Trypsin-like protease from soybean seeds. Purification and some properties

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.     

Purification and characterization of a putative proenkephalin cleaving enzyme.

A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 mM), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different Km and Vmax values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower Km and Vmax of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.     

A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates

A novel serine protease, which we have called IRCM-serine protease 1, was purified from both porcine neurointermediate and anterior pituitary lobes. The enzyme was inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, benzamidine, phenylmethyl-sulfonyl fluoride, and thiol reagents including HgCl2, p-chloromercuribenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid) and was resistant to lima bean trypsin inhibitor, alpha 2-macroglobulin, alpha 1-antitrypsin, and C1-esterase inhibitor. IRCM-serine protease 1 displayed "trypsin-like" specificity toward a number of tripeptide coumarin-containing substrates, with kcat/km values ranging from 10(4) to 10(6) M-1 S-1. The best substrate was benzyloxycarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide with a kcat/Km value of 2.27 X 10(6) M-1 S-1. IRCM-serine protease 1, Mr = 169,000-190,000 determined by gradient gel electrophoresis and gel filtration, respectively, appears to be a homologous dimer. The monomeric subunits of the enzyme are composed of an Mr = 38,000 polypeptide chain which is modifiable by 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl, disulfide-linked to another polypeptide resulting in a subunit molecular weight of 88,000.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

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.     

Proteolytic activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. 5. Role of proteases, inhibitors, and substrates

The effect of protease inhibitors and substrates on the killing of tumor cells by in vitro activated macrophages was examined. Phenylmethylsulfonyl fluoride, a serine esterase inhibitor, and the active site titrants TPCK and TLCK (chloromethyl ketone derivatives of tosyl-l-phenylalanine and tosyl-l-lysine) inhibited macrophage-mediated cytotoxicity in a dose-dependent and irreversible fashion. The synthetic protease substrates, tosyl-lysine methyl ester and tosyl-arginine methyl ester and the natural esterase inhibitors soybean trypsin inhibitor and antithrombin III had no effect. These results suggest that an activated macrophage-associated esterase may play a modulating role in the cytolytic interaction between activated macrophages and tumor cells.     

The reaction of bovine thrombin with N-butyrylimidazole. Two different reactions resulting in the inhibition of catalytic activity.

N-Butyrylimidazole has been found to be a potent inhibitor of purified bovine thrombin. The rate and extent of inhibition of thrombin by N-butyrylimidazole could be reduced by the presence of benzamidine, a competitive inhibitor, or by the ester substrate, p-tosyl-L-arginine methyl ester. Spectral studies of the reaction of N-butyrylimidazole with thrombin demonstrated the modification of approximately 1 mol of tyrosine/mol of enzyme at maximum inhibition. In addition to the reaction with tyrosine, N-butyrylimidazole also appears to react with a residue at the "active site" as judged by a decrease in the number of active sites available in the modified enzyme for titration with p-nitrophenyl-p'-guanidinobenzoate. The time course of ester hydrolysis by butyrylated thrombin showed a distinct lag phase suggesting partial reactivation of the enzyme under assay conditions. Partial reactivation of the modified enzyme also occurred spontaneously upon standing in 0.5 M NaCl but was much faster in presence of imidazole (0.03 M, pH 7.6). It is suggested that, in addition to reaction with tyrosine, there is a reaction of N-butyrylimidazole with either the histidine and/or serine residue at the active site of thrombin resulting in a derivative unstable under esterase assay conditions such as that described for the reaciton of N-acetylimidazole with trypsin (L. L. Houston and K. A. Walsh (1970), Biochemistry 9, 156).     

Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.     

Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, has been purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agrose. The purified enzyme is capable of inactivating both [8-arginine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8-Gly9-NH2 and the Leu8-Gly9-NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-trypsine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol. lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate, and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, alpha 1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon, or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The molecular weight determined by gel filtration in the presence of 1 MNaCl is approximately 100 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two identical subunits of 48 000 daltons. Each subunit consists of a heavy chain (28 000 daltons) and a light chain (19 000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the heavy chain.     

Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae)

Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.     

Isolation and characterization of a proteinase inhibitor from marama beans

A protease inhibitor was purified from the African marama bean (Tylosema esculenturm). The inhibitor is present in large amounts, representing about 10.5% of the total protein. The molecular weight is slightly larger than soybean trypsin inhibitor and was estimated at 23,000 by SDS-gel electrophoresis or 24,500 by amino acid analysis. The amino acid composition was atypical of most other plant inhibitors with a cysteine content of only one or possibly two residues/mole and a blocked amino terminus. Inhibition studies indicated virtually no inhibition of chymotrypsin activity. Elastase, however, was inhibited to the same extent as trypsin, requiring about 2 moles of inhibitor for complete inhibition of the enzyme.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Purification and characterization of diacylglycerol lipase from human platelets.

Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.     

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature-labile and had an optimal pH of 7.5. Metallo-protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability-enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability-enhancing activity in skin. This suggests that a trypsin-like protease from skin mediates the activity of the protease to enhance its vascular permeability.     

Hypodermin B, a trypsin-related enzyme from the insect Hypoderma lineatum. Comparison with hypodermin A and Hypoderma collagenase, two serine proteinases from the same source

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.     

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

The essential role of a free sulfhydryl group in blocking the cholesteryl site of cholesteryl ester transfer protein (CETP)

Cholesteryl ester transfer protein (CETP) has at least one unpaired sulfhydryl residue, which we have shown previously to be in or near the active site region. We investigated the location of this unpaired cysteine residue(s) of CETP using chemical modification with fluorescent sulfhydryl-specific reagents, limited proteolysis, and amino acid/sequence analysis. The kinetics of labeling CETP by either 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) or acrylodan were followed by observing the increase in fluorescence of the bound probes. Labeling was inhibited strongly by preincubation of the CETP with either PNU-617, a competitive inhibitor of cholesteryl ester (CE) transport, and TP2 antibody. In addition, the transfer activities of the substrate CE by the modified CETP's were also inhibited but not competitively. Finally, preincubation of the native protein with N-ethylmaleimide (NEM) resulted in inhibition of activity that was dependent upon the time of exposure of the protein to the alkylating agent. These results provide further evidence that there is a cysteine residue in the active site region of CETP and ligands that either react or bind to this residue produce steric hindrance to CE transfer activity. Finally, although not conclusive, results of the protein chemistry experiments with the modified CETP suggest that the cysteine residue at position 333 is unpaired.