Genetic Structure of Natural Populations of Wild Soybean (Glycine soja) in Beijing Region

In order to exploit the genetic resources of wild soybean (Glycine soja) which is the progenitor of cultivated soybean (Glycine max), the genic frequencies of Ti (coding trypsin inhibitors) and Sp1 (coding β-amylase isozymes) for 13 populations of wild soybean in Beijing region were determined. There are 2 alleles (Tia and Tib) in Ti locus of Beijing populations. Calculation of heterozygosity indicates Sp1 is polymorphic, while this monomorphic within a population. Based on the vatiation (from 0 to 50%) for heterozygosity of Sp1 among populations, with special reference to the values of genetic distances among populatious, and no heterozygote has been found in 1300 plants which would be heterozygotes if they were outbreeder, we suggested that wild soybean in natural populations is absolute inbreeder. The frequencies of Ti and Sp1 alleles vary from place to place extremely, however, no correlation exists between allozyme frequencies and ecological factors. Field investigation has shown that there is a threat from the reduction in available habitats, caused by building irrigation works .and urbanization. Finally, sampling strategy for conservation of genetic resources of wild soybean was discussed and some suggestions were made.     

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

大豆Kunitz型胰蛋白酶抑制剂新类型Tid的全序列分析

大豆ｋｕｎｉｔｚ型胰蛋白酶抑制剂（ＳＢＴｉＡ２）是一种大量存在于大豆（Ｇｌｙｃｉｎｅｍａｘ）中的种子贮藏蛋白．虽然对它的生化特性及结构已有较多的研究,但它在体内的主要功能仍不很清楚．国际上所发现的３个由显性等位基因Ｔｉａ、Ｔｉｂ、Ｔｉｃ编码的大豆ｋｕｎｉｔｚ胰蛋白酶抑制剂的氨基酸顺序已被明确测定,相互间有一到多个氨基酸残基的不同［１］．Ｔｉｄ是从我国１５０００余份大豆资源中筛选到的唯一一份ｋｕｎｉｔｚ型胰蛋白酶抑制剂位点的新类型,遗传分析证明它是另一个ＳＢＴｉＡ２的显性等位基因［２,３］．严…     

Two simple quantitative assays for trypsin inhibitors using immobilized trypsin.

Two new methods for quantitative assay of trypsin inhibitors, suitable for large numbers of samples, are described. The assay methods use trypsin-Sepharose conjugates incorporated into agarose gel slabs. Trypsin inhibitors are allowed to diffuse into, or are electrophoretically moved through, the slabs, and the consequent areas of inactivation of immobilized trypsin are visualized using a histochemical enzyme substrate. Quantitation of the trypsin inhibitor content of samples can be made on the basis of the inactivated areas. The limit of detection is 1–2 μg of soybean trypsin inhibitor and determinations are reproducible to 10% or better. Measured trypsin inhibitor contents of several legume species and varieties agree with spectrophotometric determinations.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.     

Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.     

Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor

The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35|MoC. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41|MoC, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.     

C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride

A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.     

Trypsin and α-amylase inhibitors are differentially induced in leaves of amaranth (Amaranthus hypochondriacus) in response to biotic and abiotic stress

Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an α -amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and α -amylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect α -amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in α -amylase inhibitor activity and AAI levels were detected in leaves of insect-damaged, MeJA- and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water- or salt-stress. A differential induction of trypsin inhibitor activity and α -amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and α -amylase inhibitors could protect A. hypochondriacus against multiple types of stress.     

Myelin protein changes with digestion of whole sciatic nerve in trypsin.

Whole mouse sciatic nerves were split and incubated in phosphate buffered saline (PBS) and in PBS containing various amounts of trypsin. After 24 hours of exposure to PBS alone there were no changes in the gel electrophoresis pattern of myelin proteins. During the same period of time, trypsin digested major amounts ofboth the main myelin protein (P_O) and the two basic proteins of myelin (P₁, P₂). The basic proteins were undetectable after 24 hours of 1% trypsin digestion while the main myelin protein was not completely digested. The amount of digestion of the myelin proteins was related to the concentration of trypsin and the time of digestion. Myelin proteins were demonstrated by staining with Coomassie blue, periodic acid Schiff (PAS) and by special indirect lighting techniques.     

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α-amylase/serine protease inhibitor which inhibits germination-specific cereal α-amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M_r ≈20500 and pI ≈7.2. The amino acid composition and N-teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.     

Nature of proteinase inhibitors released from soybeans during imbibition and germination

Proteinase inhibitors are released to a smaller extent from soybeans which have been pre-equilibrated to an atmosphere of high relative humidity (r.h.) compared to those equilibrated to low r.h. Seeds pre-equilibrated to high r.h. also exhibited better germination. Regardless of the states of seed hydration, both Kunitz and Bowman-Birk soybean trypsin inhibitors are released in parallel with respect to each other and to other proteins at germination times up to 100 hr. After 48 hr of germination, new forms of trypsin inhibitor appear in the leachate which react with anti-Bowman-Birk trypsin inhibitor antibodies. No new forms of Kunitz trypsin inhibitor were observed immunochemically during the first 100 hr of germination.     

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Molecular cloning of the barley seed protein CMd: A variant member of the α-amylase/trypsin inhibitor family of cereals

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of α-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with α-amylases of bacterial origin.     

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.     

Amino acid sequence of a crystalline seed albumin (winged bean albumin-1) from Psophocarpus tetragonolobus (L.) DC. Sequence similarity with Kunitz-type seed inhibitors and 7S storage globulins

The complete amino acid sequence of winged bean albumin-1 (WBA-1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA-1 was found to be homologous with the Kunitz-type seed trypsin inhibitors. The similarity between WBA-1 and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C-terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz-type proteins and the carboxy-terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of beta sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of beta sheet to that found for soybean trypsin inhibitor by X-ray diffraction studies.     

Purification and characterization of a non-kallikrein arginine esterase from dog urine

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.