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Biomedical subjects

S Tsunasawa

Publications and source records attributed to S Tsunasawa.

At least 91 records · Page 5Linked to original sources
PubMed1987

Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli.

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587-4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl.

Bacillus subtilis↗
PubMed1987

Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice. Its homology with pancreatic secretory trypsin inhibitor.

The trypsin-sensitive cholecystokinin-releasing peptide is a peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. We postulate that the peptide acts as a mediator of pancreatic enzyme secretion in response to dietary protein intake and that it (designated as "monitor peptide" from its role in the intestine) could be responsible for the feedback regulation of pancreatic enzyme secretion. About 20 nmol of the highly purified peptide were obtained from 800 ml of rat pancreatic juice by reverse-phase high performance liquid chromatography. It was then sequenced. The peptide comprises 61 amino acid residues (Table I). It has a sequence that closely resembles that of a highly conserved region in pancreatic secretory trypsin inhibitors (PSTIs, Kazal type inhibitor): -Ile-Tyr-Asx-Pro-Val-Cys-Gly-Thr-Asx-Gly-. However, the peptide is less related to other mammalian PSTIs than they are to each other. The additional 5 residues at the NH2 terminus make the peptide larger than the common 56-residue PSTIs. The trypsin-sensitive cholecystokinin-releasing peptide is to be classified as a Kazal-type inhibitor and may be one of the rat PSTIs or a related peptide. The present results and increasing evidence from other laboratories and ours suggest that Kazal-type inhibitors play previously unrecognized multiple physiological roles.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of a cDNA coding for human myeloperoxidase.

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.

Amino Acid Sequence↗
PubMed1987

Human B-cell differentiation factor defined by an anti-peptide antibody and its possible role in autoantibody production.

The partial amino acid sequence of the NH2 terminus of a factor named human B-cell differentiation factor or B-cell stimulatory factor 2 (BSF-2) has been determined. Antibodies raised against the synthetic peptide corresponding to residues 1-13 of the NH2-terminal sequence specifically react with BSF-2 generated by a T-cell line and by phytohemagglutinin-stimulated normal T cells. Furthermore, the antipeptide antibodies react with a BSF-2-like factor produced by cardiac myxoma as well as uterine cervical carcinoma cells. The results show that BSF-2 functions in vivo as well and suggest that the constitutive production of BSF-2 may be involved in autoantibody production, since patients with cardiac myxoma and uterine carcinoma showed autoantibody production.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus.

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.

Alcaligenes↗
PubMed1987

Overproduction and purification of the Tn3 transposase.

The Tn3 transposase accumulated to about 4% of total cell protein in a minicell-producing Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: derepression of transcription resulting from inactivation of the repressor gene (tnpR); efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, the dosage effect of the increased plasmid copy number resulting from deletion of the rom gene of the plasmid; and use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; sonication, precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, ammonium sulfate precipitation, gel filtration through Sephacryl S-300, and phosphocellulose and DNA-cellulose chromatography. Milligram quantities of pure transposase can be obtained from one gram of wet cells. A small fraction of the accumulated transposase had a blocked amino-terminus and was eluted separately from the normal protein in the chromatography.

Chromosome Deletion↗
PubMed1987

A molecular-pathologic approach to murine senile amyloidosis. Serum precursor-apolipoprotein A-II variant (Pro5----Gln) presents only in the senile amyloidosis-prone SAM-P/1 and SAM-P/2 mice.

Murine apolipoprotein (apo) A-II is a serum precursor of murine senile amyloid protein. We determined the primary structures of apo A-II in accelerated senescence-prone mice (SAM-P) characterized by a high frequency of age-associated systemic amyloidosis and accelerated senescence-resistance mice (SAM-R) in which senile amyloidosis occurred with a low incidence. Apo-A-II variant (Pro5----Gln) was found to be present only in the serum of SAM-P and not in that of SAM-R or other random bred slc:ICR mice. The apo A-II variant in the serum of SAM-P is identical to the murine senile amyloid fibril protein (ASSAM) derived from amyloid-deposited tissues of SAM-P. These findings proved that apo A-II deposits in tissues without degradation and this mutation (Pro5----Gln) probably have significant effects on the structure and function of apo A-II and would play a critical role in murine senile amyloidogenesis.

Aging↗
PubMed1986

The single proline-glutamine substitution at position 5 enhances the potency of amyloid fibril formation of murine apo A-II.

The primary structure of murine apolipoprotein A-II (apo A-II) has been determined. Apo A-II consists of a single polypeptide chain of 78 amino acid residues, of which the amino-terminus is pyrrolidone carboxylic acid. Except for residues 5 and 38, the amino acid sequence is identical to that of murine senile amyloid protein (ASSAM), which has a common antigenicity with apo A-II. Substitution of glutamine (ASSAM) for proline (apo A-II) at position 5 is distinct and may possibly be related to murine senile amyloid-ogenesis.

Amino Acid Sequence↗
PubMed1986

High homology is present in the primary structures between murine senile amyloid protein (ASSAM) and human apolipoprotein A-II.

The primary structure of a murine senile amyloid protein (ASSAM) was determined. The protein consists of a single polypeptide chain of 78 amino acid residues. The amino-terminus is blocked with pyrrolidone-carboxylic acid. The sequence differs from that of the known murine amyloid A protein and is highly homologous to human apolipoprotein (apo) A-II. The result indicates that the putative precursor of the senile amyloid protein is apo A-II in mice.

Amino Acid Sequence↗
PubMed1986

Cadherin cell adhesion molecules with distinct binding specificities share a common structure.

Ca2+-dependent cell--cell adhesion molecules, termed cadherins, are divided into subclasses with distinct tissue distributions and distinct cell-binding specificities. To elucidate the biochemical relationship of these subclasses, we compared the pattern of tryptic cleavage and the partial amino acid sequence of mouse liver E-cadherin with those of chicken brain N-cadherin. Although these two cadherins are distinct in their cell-binding and immunological specificities, they showed an identical mol. wt and a similar tryptic cleavage pattern. We isolated tryptic fragments of E- and N-cadherin, and determined the sequences of nine amino acid residues of their amino terminus. The results showed that sequences of amino acids from the amino terminus to the 7th residues are identical in these two cadherins. We thus suggest that cadherins with distinct specificities have a common genic origin.

Animals↗
PubMed1986

Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli.

The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690.

Amino Acid Sequence↗
PubMed1986

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins.

The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH2-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH2-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis. Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH2-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.

Acetyltransferases↗
PubMed1986

Reexamination of the primary structure of an antitumor protein, neocarzinostatin.

The primary structure of an antitumor protein, neocarzinostatin, has been reinvestigated by conventional and gas-phase Edman degradation procedures. Sequence analyses of tryptic peptides of both S-carboxymethylated and S-aminoethylated derivatives as well as peptic peptides of the native protein revealed a revised primary structure of neocarzinostatin. The present sequence of 113 amino acid residues thus established agrees with results obtained by fast atom bombardment and gas chromatographic mass spectrometry which were published recently [B.N. Gibson, W.C. Herlihy, T.S.A. Samy, K.S. Hahm, H. Maeda, J. Meienhofer, and K. Biemann (1984) J. Biol. Chem. 259, 10801-10806]. The assignment of four intriguing asparagine/aspartic acid residues has been also achieved.

Amino Acid Sequence↗
PubMed1986

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein.

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.

Acid Phosphatase↗
PubMed1986

Amino acid sequence of copper,zinc-superoxide dismutase from spinach leaves.

The complete amino acid sequence of Cu,Zn-superoxide dismutase (SOD) from spinach leaves has been determined on the basis of peptides obtained by cyanogen bromide (BrCN) cleavage and by enzymic hydrolyses with Achromobacter lyticus lysylendopeptidase, Staphylococcus aureus V8 protease, trypsin, and thermolysin. The spinach SOD consists of a total of 154 amino acid residues with alanine as the amino(N)-terminus and valine as the carboxy(C-)terminus. The present sequence, which has been established for the enzyme from a plant, is also highly homologous to those of the enzymes from other species. Especially, the residues essential for metal binding and enzyme activity have been extensively conserved among all of the Cu,Zn-SODs hitherto analyzed.

Acetylation↗
PubMed1985

Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor.

The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin.

Amino Acid Sequence↗
PubMed1985

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit.

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.

Glutamates↗