Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin.

Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.     

Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles

We present a study of the cleavage specificity of IRCM-serine protease 1 from frozen porcine pituitary neurointermediate lobes using polypeptide substrates representing different segments of human pro-opiomelanocortin. Using 125I-labeled ACTH(11-24) and a 125I-labeled model beta-lipotropin (beta-LPH) peptide, the preference of this protease for cleavage C-terminal to the pairs of basic residues Lys-Arg and Lys-Lys was clearly seen. This study was extended to larger unlabeled natural human polypeptides including ACTH(1-39), beta-LPH(1-89), and the N-terminal glycopeptide (1-76), which are known to serve as substrates for further cleavage in vivo. In these substrates IRCM-serine protease 1 cleaved C-terminal to all pairs of basic residues known to be cleaved in vivo. In addition, the enzyme cleaved between two pairs of basic amino acids found in NT(1-76) which are also known to be cleaved in vivo. Many potential "tryptic-like" cleavage sites were not cleaved by the enzyme. However, IRCM-serine protease 1 cleaved C-terminal to Phe-Arg in the three melanocyte-stimulating hormone sequences of pro-opiomelanocortin. In order to better understand the physiological role of IRCM-serine protease 1, differential centrifugation was used to study the subcellular distribution of the enzyme from porcine pituitary anterior lobe homogenates. We present evidence that the active enzyme form, isolated from the subcellular fractions, possesses a similar molecular architecture as the enzyme isolated from frozen tissue (Mr 38,000 catalytic domain linked via disulfide bridge(s) to another polypeptide chain(s) to form an Mr 88,000 monomeric structure). The majority of IRCM-serine protease activity is found to be associated with small vesicles (150,000 X g for 5 h) of as yet undetermined nature. In addition, a latent activity was found to be associated with a 27,000 X g (15 min) pellet containing the majority of mature secretory granules. If IRCM-serine protease 1 participates in prohormone maturation in vivo, we propose a model in which this protease is present in an enzymatically active form in small vesicles, possibly within clathrin-coated structures (prosecretory granules) which are then transformed to mature secretory granules by a process which would also inactivate most of the enzyme.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

Purification and properties of a thiol protease from lung fluke adult Paragonimus ohirai

The thiol protease was purified from adult Paragonimus ohirai by alpha 1-antitrypsin-Sepharose, Sephadex G-75 and CM-cellulose, measuring its activities to hydrolyze hemoglobin and tosyl-L-lysine alpha-naphthyl-ester. The purified protease showed a single band on polyacrylamide disc gel isoelectrophoresis as zymogram with Tos-Lys-NE and also by protein staining, and its pI was found to be 6.4. The molecular weight was calculated to be 29,000 by gel filtration and 27,000 by SDS-polyacrylamide gel electrophoresis as a single polypeptide. The protease hydrolyzed hemoglobin and Tos-Lys-NE optimally at pH 4.0 and 5.0, respectively. The both hydrolyzing activities were inhibited by alpha 1-AT and soybean trypsin inhibitor as well as thiol protease inhibitors such as antipain, E-64 and p-hydroxymercuriphenylsulfonate. These results indicate that this enzyme is a new type thiol protease.     

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.     

Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli.

The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.     

Protease specificity and heparin binding and activation of recombinant protease nexin I

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.     

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.     

Specificity and inhibition studies of Armillaria mellea protease.

The action of Armillaria mellea protease has been evaluated on a number of polypeptide substrates. It has been shown to split the Pro7-Lys8 bonds in both native and oxidised lysine-vasopressin and the Ser11-Lys12 bond in glucagon. No other splits were detected in these substrates. The enzyme also caused extensive degradation of S-carboxymethyl lysozyme, S-carcoxymethyl pepsinogen and oxidised ribonuclease. A. In each case the only new amino-terminal residue to appear was lysine. A. mellea protease was inhibited by the chelating agents 1,10-phenanthroline, alpha, alpha'-bipyridine and imidazole. The pK1 values (negative log10 of concentration required for 50% inhibition) for these three inhibitors were 3.9, 3.4 and 1.1, respectively. Lysine, S-2-aminoethylcysteine and short chain aliphatic amines also proved to be relatively good inhibitors of A. mellea protease while arginine was a poor inhibitor.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in the hedgehog, Erinaceus europaeus, and their immunological relationships to those of other mammals (rat, pig and human)

1. Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoregrams characterized for trypsin, chymotrypsin and elastase inhibiting activities with both low and high molecular weight substrates. Approximate molecular weights were also determined. 2. At least ten protease inhibitors were characterized in hedgehog plasma including three macroglobulins. 3. The hedgehog protease inhibitors were identified by immunoelectrophoresis. Four protease inhibitors showed homologies with specific human, rat or swine antisera. These were alpha 2-and beta-macroglobulins, alpha 1-protease inhibitor, and alpha 2-antithrombin.     

Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.     

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and peptidase specificity of lugworm (Arenicola cristata) protease C

1. Lugworm protease C further purified by benzamidine-affinity chromatography, exhibited peptidase specificity for arginyl and lysyl bonds. 2. Protease C consisted of a single polypeptide with a mol. wt of ca 23,000 as determined by SDS polyacrylamide gel electrophoresis, exhibited a u.v. absorption maximum at 280 with an (mg/ml) extinction coefficient of 0.93 and fluorescence spectra typical of a protein containing tryptophan, and had an amino acid composition similar to trypsins. 3. The Kms of the cleavages of the arginyl bond of oxidized insulin B chain and of the lysyl bond of the gly23-ala30 fragment were determined to be 0.72 and 0.96 mM; the corresponding kcats were 38 sec-1 and 1.5 sec-1. The Km and kcat for TAME were 0.042 mM, and 110 sec-1. 4. Lugworm protease C was confirmed to be a trypsin.     

Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.     

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Further studies of plasma protease inhibitors in the hedgehog, Erinaceus europaeus; collagenase, papain and plasmin inhibitors

Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoretograms characterized for collagenase, papain and plasmin inhibiting activities with the high mol. wt substrate casein. The three inhibitors previously identified as alpha 2-, alpha 2-beta- and beta-macroglobulins were found to inhibit all three proteases. These were the only collagenase inhibitors found in plasma. Hedgehog alpha 2-chymotrypsin inhibitor and beta-protease inhibitor were both found to also inhibit papain. Three new inhibitors specific for papain (gamma-, alpha 2- and alpha 1-cysteine protease inhibitors) and one for plasmin (alpha 2-antiplasmin) were also found, bringing the number of protease inhibitors in hedgehog plasma to 14. Immunological cross-reactivity as studied by immunoelectrophoresis showed homology between hedgehog alpha 2-macroglobulin and rat murinoglobulin I and between hedgehog alpha 2-antithrombin and rat antithrombin III.