Comparison of trypsin inhibitors from several types of corn

Trypsin inhibitors were isolated from seeds of floury-2 corn, dent corn, and popcorn and found to be similar in physicochemical and immunological properties to the inhibitor that was previously isolated from seeds of opaque-2 corn. Thus, very similar and perhaps identical trypsin inhibitors occur in genetically diverse types of corn. Our results therefore suggest that the differences between the opaque-3 inhibitor and the amino acid sequence reported by Hochstrasser et al. for a trypsin inhibitor from an unspecified type of corn, do not stem from variations in source material.     

An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants

An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others.     

Superoxide dismutases: I. Occurrence in higher plants

Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms.     

Superoxide Dismutases: II. Purification and Quantitative Relationship with Water-soluble Protein in Seedlings

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F → E → C → A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.     

Amino acid sequence of thePhaseolus vulgaris var. “Fogo na Serra” inhibitor and interactive surface modeling for the enzyme-inhibitor complex

A trypsin and chymotrypsin inhibitor from seeds ofPhaseolus vulgaris var. “Fogo na Serra” (PFSI) was purified and its complete amino acid sequence was determined using Edman degradation methods. The inhibitor was found to belong to the Bowman-Birk family of enzymatic inhibitors; it has 82 amino acid residues and a 8.985-kDa molecular mass. The PFSI/α-chymotrypsin binary complex has been modeled using the Turkey ovomucoid inhibitor third domain (OMTKY3) bound toα-chymotrypsin [Fujinagaet al. (1987),J. Mol. Biol.,195, 397–418. template. The model allowed identification of the binding surface.     

Identification and Partial Characterization of Trypsin Inhibitory Activity in Seed of Some Fruit Plants

Plant proteinase inhibitors are natural plant defense agents against pest and predators. Many plant serine proteinase inhibitors have been purified and characterized particularly from the seeds of Leguminosae family. In this study, some common fruit plant seeds were evaluated for proteinase inhibitory activity. The seed extract of six fruit plants (Prunus domestics, Prunus persica, Prunus amygdalus, Prunus armeniaca, Citrus aurentium and Aegle marmilos) showed significant inhibitory activity against trypsin. The seed extract of P. domestica showed highest trypsin inhibitory activity (133.81 TIU mg^?1 protein).The highest protein content was found in P. persica and P. armeniaca (106.90 and 105.52 mg g^?1 flour respectively). Zymogram analysis showed variable number of trypsin inhibitor isoforms ranging from single band for A. marmilos to four isoforms for P. domestica and P. armeniaca. The seed extract of all plants, except C. aurentium, exhibited trypsin inhibitory activity over a broad range of pH and temperature.The inhibitory activity in seed extract of A. marmilos was found to be the most stable at higher temperature retaining almost 60% of inhibitory activity at 90 °C.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.     

Isolation and characterization of an inter-α-trypsin inhibitor-IgG complex from human serum

Inter-α-trypsin inhibitor is a human serum protease inhibitor of M_r 180 000 which may release physiological derivatives. A complex between IgG and an inter-α-trypsin inhibitor derivative of M_r 30000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-α-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-α-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-α-trypsin inhibitor antibodies. This complex contained M_r 180 000 inhibitor at least to some extent. It migrated in the β-γ zone in agarose; its molecular weight was estimated to be 1500 000 or more; part of it displayed covalent bonding between inter-α-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-α-trypsin inhibitor and anti-γ radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

Studies on Trypsin Inhibitor in Bean (Phaseolus vulgaris L) Cultivars of Himalayan Region

Eight Phaseolus vulgaris L cultivars of Himalayan region were analyzed for trypsin inhibitor activity and inhibition of gut trypsin enzyme extracted from Spodoptera littoralis larvae. Trypsin unit inhibited per gram seed weight was maximum in local yellow cultivar. The trypsin inhibitor was purified to 65.9-fold with 55.6% recovery from seeds of selected cultivar. The purified protein had a molecular weight of 14,130 Daltons and was found to be a monomer by SIDS-PAGE. It was heat stable at 100°C for 10 minutes and had a pH optimum of 7.5. Hence, the purified inhibitor appears to be of Bowman-Birk type. It lost its activity on exposure to 0.2M 2-mercaptoethanol. The inhibition pattern was of non-competitive type and the Ki value was 0.8μM. The KM value of trypsin enzyme for the substrate BApMA was 2.2mM.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

Population Dynamics of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) on Host Plants

The stink bug Dichelops melacanthus (Dallas) has become one of the major pests of corn and wheat in Brasil, mainly after a shift from the conventional tillage system to the no tillage cultivation system. This fact may be due to the simultaneous occurrence of second planting corn with wheat cultivation, and the presence of wild hosts. This study aimed to evaluate the population dynamics of D. melacanthus on wild hosts adjacent to areas cultivated with corn, wheat, and soybean during the season and off-season of soybean cultivation. Weekly surveys were conducted in the region of Londrina, PR, Brasil from the beginning of July 2007 up to the end of June 2008 using the square meter method. Corn (Zea mays), soybean (Glycine max), tropical spiderwort (Commelina benghalensis), hairy indigo (Indigofera hirsuta), crotalaria (Crotalaria pallida), wheat (Triticum aestivum), and signal grass (Brachiaria decumbens) were identified as hosts of D. melacanthus. Signal grass was the host in which stink bug adults were found in higher numbers, while nymphs and adults were consistently collected on tropical spiderwort. Although nymphs completed their development on tropical spiderwort seeds, this host was found less suitable than soybean seeds.     

Validation of SSR markers linked to null kunitz tryspin inhibitor allele in Indian soybean [Glycine max (L.) Merr.] population

Kunitz trypsin inhibitor, a proteinaceous antinutritional factor present in soybean seeds, is responsible for inferior nutritional quality of raw soybean and incompletely processed soy products. The objective of the present investigation was to validate the SSR markers (Satt228 and Satt409) reported to be linked to Ti locus in an Indian soybean population generated from the cross between soybean cultivar LSb1 (TiTi) and PI542044 (titi). Parental polymorphism was surveyed using Satt409, Satt228 and 5 SSR markers in the neighbouring genomic region of Ti locus. A portion of the cotyledon of F₂ seeds was used for analyzing the presence or absence of kunitz trypsin inhibitor polypeptide electrophoretically while the remaining portion containing the embryo was used for raising the F₂ plants (104) for the development of mapping population. The SSR marker Satt228 reported to be tightly linked with Ti locus was not found to be polymorphic for the parents used in our study. Satt409 was found to be linked with Ti locus at 4.7 cM. Besides, a new marker Satt538 was found to be linked with Ti locus at a distance of 17.8 cM. Thus, the SSR marker Satt409 can be useful for Marker Assisted Selection for transferring titi allele in the background of Indian soybean genotypes.     

Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.     

The secretory nature of the excretory gland cells of Strephanurus dentatus. 3. Proteinase inhibitors

A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.     

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.     

Metabolism of trypsin-inhibitory proteins in the germinating seeds of kidney bean (Phaseolus vulgaris)

Summary A number of proteins with trypsin-inhibitory activity was separated by isoelectric focusing and their amounts measured in the extracts of the seeds of kidney bean at various stages of germination up to 16 days.The total trypsin inhibitor content of the dormant seed, 2.2 mg per g bean rose to about 3.6 mg by the seventh day and declined slowly after the tenth day of germination. The individual trypsin inhibitors however, appeared to change independently of each other and some components disappeared almost completely with the progress of germination. The emergence of an inhibitor not found in the dormant seed was also observed. Some of the inhibitor proteins attained a maximum concentration by the 7–8th day of germination. This coincided with a similar maximum in the general protein and proteolytic enzyme content of the germinating bean seeds. The results obtained suggested that the main function during germination of these protein components might not be related to their trypsin-inhibitory activity.