Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.     

Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

Role of Asp102 in the enzymatic reaction of bovine β-trypsin: A molecular orbital study

Using the X-ray structure of the complex of bovine β-trypsin with the basic pancreatic trypsin inhibitor, the hydrogen-bond structure consisting of Ser195, His57 and Asp102 is clarified in relation to the mechanism of the enzymatic reaction from an ab initio quantum chemical point of view. Under the influence of the inhibitor, of the three hydrogen bonds involving Ser214, His57 and Ala56 around Asp102, and of the other ionic amino acid residues, Asp102 plays a significant role in lowering the barrier height of the proton transfer from Ser195 to His57 without accepting a proton from His57. The principal cause of the barrier height lowering is the electrostatic interaction.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 Å resolution: Structure solution,crystallographic refinement and preliminary structural interpretation

The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex (Papamokos et al., 1982). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981). The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex (Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.     

Trypsin isoinhibitors from Momordica repens seeds

Four trypsin isoinhibitors (CM-1 to CM-4) were purified from Momordica repens seeds by gel filtration on Sephadex G-50 followed by ion exchange chr     

Cloning,characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman–Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5 h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.     

Proteolysis of soybean bowman-birk trypsin inhibitor during germination

The Bowman—Birk type trypsin inhibitor, BBSTI-D, which appears in the cotyledons of germinated soybeans (Glycine max), was isolated in homogeneous form. BBSTI-D has an amino acid composition identical to the native Bowman—Birk soybean trypsin inhibitor (BBSTI-E) except for the loss of one glutamyl/glutaminyl residue and one aspartyl/asparaginyl residue. The amino-terminal sequence of BBSTI-D was identical to that of BBSTI-E. These data, as well as the compositions of the tryptic peptides from reduced carboxymethylated BBSTI-D, indicate that BBSTI-D is derived from BBSTI-E by the loss of the carboxyl-terminal residues Glu⁷⁰—Asn⁷¹.     

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.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Isolation and Characterization of Two Novel Nematicidal Depsipeptides from an Imperfect Fungus,Strain D1084

Two novel nematicidal cyclodepsipeptides, designated bursaphelocides A and B, were isolated from the culture filtrate of an imperfect fungus, strain D1084, belonging to Mycelia sterilia. Bursaphelocide A (1), containing 2-hydroxy-3-methylpentanoic acid, proline, isoleucine, N-methylalanine, N-methylvaline, and β-alanine in sequence, and bursaphelocide B (2), comprising 4-methylproline instead of proline in 1, are novel 2-hydroxy-3-methylpentanoic acid analogues of insecticidal destruxins.     

Engineered Cystine Knot Miniproteins as Potent Inhibitors of Human Mast Cell Tryptase β

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase β, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase β tetramer with an overall equilibrium dissociation constant K_i of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase β described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.     

Fermentation of a composite finger millet-dairy beverage

Fermented composite beverages of finger millet and milk are popular, nutritious, traditional foods in many parts of Zimbabwe. With the aim of commercial production, we determined what type of microbial cultures can be used to ferment a composite finger millet and skimmed milk powder gruel and the optimum conditions for its production. Composites containing between 0 and 100% finger millet gruel by volume were inoculated and incubated at various temperatures. The desired pH of 4.5 or less was obtained with incubation at 30 to 45 °C (but not at lower temperatures) with lower pH values being obtained as the temperature increased. YC380 (a yoghurt type bacterial starter culture) produced a pH of 4.5 or less only when skim milk was also present; V2 (another yoghurt type bacterial starter culture) did so at all levels of finger millet gruel and JC (a mixed strain culture developed to ferment cereals) only when finger millet gruel was present. A clear relationship between incubation temperature and syneresis could not be established but syneresis decreased significantly (p < 0.05) with increasing proportions of finger millet gruel. A thick product with a set consistency was obtained with YC380 at an incubation temperature of 45 °C and a storage temperature of 7 °C regardless of proportion of finger millet gruel. V2 produced a thick product with a set consistency at an incubation temperature of 45 °C, and storage temperature of 7 °C and when the proportions of finger millet gruel were between 0 and 50%. It appears that yoghurt type bacterial cultures can be successfully used to produce a composite fermented beverage from finger millet and skim milk, but cultures developed for fermentation of cereals are not suitable.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Phenotypic cost to plants of an extra gene

In lines of transgenic tobacco plants containing cowpea trypsin inhibitor gene constructs, the cost to various phenotypic characteristics has been measured in plants which express the gene at a high level and in plants which possess, but do not express, the cowpea sequences. Small, but in some cases significant, differences between transgenic and untransformed control plants were found in various parameters. There was no additional difference between transgenic plants which expressed cowpea trypsin inhibitor and those which did not. Thus, although the processes of transformation/regeneration may have some small effects on non-targeted phenotypic characteristics, the expression at high levels of this ‘foreign’ protein imposed no additional yield penalty on the plants.     

Variation in response of accessions of minor millets,Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and Eragrostis tef (Zucc.) Trotter (tef) to salinity in early seedling growth

The response to increasing NaCl concentration of seedlings of 25 accessions of Ethiopian land races of each of Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and 15 accessions of Eragrostis tef (Zucc.) Trotter (tef), was examined after two week's growth in NaCl solution culture. Although increasing NaCl concentration significantly reduced seedling root lengths, there was considerable variation within, and between accessions within each species.Analysis based upon a non-linear least square inversion method, using root length data, revealed significant differences in accessions of P. americanum and E. tef on the basis of the estimated salinity threshold, C _t , the NaCl concentrations at which root length begins to decrease. C _t did not differ significantly between E. coracana accessions. Estimates of C₅₀ and C₀, mininum concentrations causing a 50% decrease in root length, and zero root growth respectively, revealed differences between and within accessions for all three species. Overall, finger millet was more tolerant than tef, which was more tolerant than pearl millet. There is clear evidence that differences in tolerance are genetically based from broad sense heritability estimates.     

Genetic control of a novel series of trypsin inhibitors in wheat and its relatives

The aneuploids of Chinese Spring wheat have been used to locate the genes(Ti-2) coding for a novel series of trypsin inhibitors to the long arms of the homoeologous group 5 chromosomes. Three allelic variants at the 5D locus were detected in a limited survey among wheat varieties, but no variation at the loci on either chromosome 5A or chromosome 5B was detected. Homoeoloci were found in a number of alien relatives, and in the majority of cases, these were present on the group 5 homoeologue. However, inAegilops umbellulata, theTi-U2 locus was located on a chromosome presumed to belong to homoeologous group 1. NoHordeum vulgare orH. chilense Ti-2 gene was expressed in a wheat background. This new marker will be especially useful as a screening mechanism for nullisomy of chromosome 5B in work aimed at introgression of alien chromatin into wheat.The Agricultural Genetics Company is thanked for financial support.