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The K m and k cat values are for α-N- benzoyl- l-arginine ethyl ester (BAEE) 7.7 × 10 −5M and 43.8 sec −1, for p- tosyl- l-arginine methyl ester (TAME) 3.6×10 −4M and 39.8 sec −1 (pH 8.5, 25°C, and for α-N- benzoyl- dl-arginine -4- nitroanilide (BAPNA) 1.8×10 −4M and 0.94 sec −1 (pH 8.3, 25°C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys 12-Tyr 13, Arg 17-Arg 18 and Arg 18-Ala 19. In fibrinogen it cleaves Bβ-chain first and later also the Aα-chain.

The influence of pH on aggregation of human washed platelets induced by thrombin or collagen

The anti-aggregating activity of L-arginine and its analogues has been investigated in washed human platelets. The ability of these compounds to inhibit platelet aggregation induced by thrombin or collagen was mimicked by a change in the external pH of the buffer from pH 8.0 to pH 7.4. Of the several analogues tested, only benzoyl-L-arginine ethylester (BAEE) inhibited thrombin- but not collagen-induced platelet aggregation. In platelet-rich plasma, BAEE also inhibited platelet aggregation without inhibiting fibrin clot formation. These results suggest that the marked sensitivity of washed human platelets to small changes in the external pH may lead to misinterpretation of the anti-aggregatory potency of protonated test drugs. For work with human washed platelets a physiological salt solution with more buffering capacity than Krebs-bicarbonate (such as Tyrode-HEPES) is recommended.

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z Gly Pro Arg pNA, Z Gly Arg AMC, benzoyl- l-arginine ethyl ester (BAEE) and tosyl- l-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z Gly Pro pNA or AMC) were used. Only the K m value increased, while the V max value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent K m and V max values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. 1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. 4. Specific esterolytic activities of the kallikrein-like enzyme on N- tosyl- l- arginine methylester (TAME) and N- benzoyl- l- arginine ethylester (BAEE) are 109.5 and 23.6 μmol/min/mg, respectively. 5. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.

Purification and some properties of three serine carboxypeptidases from aspergillus niger

Three enzymes exhibiting peptidyl- l-amino acid hydrolase and esterase activities have been purified by immobilized metal-ion affinity chromatography and ion-exchange chromatography. The three enzymes were entirely free of the acid protease activity that normally exists along with them in the crude culture filtrates of Aspergillus niger. Although all three exo-peptidases possessed nearly identical molecular weights ( ca. 140 000), isoelectric points ( ca. 5.0) and other properties, their affinities for the two substrates tested, carbobenzoxy- l-Glu- l-Tyr and benzoyl l-arginine ethyl ester, differed. All three peptidases were inhibited by phenylmethanesulphony fluoride, indicating that they are serine carboxypeptidases. They were also inhibited by tosyl phenylalanine chloromethyl ketone, suggesting the presence of a histidyl residue in their active sites. The differences in the number of accessible histidyl residues on the enzyme surfaces could explain the differences in their retentions on Cu 2+—iminodiacetate—Sepharose 6B.

Characterization of two arginine ester hydrolases from Mojave rattlesnake ( Crotalus scutulatus scutulatus) venom

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0 – 8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or α- N-benzoyl- dl-arginine -p- nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed α-N- benzoyl- l-arginine ethyl ester (BAEE), α-N- benzoyl- l-arginine methyl ester (BAME), p- tosyl- l-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The K m values for each enzyme with α-N-benzoyl- l-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.

AM 1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities

The previously isolated female submandibular glycoprotein AM 1 (Nieuw Amerongen, A.V.; Vreugdenhil, A.P. and Roukema, P.A. (1977) Biochim. Biophys. Acta 495, 324–335) has been shown to have hydrolytic activity using N-α- benzoyl- l-arginine ethylester (BAEE) and Chromozym R PK as a substrate. AM 1 can be secreted in vivo by isoproterenol, and to a lesser extent by carbamylcholine and phenylephrine. Based on BAEE as a substrate, AM 1 has an optimum pH of 7.8. In female submandibular glands, about one-third of total esterolytic activity resided in glycoprotein AM 1, but in male submandibular glands, less than 3%. The K m value of glucoprotein AM 1 is 50 μM and its V max is 117 μmol/min per mg glycoprotein AM 1. The enzymatic activity is not inhibited by Ca 2+, Mg 2+ and Na +, slightly by Cu 2+ and strongly by Hg 2+ and phenylmethylsulfonyl fluoride, Glycoprotein AM 1 is capable of hydrolyzing Chromozym R PK with a turnover value 20-fold lower than that for BAEE. With Chromozym R PK as a test substrate, glycoprotein AM 1 was purified by a factor 11. With this substrate, glycoprotein AM 1 has an optimum pH between 6.6 and 7.6 Also with chromozym R PK as a substrate, the submandibular glands of female mouse showed a much higher activity of glycoprotein AM 1 than the submandibular glands of the male mouse. About 75% of all enzymatic activity of female submandibular glands resided in glycoprotein AM 1 and in male submandibular glands 22%. The K m value is 57 μM and its V max 6.7 μmol/min per mg glycoprotein AM 1. From the biochemical characteristics and the localization of glycoprotein AM 1, it has been conclude that glycoprotein AM 1 is not identical to any of the other described murime submandibular esteroproteinases, such as kallikrein, γ-subunit of the nerve growth factor, proteinase A and proteinase F.

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 N- benzoyl- l - 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.

Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase

1. 1. A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake ( Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. 2. 2. The Aα chain of fibrinogen was first split and the Bβ chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in Aα and Bβ chains of fibrinogen must be different from thrombin sites. 3. 3. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME ( p- toluenesulfonyl- l-arginine methyl ester), BAEE ( N- benzoyl- l-arginine ethyl ester). TLME ( N-tosyllysine methyl ester) but not BAA ( N-benzoylarginine amide), TAA ( N-tosylarginineamide) or ATEE ( N-acetyltyrosine ethyl ester). 4. 4. The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). 5. 5. Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet capillary permeability increases as shown by use of Evans blue dye.

Modification of papain with tetranitromethane.

Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BAEE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.

Human Leukocyte Inhibitory Factor (LIF): Use of Benzoyl L-Arginine Ethyl Ester to Detect LIF Activity

Abstract Human leukocyte inhibitory factor (LIF), a mediator elaborated by activated lymphocytes, has recently been shown to have the properties of an esterase. Further more, the LIF preparation contains an esteratic principle that hydrolyzes the amino acid ester, benzoyl arginine ethyl ester (3H-BAEE). In the present experiments, we sought to determine whether the esterase and biologic activities in the LIF preparation were due to the same or different molecules. This was accomplished by means of two series of experiments. With one approach, LIF was initially subjected to gel filtration and subsequently to polyacrylamide gel electrophoresis, sucrose density gradient electrophoresis, and affinity chromatography. The fractions obtained from each procedure were analyzed for biologic and esterase activities. The results indicate that both activities co-chromatographed with each technique. In addition, there are contaminating esterases present in the LIF preparation that can hydrolyze 3H-BAEE, some activity(ies) of which are eliminated by purification. The other approach directly demonstrated whether BAEE could serve as a substrate for LIF by substrate-competition experiments. BAEE and other structurally related amino acid esters were co-incubated with LIF and the ability of the former to protect the latter against inactivation by esterase inhibitors, or block biologic LIF activity on polymorphonuclear leukocytes was ascertained. It was shown that BAEE specifically prevented the destruction of LIF by diisopropylphosphofluoridate (DFP) and phenyl methyl sulfonyl fluoride (PMSF). Moreover, BAEE in a dose-dependent fashion, blocked LIF activity on PMN leukocytes in vitro when added to the culture supernatants. Taken together, these results strongly suggest that BAEE may serve as a substrate for LIF and point to the eventual development of a biochemical assay for this lymphocyte mediator.

Effect of kininogen on the activity of the kallikreinogen-kallikrein system of human blood serum

In experiments with kallikreinogen, kallikrein and kininogen preparations from human blood serum partially purified on QAE-Sephadex A-50, CM-Sephadex and Sephadex G-200 it was established that N- benzoyl- l- arginine ethyl ester (BAEE)-esterase activity of kallikrein and the process of autoactivation of kallikreinogen are inhibited by kininogen, especially by its high-molecular form.

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

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 K m for benzoylarginine ethyl ester. At pH 5.3, 2 mM calcium ions reduce the K m for benzoylarginine ethyl ester from 0.31 mM to 0.26 mM while at pH 6.5 the K m increases four-fold from 0.035 mM to 0.12 mM in the presence of calcium ions. Enteropeptidase activity is not inhibited by ethylenediaminetetraacetate indicating that calcium ions are a non-essential cofactor for benzoylarginine ethyl ester hydrolysis.