Search

The article presents the study which focuses on the purification and characterization of a major collagenase. It states that the purification of the collagenase was taken from the crude enzyme sample of Streptomyces parvulus through successive phases of chromatography. Accordingly, the purified enzyme revealed a relative molecular mass and it was strongly inhibited by metal-chelating agents. Moreover, purified collegenases from Streptomyces species have been characterized partially only.

The article presents a study on the structural evidence for establishing the relationship of scytalidolisin with the sedolisin family of serine-carbonyl peptidases. It details the elution as well as the effuluent monitoring of the enzyme by measuring their absorbance. Peptide peak fractions were collected and lyophilized while peptide residues are identified. The amino acid sequences alignment of major peptides and sequences of typical serine carboxyl peptidases are also presented.

Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol Chem 269, 32985–32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes. [ABSTRACT FROM AUTHOR]

One of the bromelain inhibitors, isoinhibitor VI (BI-VI), was purified from pineapple stem powder and its complete amino acid sequence was determined by conventional protein sequencing. These results revealed that the protein consists of an 11-residue light chain and a 41-residue heavy chain, cross-linked to each other by disulfide bonds to form the native inhibitor of 52 residues (Mr) = 5888). The secondary structure of BI-VI was analyzed based on the sequence-specific 1H resonance assignment of its two-dimensional NMR spectra. BI-VI was shown to be composed of two domains (A and B) which are formed by antiparallel β-sheets, but has no α-helix. These results were consistent with the CD spectra of BI-VI. Residues Lys27-Ile29 (heavy chain) form a triple-stranded antiparallel β-sheet with residues Asp9-Tyr11 and Lys22-Glu24 (heavy chain) in the A domain and residues Cys5-Cys7 (heavy chain) form another triple-stranded β-sheet with residues Cys6-Cys8 (light chain) and Asp32-Ile34 (heavy chain) in the B domain. The secondary structure as well as the primary structure of BI-VI was distinctly different from that of the other cysteine protease inhibitor, cystatin, and from that of basic pancreatic trypsin inhibitor. [ABSTRACT FROM AUTHOR]

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thumus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DLnorleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution. [ABSTRACT FROM AUTHOR]

Eight bromelain inhibitor (BI) isoform fractions were isolated from pineapple stem, and then submitted to conventional amino acid sequencing after performic acid oxidation and subsequent separation of the resulting light and heavy chains. The results revealed that all fractions exhibited microheterogeneity, containing at least two major components, but that all the isoinhibitors have a common double-chain structure (Mr =ca. 5, 700-5, 900) with five disulfide bonds and similar amino acid sequences. Notably, Fraction BI-VIII exhibited less than 40% of the specific inhibitory activity toward stem bromelain as compared with the other inhibitor fractions. This fraction was a mixture of two isoforms, BI-VIII(1) and BI-VIII(2), the latter lacking the arginine or glutamine residue at the COOH-terminus of the light chain. Furthermore, the oxidized light chain of BI-III, used as a representative normal isoinhibitor, was found to exhibit significant inhibitory activity, whereas the oxidized light chain of BI-VIII(2) lacking the COOH-terminal Arg or Gin showed only very low inhibitory activity. Therefore, the major bromelain-inhibitory site was indicated to be the COOH-terminal residue, Arg or Gin, of the light chain. This is consistent with the three dimensional structure model constructed by computer modeling for the hypothetical complex between BI-VI and papain, a close homolog of bromelain. [ABSTRACT FROM AUTHOR]

The complete primary structure of the major component of tuna pepsinogens was determined by conventional protein chemistry methods. It was composed of a prosegment of 37 residues and a pepsin moiety of 323 residues, having a relative molecular mass of 39,364. The essential aspartyl residues in the active site and the three disulnde bonds common to other pepsinogens were conserved; however, several unique substitutions and/or deletions characteristic of tuna pepsinogen were found at various positions, especially in the prosegment and subsite regions, as compared with the sequences of other pepsinogens, which may affect the rate of activation of the zymogen, and/or the catalytic function and substrate specificity of the enzyme. Tuna pepsinogen is the least acidic among pepsinogens. The sequence identity between tuna pepsinogen and other pepsinogens ranged from 45 to 52%. A phylogenetic tree based on the primary structures suggested that tuna pepsinogen diverged from the pepsinogen A and prochymosin groups in an early period of pepsinogen evolution. [ABSTRACT FROM AUTHOR]

Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trwnyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl- L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymoain. [ABSTRACT FROM AUTHOR]

The article discusses a study which determines the presence of cysteine endopeptidase in the digestive fluid of Dionaea muscipula or Venus's Fly-trap. It states that the identification of the enzyme's occurrence was done through inhibitor studies and amino acid sequencing of band protein. It proposes the name dionain to characterize the enzyme.

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates. • PEP is the prolyl endoprotease obtained from the culture filtrate of Aspergillus niger. • The X-ray structure of PEP was determined by the molecular replacement method using a model structure generated by AlphaFold. • PEP has a characteristic wide-open catalytic pocket compared to homologs. • The wide-open catalytic pocket is important to recognize large protein substrates. [ABSTRACT FROM AUTHOR]

Abstract: The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1′-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology. [Copyright &y& Elsevier]