Proteinaceous Protease Inhibitors Research Articles

Substrate-Gel Electrophoresis for Composition and Molecular Weight of Proteinases or Proteinaceous Proteinase Inhibitors

A rapid, sensitive, and generally applicable substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for detection of proteinases or proteinaceous protease inhibitors in biological preparations is described. Electrophoretic separation of proteinases or proteinaceous proteinase inhibitors in the sample using sodium dodecyl sulfate-polyacrylamide gel slabs is followed by immersion of the gel in (1) a protein substrate solution for detection of proteinases or (2) an appropriate proteinase solution, and then in a protein substrate solution for detection of proteinase inhibitors. Some advantages of the reported method over previously described techniques that incorporate substrate into the gel matrix are: (1) the development of bioactive bands, as well as staining and washing, is accomplished more quickly, i.e., 4 to 6 h; (2) the trailing of proteolysis which often appears in copolymerized substrate systems is eliminated; (3) the method is applicable to assay at pH values other than those used for electrophoresis; (4) molecular weight markers can be visualized on the same gels; (5) proteinaceous proteinase inhibitors can also be examined in the biological samples; and (6) the sensitivity is several times higher than that of former assays.

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.

Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene

To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into ‘leaky’ E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH 2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, ▪ and ▪, of pre-SSI. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN′ was almost the same as that of authentic SSI.

Functionally Distinct Agonist and Receptor-binding Regions in Human Chorionic Gonadotropin: Development of a tertiary structure model

Abstract The histidine residues in human chorionic gonadotropin (hCG) were chemically modified using diethyl pyrocarbonate. Derivatives of hCG with an average of 0.5-3.5 histidines modified (maximum of 4 per hCG) had reduced receptor-binding and cell-stimulating activities. Acylation of hCG at progressively lower pH values (conditions in which 1 of the 2 absolutely conserved histidines alpha His-83 is not titratable, whereas alpha His-94 becomes increasingly protonated and resistant to modification) produced hCG derivatives with a greater retention of receptor-binding activity than cell-stimulating activity. The involvement of alpha His-94 as part of the receptor-binding region of the hormone and of alpha His-83 as a putative active site residue was inferred. Proteinaceous protease inhibitors were shown to neutralize the agonist activity of hCG and to reduce the binding of hCG to its receptor and also to specific antisera. It was presumed that an inhibitor-hormone complex was formed which was analogous to the complexing of inhibitor with the of a serine protease. The discovery of primary sequence analogies between hCG and the serine protease chymotrypsin enabled the prediction of hCG structure using the enzyme as a folding template. Solvent-exposed and buried core regions of the peptide chain were delineated using smoothed hydrophobicity profiles in combination with Chou-Fasman secondary structure predictions. Hypervariable hydrophobicity indices between residues 38 and 80 of the human beta subunits reflected different folding arrangements which presumably conferred the individual receptor specificities. When mapped to the putative structure these receptor-determinant loops were adjacent to an area of the alpha subunit analogous to the substrate pocket of serine proteases. Disulfide bond assignments and intersubunit contact regions were identifiable. The proposed tertiary structure for hCG manifests the topographical epitopes defined using monoclonal antibodies and satisfies the currently available data on specific modification and its effects upon hormonal structure and function. This paper is considered to be the first report of a differential effect upon the agonist and receptor binding abilities of a glycoprotein hormone after modification of the proteinaceous, as opposed to the glycosylated, moiety of the molecule.

Isolation and characterization of a proteinaceous protease inhibitor from Bacillus subtilis.

A proteinaceous protease inhibitor which might have an intracellular role in modulating protease activity during sporulation was isolated from B. subtilis IFO 3027 by trichloroacetic acid and ethanol precipitations, and column chromatographies on SP-Sephadex, DEAE-Sephadex, DE AE-Toyopearl and Sephadex G-75.The molecular weight of the inhibitor was estimated by gel filtration and SDS polyacrylamide gel electrophoresis to be about 16,000. The isoelectric point was determined as pH 4.8. The inhibitor is an acid and thermostable protein. The-amino acid sequence in the amino terminal region was determined to be (Met)-Glu-Asn-Gln-Glu-Val-Val-Leu-X-X-Asp-Ala-Ile-Gln-Glu- ··· (X, unidentified).In addition to cytoplasmic serine proteases of the inhibitor-producing strain, the inhibitor inhibits various microbial serine proteases.

Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular proteases from the same strain.

Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular proteases from the same strain.

Natural toxicants in selected leguminous seeds with special reference to their metabolism and behaviour on cooking and processing

A brief survey is presented of the most relevant heat labile and heat stable natural toxicants found in syobean and dry bean (Phaseolus vulgaris). Investigators, aided by rapid development of biochemical instrumentation and techniques, have concentrated upon the elucidation of the chemistry of proteinaceous protease inhibitors and glycoproteins with lectin character. It appears that the low M.W protease inhibitor (abt. 10000) in raw legume meal is in part responsible for the pancreatic hypertrophy observed in rats and chicks fed raw legume meal as the principal source of dietary protein, whilst the large M.W. lectin (abt. 100000) from raw navy bean has been found to be lethal to conventional but not to germfree quail. No real explanation has been advanced for the poor growth observed when these small animals feed on protein from raw legumes.