Purification and characterization of ostrich pancreatic secretory trypsin inhibitor

Ostrich pancreatic secretory trypsin inhibitor was isolated and purified using acid extraction, salt fractionation, SP-Sephadex C-50 and QAE-Sephadex A-25 chromatography and RP-HPLC. The amino acid sequence of ostrich PSTI showed it is a single peptide chain containing 69 amino acid residues with the highest homology between ostrich and chicken PSTI. The molecular weight, as determined by electronspray mass spectrometry and from amino acid sequence data, is 7650 Da. The isoelectric point of ostrich PSTI was found to be 5.7. Ostrich PSTI specifically inhibited ostrich and commercial bovine trypsin with K_i values of 8.0 × 10^?9 and 2.4 × 10^?7M, respectively, while no inhibitory effects were observed with other serine proteases. © Munksgaard 1996.     

Isozymes of rat urinary kallikrein.

Two isozymes of rat urinary kallikrein (A and B) have been purified and studied in detail. The enzymes were separated by chromatography on DEAE-cellulose and distinguished by electrophoresis and electrofocusing in polyacrylamide slab gels. The electrophoretic mobilities were not altered by treatment with neuraminidase but were shifted toward the cathode after complexing with aprotinin. Molecular weights of rat urinary kallikreins A and B were estimated to be 36,500 and 35,500, respectively, by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. The enzymes did not differ in pH optimum, specific activity or substrate specificity, but differed slightly in heat stability. They were not distinguishable biologically, nor immunologically using an antiserum generated in a sheep against kallikrein B. Both kallikreins were activated by deoxycholate and several nonionic detergents. α-N-tosyl-L-arginine methyl ester (Tos-L-Arg OMe) hydrolysis by both forms was inhibited by aprotinin and stimulated by ovomucoid and lima bean trypsin inhibitors, whereas Tos-L-Arg-OMe hydrolysis was stimulated by soybean trypsin inhibitor at low concentrations and inhibited at high concentrations. Kinetic measurements of α-N benzoyl-L-arginine ethyl ester (Bz-L-Arg-OEt) hydrolysis by the two enzymes showed no significant differences in substrate affinity or in maximal velocity.     

Inhibition of serine proteases by steroids

Proteolysis of ¹⁴C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with K_i values of 9.9 × 10^?5 M for triamcinolone (9-fluoro-11β, 16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 × 10^?4 M for cortisol (11β,17α,21-trihydroxypregn-4-ene-3,20-dione), 3.7 × 10^?4 M for testosterone (17β-hydroxy-4-androsten-3-one), 5.0 × 10^?4 M for dexamethasone (9-fluoro-11β,17,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione), and 1.0 × 10^?4 M for epicortisol (11α,17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind ³H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (l-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.     

ISOLATION OF A TRYPSIN-LIKE ENZYME FROM. STREPTOMYCES PAROMOMYCINUS (PAROMOTRYPSIN) BY AFFINITY ADSORPTION THROUGH KUNITZ INHIBITOR-SEPHAROSE

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 u mg^-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000–25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Arg-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhibited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.     

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 alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-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 Km values for each enzyme with alpha-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.     

Inhibition of bovine trypsin by the phosphorylated N-terminal part β(1–105) of β-casein

The sensitivities of the R₂₅-I₂₆ bond on bovine β-casein and on its N-terminal fragment β(1–105) to trypsin digestion were compared by monitoring the liberation of the β(1–25) product. It was shown that this peptide bond was poorly and slowly hydrolysed on β(1–105), while it is highly susceptible to trypsin attack when whole protein is used as substrate. The marked resistance of β(1–105) is linked to its inhibitory effect on trypsin activity (apparent K′_i= 1.2 × 10^?6, M), as demonstrated by using a related chromogenic substrate. Indeed, a preincubation step of trypsin with β(1–105) leads to a more pronounced inhibitory effect. The progress curves obtained with and without preincubation show that β(1–105) acts as a slow binding inhibitor on trypsin activity. These findings promise further insight into the action and the regulation of proteolytic enzymes. © Munksgaard 1997.     

Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.     

Plasma kallikrein-generating activity evoked by rat peritoneal-fluid mast cells following treatment with epinephrine, 8-bromo-cyclic 3',5'-guanosine monophosphate or compound 48/80

Acting in a dose-dependent fashion, l-epinephrine caused rat peritoneal-fluid cells to rapidly deplete rat plasma kininogen in vitro; 8-bromo-cyclic 3′,5′-guanosine monophosphate (8-Br-cGMP) behaved similarly; N⁶-2¹-O-dibutyril-cyclic 3′,5′-adenosine monophosphate (di Bu-cAMP) inhibited this effect of epinephrine or 8-Br-cGMP. After fractionation of peritoneal-fluid cells by differential centrifugation, this kininogen-depleting activity was observed only in mast cells; eosinophils, lymphocytes, and monocytes were inactive. Epinephrine-treated mast cells were ablt to hydrolyze the trypsin substrates N-p-toluenesulfonyl-arginine-methyl-ester (TAME) or N-benzoyl-arginine-ethyl-ester and to generate the capacity to hydrolyze these substrates in rat plasma; because this activity accompanied kininogen depletion, it was attributed to plasma kininogenase (plasma kallikrein). Diisopropyl-fluorophosphate (DFP) inhibited the mast cell esterase activity toward TAME but did not prevent activated cells from depleting plasma kininogen. Thus, mast cell-bound argininase ester esterase may not have been necessary for the activation of plasma kininogenase. Mast cell heparin, exposed following epinephrine or 8-Br-cGMP treatment, may have been the activator of plasma kallikrein. Unlike DFP, Trasylol [polyvalent bovine proteinase inhibitor (BPTI)] inhibited both mast cell esterase and kininogen-depleting activity. This inhibitor may have acted on mast cells both as a heparin antagonist and and a non-specific esterase inhibitor. Compound $\text{48}\text{80}$, at concentrations causing 40 per cent release of mast cell histamine, failed to cause mast cells to exhibit the ability to active plasma kallikrein. At high concentrations it activated the kininogen depleting action of mast cells, but to a lesser degree than did epinephrine or 8-Br-cGMP. These compounds did not release histamine; it may be concluded, therefore, that the ability to activate plasma kininogenase was present in non-histamine releasing mast cells.     

Synthesis and properties of cholecystokinin-releasing peptide (monitor peptide), a 61-residue trypsin inhibitor

A 61-residue cholecystokinin-releasing peptide (monitor peptide), which was obtained from rat pancreatic juice and found to stimulate pancreatic enzyme secretion, was recently reported to inhibit bovine trypsin and to possess epidermal growth factor (EGF)-like activities, at a concentration of about 10nM. However, monitor peptide is structurally different from the EGF family of growth factors. To investigate whether monitor peptide contains the supposed EGF-like activities, it has been synthesized together with its [Ala23, Ala47] analog. The purified peptides, which were fully characterized by a range of methods including Cf-252 ionization mass spectrometry and enzymatic digestion to establish the locations of disulfide linkages, were shown to belong to the pancreatic secretory trypsin inhibitor family and not to the EGF family. Neither synthetic monitor peptide nor its analog were able to compete with ¹²⁵I-EGF in A-431 cells or to stimulate growth of Swiss 3T3 and NRK 49F cells, up to 1 μM concentration. However, synthetic monitor peptide was as effective as the native product in the inhibition of trypsin. Replacement of the essential Arg23 in the [Ala23, Ala47]-analog led to loss of trypsin inhibition activity.     

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster)

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.     

Predicted three-dimensional structural models of venom serine protease inhibitors and their interactions with trypsin and chymotrypsin

Three homology models of trypsin and chymotrypsin inhibitor polypeptides from snake venom of Naja naja naja and Leaf-nosed viper in the unbound state and in complex with trypsin and chymotrypsin were built based on homology to bovine pancreatic trypsin inhibitor (BPTI). These venom inhibitors belong to the Kunitz-type inhibitor family, which is characterized by a distinct tertiary fold with three-conserved disulfide bonds. The general folding pattern in these trypsin and chymotrypsin inhibitor homology models is conserved when compared to BPTI. The respective orientations of the inhibitors bound to trypsin/chymotrypsin are similar to that of BPTI bound to bovine trypsin/chymotrypsin. The principal binding loop structure of the inhibitors fills the active site of enzymes in a substrate-like conformation and forms a series of independent main-chain and side-chain interactions with enzymes. In order to provide the possible fingerprints for molecular recognition at the enzyme-inhibitor interface, a detailed theoretical analysis of the interactions between the principal binding loop of these inhibitors and active site of trypsin/chymotrypsin is performed based on available crystal structural, site-directed mutagenetic, kinetic, and sequence analysis studies. Despite the variations present at different positions of the principal binding loop of trypsin and chymotrypsin inhibitor models from Leaf-nosed viper and cobra Naja naja naja, respectively (designated as LnvTI and NCI), there are favorable subsite binding interactions which are expected to exhibit equally potent inhibitory activity as BPTI. On the contrary, significant mutations at several secondary specificity positions in the Naja naja naja trypsin inhibitor (designated as NTI) are likely to affect different inhibitor-enzyme-subsites interactions. This may explain the observed increased inhibitory activity of this polypeptide on a structural basis.     

抑肽酶基因的克隆和表达

抑肽酶作为天然非特异性丝氨酸蛋白酶抑制剂，用途广泛。目前抑肽酶制品主要从牛肺中提取。用基因重组技术，将抑肽酶结构基因导入表达载体pGrxA，并在抑肽酶基因上游引入FXa识别位点。将重组质粒转化至Origami^TM中，用异丙基硫代β-D半乳糖苷(IPTG)诱导表达目的蛋白，产物以可溶性形式存在于胞内。将菌体超声破壁和离心，融合蛋白经分子筛层析和离子交换层析纯化后，用FXa切割该融合蛋白可得到N端不含多余氨基酸残基且与天然构像一致的活性抑肽酶。     

纳豆激酶的分离纯化及酶学性质研究

目的研究纳豆激酶分离纯化工艺及酶学性质。方法纳豆激酶发酵液的粗提物经Superdex 75凝胶色谱和聚丙烯酰胺凝胶电泳(PAGE)分离纯化,采用TAME法测定酶的活性,通过SDS-PAGE对纯化结果进行了检验。结果SDS-PAGE中显示单一色带,相对分子质量28000,以TAME为底物时纳豆激酶的米氏常数(Km)为35.47mmol/L,最适宜的温度37℃,最适宜pH为8.6。结论该分离纯化方法可以得到较纯的纳豆激酶。     

重组牛胰蛋白酶抑制剂对小鼠急性肝损伤的保护作用

目的观察重组牛胰蛋白酶抑制剂(rBPTI)对小鼠急性肝损伤的保护作用。方法小鼠60只随机分成6组:正常对照组、模型组、rBPTI的3个剂量组(分别为3、6、12×104kIU·kg-1)、抑肽酶组(6×104kIU·kg-1)。每天腹腔注射给药,7d后体外注射CCl4建立小鼠急性肝损伤模型。末次给药后眼眶取血测定小鼠血清ALT、AST活性,观察肝脏组织病理改变。结果各剂量rBPTI可不同程度降低小鼠血清ALT、AST活性,降低肝组织MDA含量,与模型组比较差异有统计学意义(P<0·05、P<0·01),同时不同程度减轻肝组织病理损伤。结论rBPTI对体外CCl4诱导的小鼠急性肝损伤具有保护作用,作用效果同天然BPTI相当。     

Involvement of Nitric Oxide and Peptides in the Inhibitory Non-Adrenergic,Non-Cholinergic (NANC) Response in Bovine Mesenteric Artery**

Abstract: The cyclic GMP mediated non-adrenergic non-cholinergic (NANC) relaxation in field stimulated bovine mesenteric artery and its modulation by various factors was studied. Electrical field stimulation of precontracted (Phe 2.5 μM or histamine 5 μM) bovine mesenteric arteries resulted in relaxations varying between 10-70% in different preparations. Tetrodotoxin (3 μM) completely blocked the inhibitory NANC response. Preincubation with high concentrations (100 μM ? 1 mM) of N^G-nitro-L-arginine for 15 min. significantly reduced the relaxation induced by electrical field stimulation. Blockade of cyclooxygenases and prostaglandin synthesis by indomethacin had no effect on the relaxatory response to electrical field stimulation. Neither the α₂-adrenoceptor selective antagonist yohimbine (1 μM) nor the α₂-adrenoceptor selective agonist UK 14,304 (1 μM) had any significant effect on the electrical field stimulation-induced relaxation. Pertussis toxin (100 ng/ml) was without effect on relaxations elicited by electrical field stimulation. GTP in the concentration range 10 μM ? 1 mM slightly potentiated the relaxant response. N-carboxymethyl-Phe-Leu (an inhibitor of enkephalinase) or aprotinin (an inhibitor of several proteases) had no significant effect on the electrical field stimulation response. Addition of trypsin (100 U/ml) in combination with chymotrypsin (20 U/ml) significantly reduced the electrical field stimulation-induced relaxation. In the present study we have found indications for the involvement of nitric oxide and possibly also peptides in mediating the inhibitory NANC response (relaxation) in bovine mesenteric arteries.     

Photoreactive derivative of Kunitz's soybean trypsin inhibitor.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1–114 and 115–181.Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [³H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.     

超声波对胰蛋白酶活力影响的机理研究

目的探索超声波对胰蛋白酶催化的影响效果和作用方式。方法研究胰蛋白酶经不同频率、不同功率、不同时间的超声波处理后酶活力的变化;用动力学参数变化和光谱学探索其影响催化的机理。结果经超声波处理后酶活力普遍升高。用15 kHz5、0 W超声波处理胰蛋白酶3 min,酶活力可提高45.4%。将粗酶用SephadexG75凝胶色谱,分部液在聚丙烯酰胺凝胶电泳检验显示出一条带,说明酶被纯化到电泳纯程度。对纯酶进行动力学分析,结果表明经超声波处理后Km值变小,Vmax值也降低,说明超声波处理使酶对底物的亲和力增大。超声波处理不会改变胰蛋白酶分子的构型,但会改变酶分子的构象。结论超声波对胰蛋白酶催化活性的提高可能是由于改变酶分子的构象、增强酶分子对底物的亲和力的结果。     

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.     

STUDIES ON TRYPSIN INHIBITORS Part IX. Synthesis and Trypsin Inhibitory Activity of the Duopentacontapeptide Corresponding to the Amino Acid Sequence of Porcine Pancreatic Secretory Trypsin Inhibitor II (Kazal)*

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence 1–52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1–14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15–52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1M piperidine and mercuric acetate. Folding and formation of the disulfide bonds was accomplished by air oxidation in 0.02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio, in inhibiting the tryptic hydrolysis of Nα-benzoyl-dl -arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.     

Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.