Amino acid sequence of rat epidermal thiol proteinase inhibitor

The complete amino acid sequence of rat epidermal thiol proteinase inhibitor was determined. The unique 103-residue sequence was derived by analysis of two peptides generated by limited proteolysis of the native inhibitor with Staphylococcus aureus V8 protease and of three cyanogen bromide fragments. The protein has a high degree of sequence homology to either rat liver or human leucocyte inhibitor but is not identical and may represent a new type of low molecular weight thiol proteinase inhibitors.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Neurotoxins from the venom of the Central Asian scorpion Buthus eupeus

Three main polypeptide neurotoxins M9, M10, M14, possessing paralytic activity in mice, have been isolated from the venom of the Central Asian scorpion Buthus eupeus. The amino acid composition of these toxins was determined. Toxins M9 and M14 were digested with trypsin, chymotrypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The complete amino acid sequences of the toxins M9 and M14 were established.     

Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.

The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen.     

Proteinases in the study of amino acid sequences in proteins

The proteolytic action of trypsin, chymotrypsin, submaxillary gland proteinases, Lys-C, Staphylococcus aureus st. V8, Armilarria mellea, Mixobacter AL-2 proteinase II, thermolysin and alpha-lytic proteinase is elucidated from the analysis of te data available on the amino acid sequence studies for above 70 proteins. Properties of a series of commercial enzymic preparations and the way of preferential application of proteinases for studying the amino acid sequence are discussed.     

The primary structure of Aspergillus niger acid proteinase A

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.     

Amino acid sequence of 2 neurotoxins from the scorpion Buthus eupeus venom

Three polypeptides, M10, M14 and M9, toxic to mammals were isolated from the venom of the Central Asian scorpion Buthus eupeus. All the toxins were shown to be homogeneous according to disc-electrophoresis and N-terminal group analyses. The toxin M9 was digested with trypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The toxin M14 was subjected to tryptic and chymotryptic hydrolyses. The complete amino acid sequences of the toxins M9 and M14 were established and it was shown that each of them consists of 66 amino acid residues with four intramolecular disulfide bonds.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.     

Isolation and amino acid sequence of two trypsin isoinhibitors from duck pancreas

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.     

The complete primary structure of bovine stefin B.

A new stefin B-type low-Mr CPI was isolated from bovine thymus and subjected to structural analysis. The inhibitor consisted of 98 amino acids and its Mr was calculated to be 11,178. The NH2-terminal amino acid residue was blocked. The sequence was determined by automated sequencing of peptides derived by cleavage with cyanogen bromide and fragments of the inhibitor resulting from enzymatic digestion with beta-trypsin and Staphylococcus aureus V-8 proteinase. The NH2-terminal blocking group was established with mass spectrometry. The inhibitor exhibits considerable sequence homology with inhibitors from the stefin family. Furthermore, a highly conserved QVVAG region within the stefin family is for the first time replaced by the QLVAG sequence.     

Purification and characterization of staphylococcin BacR1, a broad-spectrum bacteriocin.

The bacteriocin BacR1 was purified from culture supernatant of Staphylococcus aureus UT0007 by sequential ammonium sulfate precipitation, cation-exchange chromatography, and C4 reverse-phase chromatography steps. Mass spectrographic analysis indicated that the purified peptide has a molecular mass of 3,338 Da. It is resistant to environmental conditions, retaining full biological activity after exposure to pH extremes (pHs 3 to 11), heating at 95 degrees C for 15 min, and exposure to strong chaotropic agents. BacR1 was destroyed with a complete loss of biological activity after digestion with trypsin and proteinase K. Amino acid sequence analysis revealed a high concentration of Asx, Gly, and Pro residues and a high proportion of hydrophobic amino acids. The peptide is bactericidal and kills in a dose-dependent manner, but it does not lyse log-phase cells of Corynebacterium renale, the routine indicator organism for bacteriocin assay. A specific receptor for binding was detected on sensitive cells but not on insensitive cells. Competition assays showed that UV-inactivated cells could protect susceptible cells from antibacterial action. A partial inhibitory spectrum revealed that organisms from the following genera are susceptible: Staphylococcus, Streptococcus, Corynebacterium, Haemophilus, Bordetella, Moraxella, Pasteurella, Neisseria, and Bacillus.     

A comparative study of the structure of egg-white riboflavin binding protein from the domestic fowl and Japanese quail.

1. The riboflavin binding proteins from domestic fowl and Japanese quail have been isolated and their structures compared by circular dichroism, fluorescence and peptide mapping. 2. The two proteins have similar secondary structures, but differ in their tertiary structures as reflected in the environments of aromatic amino acid side chains. 3. Differences in amino acid sequence between the proteins are indicated by the digestion patterns obtained with thermolysin, chymotrypsin and V8 proteinase from Staphylococcus aureus. Both proteins are resistant to digestion by trypsin.     

Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus.

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30 degrees C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.     

Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.     

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.     

Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.     

Pseudonajatoxin b: unusual amino acid sequence of a lethal neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis

The complete amino acid sequence of pseudonajatoxin b, a basic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, was determined by automated Edman analysis of the reduced carboxymethylated polypeptide and of peptides derived by digestion of it with Staphylococcus aureus V8 proteinase. Pseudonajatoxin b consists of a single polypeptide chain of 71 amino acids with Mr 7762. The amino acid sequence showed considerable homology with postsynaptic long neurotoxins, but there were striking differences. Pseudonajatoxin b displayed relatively high lethality, LD50 15 micrograms/kg in mice.