Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean.

Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.     

Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis).

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

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.     

Trypsin inhibitors of buffalo seminal plasma.

Two trypsin inhibitors from acid-treated buffalo seminal plasma were purified by gel filtration and affinity chromatography. These acid-stable trypsin inhibitors having charge heterogeneity were homogeneous with respect to size as revealed by gel filtration and SDS-PAGE. Gel filtration data suggest molecular weight value of 9,900 Da for inhibitor I and 10,900 Da for inhibitor II. Molecular weight estimated by SDS-PAGE was found to be 10,600 Da and 11,200 Da for inhibitors I and II, respectively. The hydrodynamic properties such as Stokes radii (1.58 nm and 1.62 nm); intrinsic viscosity (2.5725 ml/g and 2.5025 ml/g) and diffusion coefficient (13.499 x 10(-11) m2/sec. and 13.166X10(-11) m2/sec) respectively for inhibitor I and II were determined by analytical gel filtration. These inhibitors were fairly thermostable and could not be stained by PAS reagent. Both the inhibitors were found to inhibit buffalo acrosin but not bovine chymotrypsin.     

Proteinase inhibitors from the excretory gland cells of Stephanurus dentatus. Purification and properties of three secretory proteinase inhibitors

Three proteinase inhibitors designated as I, II, and III were isolated from the excretory gland cells of the swine kidney worm, Stephanurus dentatus. The inhibitors, which were trichloroacetic acid-soluble, were purified by affinity chromatography and ion exchange chromatography. The homogeneity of each inhibitor was shown by polyacrylamide gel electrophoresis and electrofocusing. The molecular weights of the inhibitors estimated by sodium dodecyl sulfate gel electrophoresis fell within a limited range of 9300 to 9700, and the isoelectric points were 6.45, 6.20, and 5.34 for Inhibitors I, II, and III, respectively. The inhibitors formed complexes with trypsin having apparent dissociation constants (Ki) of 2.9 X 10(-11), 7.6 X 10(-11), and 6.4 X 10(-11) M, respectively. Each inhibitor inhibits the esterolytic and proteolytic activities of both trypsin and chymotrypsin. A proteinase inhibitor present in the reproductive organs, intestines, body walls, and esophagi was identical with Inhibitor II found in the excretory gland cells. Culture medium collected after 24-h incubation with adult worms contained the same three inhibitors as the excretory gland cells. These data suggest that the gland cells may secrete the inhibitors internally and externally.     

Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).     

Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.     

Purification and Properties of the Trypsin Inhibitors from Buckwheat Seed

The trypsin inhibitors in buckwheat seeds were isolated by affinity chromatography on trypsin-Sepharose 4B, and the components were fractionated by chromatography on DEAE-Sepharose CL-6B. The major components, inhibitors I, II and III, were found to be homogeneous proteins with molecular weight of about 8,000. Trypsin inhibitory activity was more pronounced than the chymotrypsin inhibitory activity in all the inhibitor preparation obtained. The three major inhibitors had similar amino acid compositions and had no detectable amounts of tryptophan and carbohydrate. A high level of acidic and basic amino acid residues and a low level of methionine, tyrosine and phenylalanine residues characterized the inhibitors. Although the inhibitors I and II were particularly thermostable, inhibitor III, the most abundant component, was shown to be relatively heat-labile.     

苦荞胰蛋白酶抑制剂的纯化及特性研究

利用固相胰蛋白酶亲和色谱及离子交换色谱,从苦荞种子中分离纯化了三个具有抑制胰蛋白酶活力的组份(BTI-Ⅰ,Ⅱ,Ⅲ)。经聚丙稀酰胺凝胶等电聚焦测定,三种抑制剂的等电点为6.87,6.7,5.14。根据Sephadex G-75凝胶过滤,SDS-PAGE测定,以及由当量抑制比值和氨基酸组份的推算,它们的分子量范围分别为5200-5700(Ⅰ),5500—6000(Ⅱ),5000—5600(III)。三者对胰蛋白酶的抑制常数分别为2.12×10~(-8),4.78×10~(-9),1.22×10~(-8)。氨基酸分析结果表明,它们均含有很高的极性氨基酸。在酸性条件下,它们均具有很高的热稳定性。化学修饰的结果表明,它们活性中心的氨基酸残基为Lys。     

Inhibitory Effect of di- and Tripeptidyl Aldehydes on Calpains and Cathepsins

Abstract

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of α-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B < calpain II ≈ calpain I < cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (K_i) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (K_i, 36 nM) and calpain II (K_i, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (K_i, 0.5nM); acetyl-Leu-Leu-Met-H for cathepsin B (K_i, 100nM).     

A crystalline protein-proteinase inhibitor from pinto bean seeds.

A crystalline protein-proteinase inhibitor has been isolated from seeds of Pinto bean (Phaseolus vulgaris cultvar. Pinto). It has an average molecular weight of 19 000 as estimated by gel filtration. This crystalline inhibitor is highly active against both bovine pancreatic trypsin and alpha-chymotrypsin. Complexes of both trypsin-inhibitor and alpha-chymotrypsin-inhibitor have been isolated. The inhibitor which was derived from the dissociated trypsin-inhibitor complex was only 62% as effective as the original compound against either enzyme. In contrast, the inhibitor obtained from alpha-chymotrypsin-inhibitor complex retained its full original inhibitory activity for trypsin, but only 25% of its original activity against alpha-chymotrypsin. The dissociated inhibitor from alpha-chymotrypsin-inhibitor compex, despite its full inhibitory activity, had been modified to such an extent that it could no longer form any precipitable complex with trypsin. The crystalline protein-proteinase inhibitor is not homogeneous and has been resolved into two distinct inhibitors in terms of their physical and chemical properties. These two inhibitors are designated as Pinto bean proteinase inhibitor I and II and their respective minimum molecular weights are 9100 and 10 000. They differ most strikingly in their amino acid composition in that inhibitor II is void of both valine and methionine.     

Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots

A simple purification protocol, involving ion exchange chromatography on DEAE-cellulose and CM-cellulose and fast protein liquid chromatography-gel filtration on Superdex 75, was employed to isolate a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Both the trypsin inhibitor and the lectin were unadsorbed on DEAE-cellulose and adsorbed on CM-cellulose. They could be separated from one another by gel filtration on Superdex 75 in which the 36-kDa lectin appeared as the first peak and the 20.5-kDa trypsin inhibitor as the second peak. P. heterophylla trypsin inhibitor exhibited a trypsin inhibitory potency similar to that of soybean trypsin inhibitor. It also demonstrated antifungal activity toward Fusarium oxysporum like aprotinin and Kunitz-type trypsin inhibitors from soybeans and lima beans. P. heterophylla lectin was devoid of antifungal activity and exhibited low thermostability and also lability in the presence of acid and alkali. The novel aspects of the present report include demonstration of antifungal activity in Kunitz-type trypsin inhibitors and isolation of a novel lectin as well as a trypsin inhibitor from roots.     

Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice

Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

The presence of two heat-stable inhibitors of phosphoprotein phosphatases in the dimorphic fungus, Mucor rouxii

Two heat-stable inhibitors (a and b) of phosphoprotein phosphatases I and II from Mucor rouxii were isolated from mycelium of the fungus. They were partially purified from extracts by heating, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of inhibitors a and b, estimated by gel filtration, are 5,000 and 20,000 respectively. Inhibitor a acts similarly on both enzymes while inhibitor b is relatively more active on enzyme II. Storage of inhibitor b at -20 degrees C for several weeks resulted in a partial conversion to a lower-molecular-weight form with properties similar to those of inhibitor a.     

Human granulocyte elastase is inhibited by the urinary trypsin inhibitor

Two forms of urinary trypsin inhibitor, A and B, were purified from the urine of pregnant women. Form A was the only inhibitor present in fresh urine and inhibitor B arose from degradation of A upon storage of urine. The molecular masses of A and B were about 44 and 20 kDa, respectively, as judged from dodecyl-sulfate polyacrylamide gel electrophoresis, but about 60 kDa and 30 kDa, respectively, as judged from gel filtration analysis. The discrepancy can perhaps be explained by the carbohydrate content amounting to about 10% of each inhibitor. After reduction with mercaptoethanol, inhibitor A and inhibitor B had identical apparent molecular masses of about 20 kDa on dodecyl-sulfate gel electrophoresis. These results and the results of amino acid analysis suggest that one molecule of inhibitor A yields two molecules of inhibitor B. On agarose gel electrophoresis inhibitor A migrated as a rather broad band in the prealbumin region and inhibitor B as 3 well defined bands in the beta-region. Specific antisera were raised against inhibitor A and B. The two inhibitors showed the immunologic reaction of identity with each other and with the plasma inter-alpha-trypsin inhibitor, when using either antiserum. The inhibitors both gave quantitative inhibition of bovine trypsin, the results indicating a 4/1 trypsin/inhibitor molar ratio for A and a 2/1 ratio for B. The two substances also effectively inhibited granulocyte elastase. No inhibition of porcine pancreatic elastase was demonstrable.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

黑豆种子中一种耐热型胰蛋白酶抑制剂的分离及性质表征

应用硫酸铵分级沉降、弱阳交换色谱CM-Sephadex C-50、凝胶过滤 色谱Sephacryl S-200HR、强阳离子高效液相色谱POROS HS-20,从黑豆种 子中分离纯化一种耐热型蛋白酶抑制剂,命名为TSTI.该蛋白的Ｎ末端序列 为DEYSKPCCDLCMCTRRCPPQ,与豆科植物Bowman-Birk型胰蛋白酶抑制剂具有 高度同源性,推测其属于Bowman-Birk型胰蛋白酶抑制剂.SDS－PAGE和IEF 测出TSTI分子量和等电点分别为23.9 kD 和6.2.TSTI对胰蛋白酶有很强的 抑制作用,当二者摩尔比达到1时,胰蛋白酶活力被完全抑制.此外,该蛋 白酶抑制剂具有很强的热稳定性和pH稳定性,在高达100 ℃温度及pH2-12 范围内处理,其活性均不会受到太大影响.TSTI对植物致病菌苹果轮纹病菌 、瓜果腐霉病菌、白菜黑斑病菌、甜瓜枯萎病菌和葡萄灰霉病菌具有抑制 作用.