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A Tsugita

Publications and source records attributed to A Tsugita.

At least 19 recordsLinked to original sources
PubMed1997

The Protein Information Resource (PIR) and the PIR-International Protein Sequence Database.

From its origin, the PIR has aspired to support research in computational biology and genomics through the compilation of a comprehensive, quality controlled and well-organized protein sequence information resource. The resource originated with the pioneering work of the late Margaret O. Dayhoff in the early 1960s. Since 1988, the Protein Sequence Database has been maintained collaboratively by PIR-International, an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. The work of the resource is widely distributed and is available on the World Wide Web, via FTP, E-mail server, CD-ROM and magnetic media. It is widely redistributed and incorporated into many other protein sequence data compilations including SWISS-PROT and theEntrezsystem of the NCBI.

Amino Acid Sequence↗
PubMed1997

Bond-specific chemical cleavages of peptides and proteins with perfluoric acid vapors: novel peptide bond cleavages of glycyl-threonine, the amino side of serine residues and the carboxyl side of aspartic acid residues.

Peptide bond cleavages by vapors composed of various from aqueous solutions of perfluoric acid were studied using synthetic peptides and proteins, and specific conditions were established for peptide bond cleavages including a novel cleavage of the glycyl-threonine bond. The peptide bonds on the aminosides of serine residues were cleaved by exposure to a vapor of 75% aqueous heptafluorobutyric acid at 30 or 50 degrees C for 24 h. Glycyl-threonine peptide bonds were cleaved with vapors of various concentrations (5, 75, and 90%) of heptafluorobutyric acid at 30-40 degrees C for 24 h. The peptide bonds on the carboxylsides of aspartic acid residues were cleaved by exposure to a vapor of 0.2% heptafluorobutyric acid at 90 degrees C for 4 to 24 h. The same vapor cleaved aspartyl-proline bonds under milder conditions such as at 60 degrees C for 16 h, under which the other aspartyl bonds were uncleaved. These specific chemical cleavages were applied to several proteins including newly characterized proteins.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of a molecular chaperone, gp57A, of bacteriophage T4.

A molecular chaperone of bacteriophage T4, gp57A, which facilitates the formation of the long and short tail fibers, was isolated and characterized by peptide analysis, sedimentation equilibrium, and circular dichroism (CD). Sequence analysis confirmed the predicted sequence of 79 amino acids from the nucleotide sequence of the gene with the N-terminal methionine removed. The result led to the conclusion that the apparent smaller molecular weight of 6,000 from Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the expected molecular weight of 8,710 was due to its abnormal electrophoretic behavior instead of cleavage or processing of the gene product. Estimation of the secondary structure from far-UV CD indicated a 94% alpha-helix content, which was in accord with the prediction from the primary structure. A sedimentation equilibrium study, on the other hand, revealed that gp57A assumes a tetrameric subunit structure.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence of the maize ferredoxin:thioredoxin reductase variable subunit.

The ferredoxin:thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N-terminus, one in the middle of the subunit and one near the C-terminus. We have obtained some evidence indicating that the N-terminal consensus domain is possibly involved in the interaction with the catalytic subunit.

Amino Acid Sequence↗
PubMed1996

The PIR-International Protein Sequence Database.

From its origin the Protein Sequence Database has been designed to support research and has focused on comprehensive coverage, quality control and organization of the data in accordance with biological principles. Since 1988 the database has been maintained collaboratively within the framework of PIR-International, an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. The database is widely distributed and is available on the World Wide Web, via ftp, email server, on CD-ROM and magnetic media. It is widely redistributed and incorporated into many other protein sequence data compilations, including SWISS-PROT and the Entrez system of the NCBI.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence of peptidyl-prolyl isomerase a of Fusarium sporotrichioides.

Peptidyl-prolyl-cis-trans isomerase catalyzes the interconversion of the cis and trans isomers of the proline-containing polypeptides and the folding process of proteins. This protein was known to be cyclophilin which has high binding affinity for cyclosporin A, a cyclic undecapeptide of fungal origin with potent immunosuppressive property agent. The two cytosolic peptidyl-prolyl-isomerases were found from Fusarium sporotrichioides. The amino acid sequence of the major peptidyl-prolyl isomerase a was determined by conventional sequencing methods; the protein with a calculated molecular mass of 19.7 kDa consisting of 179 amino acids. The comparison of the amino acid sequence of peptidyl-prolyl-isomerase from Fusarium with that of Nucerospora crassa revealed a significant degree of amino acid sequence homology (82.2%).

Amino Acid Isomerases↗
PubMed1996

Two-dimensional electrophoresis of plant proteins and standardization of gel patterns.

Proteins of two plants, Arabidopsis thaliana and rice (Oryza sativa) were subjected to two-dimensional electrophoresis analysis with two modifications: (i) comigration of external standard marker proteins with resultant horizontal and vertical stripes in the gel, and (ii) deblocking with a vapor of aqueous heptafluorobutyric acid for N-acetylserine. Approximately 5000 protein spots were separated from both the five tissues of Arabidopsis and the nine tissues of rice. Over one hundred spots were electroblotted for N-terminal sequencing. Among the newly sequenced proteins, 62 were from Arabidopsis and 51 from rice.

Amino Acid Sequence↗
PubMed1996

Isolation of a venom factor devoid of proteolytic activity from Taiwan habu (Trimeresurus mucrosquamatus): N-terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin.

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated from Trimeresurus flavoviridis and botrocetin from Bothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated from Bothrops jararaca venom. We have therefore named this novel venom factor trimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.

Amino Acid Sequence↗
PubMed1995

Characterization of multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus): protein microsequencing coupled with cDNA sequence analysis.

Three fibrinogenolytic proteases were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases of approximately 24 kDa, possessing strong cleaving activity mainly on the A alpha and less on B beta and gamma chains of fibrinogen subunit chains. Enzyme activities were strongly inhibited by EDTA or 1,10-phenanthroline and not by phenylmethanesulfonyl fluoride, suggesting that these fibrinogenases belong to the family of metalloproteinases and not thrombin-like serine proteases. N-Terminal sequence analysis of these proteases failed to show any free amino-terminal residues, thus hampering the sequence determination by conventional sequencing strategy. Microsequencing on the electroblotted fragments of CNBr-treated proteases separated on SDS-PAGE was then used to determine the partial sequences. Sequence comparison of the determined partial sequences of these proteins with published sequences of the protein data bank revealed that they showed sequence homology with H2-protease. HR2a and protrigramin, which were all shown to belong to metalloproteinases present in various snake venoms. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate cloning and sequencing of these proteases. Sequencing several positive clones containing amplified cDNAs revealed the existence of one fibrinogenase in the Taiwan habu, which was contained within one complete cDNA encoding the preproproteinase precursor of hemorrhagic metalloproteinases.

Amino Acid Sequence↗
PubMed1995

Amino acid sequence of spinach ferredoxin:thioredoxin reductase catalytic subunit and identification of thiol groups constituting a redox-active disulfide and a [4Fe-4S] cluster.

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

Amino Acid Sequence↗
PubMed1995

Carboxy-terminal degradation of peptides using perfluoroacyl anhydrides. A C-terminal sequencing method.

An accurate carboxy-terminal sequencing method has long been sought to complement the Edman degradation procedure for amino-terminal amino acid sequence analysis. The method presented here is a unique and simple method to partly fulfill the needs. Exposure of a polypeptide to perfluoroacyl anhydride vapor at -20 degrees C for 0.5-1 h causes sequential chemical degradation of the molecule from the C-terminus. Fast-atom-bombardment mass spectrometric analysis of the resultant mixture of C-terminally truncated molecules permits the determination of the C-terminal sequence by simple calculation of the mass differences in molecular ions. Experiments suggested that this C-terminal degradation proceeds by active intermediates such as oxazolone at the C-terminal carboxyl residues.

Acetic Anhydrides↗
PubMed1995

Separation and characterization of Arabidopsis thaliana proteins by two-dimensional gel electrophoresis.

Arabidopsis (Arabidopsis thaliana) proteins were isolated from five tissues (leaf, stem, root, seed and callus), and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out by immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. With the aid of comigrated five-marker proteins, the patterns of 2-DE gels for each tissue were graphically combined by a computer into a single synthetic image for the integrated Arabidopsis protein spots. The protein spot images, altogether 4763, were characterized by both molecular mass and isoelectric point. Partial amino(N)-terminal sequences of 101 protein spots were analyzed by Edman degradation. Fifty seven proteins were partially sequenced and 46 proteins appeared to have blocked N-termini. Deblocking by hydrazine vapor was carried out on 14 proteins and two of them were found to be pyroglutamyl-blocked N-termini. Forty seven new proteins were found by the present investigation.

Amino Acid Sequence↗
PubMed1994

Identification as beta-adducin of a protein interacting with rabphilin-3A in the presence of Ca2+ and phosphatidylserine.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein implicated in neurotransmitter release. We have previously identified a Rabphilin-3A-interacting protein with a Mr of about 115 kDa in bovine brain. We have attempted here to purify this protein and to determine its primary structure. Amino acid sequence analysis has revealed that this protein is a bovine counterpart of human beta-adducin which is known to be a good substrate for protein kinase C. The Rabphilin-3A-interacting protein also binds to protein kinase C in the presence of Ca2+ and phosphatidylserine. These results indicate that Rabphilin-3A binds to beta-adducin in the presence of Ca2+ and phosphatidylserine.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Amino acid sequence of spinach ferredoxin:thioredoxin reductase variable subunit.

Ferredoxin:thioredoxin reductase (FTR) is an iron-sulfur protein, which, in the presence of ferredoxin and thioredoxin, catalyses the light-dependent activation of several photosynthetic enzymes. Spinach FTR consists of two dissimilar polypeptide chains, A and B, present in equal amounts. Whereas subunit B seems to be responsible for the catalytic activity, subunit A has no known catalytic function. We found earlier that the N-terminus of subunit A, also called the variable subunit, shows terminal redundancy and that 2-3 of its serine residues are phosphorylated [Tsugita, A. Yano, K., Gardet-Salvi, L. & Schürmann, P. (1991) Protein Sequence Data Anal. 4, 9-13]. We now report the complete amino acid sequence of subunit A, determined by conventional protein sequencing methods. The polypeptide chain with a calculated molecular mass of 12,669 Da consists of 112 amino acids and has a calculated isoelectric point of 5.4. The analysis of the sequence supports the idea that this subunit has no catalytic function. The comparison with a known cyanobacterial FTR reveals about 58% similarity and the striking presence of a N-terminal extension in the spinach protein. This extension may be responsible for the reported size variability of this subunit.

Amino Acid Sequence↗
PubMed1994

Separation and characterization of rice proteins.

Rice proteins from nine tissues and one organelle (leaf, chloroplast, stem, root, germ, dark germinated seedling, seed, bran, chaff and callus) were isolated and then separated by two-dimensional gel electrophoresis (2-DE). The protein spots were characterized according to molecular weight, isoelectric point and partial amino-terminal sequence. Electrophoresis was carried out by isoelectric focusing (IEF), nonequilibrium pH gradient electrophoresis (NEPHGE) and immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. With the aid of nine marker proteins, the patterns of IEF, NEPHGE and IPG 2-DE gels were graphically combined by computer into a single synthetic image for each tissue, respectively, and these images for the nine tissues and one organelle were again combined into a single 2-DE image for the integrated rice protein spots. The rice 2-DE gel image resolved 4892 proteins. About 3% of the spots are characterized by amino-terminal sequencing.

Amino Acid Sequence↗
PubMed1994

Specific chemical cleavage of asparaginyl and glycyl-glycine bonds in peptides and proteins by anhydrous hydrazine vapor.

Hydrazinolysis of peptide or protein has been used for C-terminal amino acid determination by Akabori et al. (1952). In this study, proteins were reacted with anhydrous hydrazine vapor at 20 degrees C for 16 h. Asparaginyl linkages were cleaved. Asparagine and glutamine were converted to their hydrazides, beta-hydrazidyl aspartic acid and gamma-hydrazidyl glutamic acid, respectively, even under milder conditions. The former hydrazide cyclizes to a 6-membered ring, asparaginyl bond at the carboxyl side. Other cleavages, including the glycyl-glycine bond, were also observed.

Amino Acid Sequence↗
PubMed1994

The PIR-International Protein Sequence Database.

PIR-International is an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. A major objective of PIR-International is to continue the development of the Protein Sequence Database as an essential public resource for protein sequence information. This paper briefly describes the architecture of the Protein Sequence Database and how it and associated data sets are distributed and can be accessed electronically.

Amino Acid Sequence↗