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Biomedical subjects

F Sakiyama

Publications and source records attributed to F Sakiyama.

At least 73 records · Page 4Linked to original sources
PubMed1989

Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties.

The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.

Amino Acid Sequence↗
PubMed1989

The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease.

The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.

Alcaligenes↗
PubMed1989

Identification of glutamic acid 186 affinity-labeled by 2,3-epoxypropyl alpha-D-glucopyranoside in soybean beta-amylase.

Soybean beta-amylase was modified with 2,3-epoxypropyl alpha-D-[U-14C]glucopyranoside ([14C]alpha-EPG), a radioactive affinity-labeling reagent for beta-amylase, until it lost 95% of its enzyme activity. After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC. A radioactive peptide was finally isolated and its amino acid sequence was determined to be 181Leu-Gly-Pro-Ala-Gly-Glu186. Radioactivity derived from [14C]-alpha-EPG was found exclusively at Glu-186, the gamma-carboxyl group of which is esterified with the affinity label. It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean beta-amylase.

Affinity Labels↗
PubMed1989

The primary structure of porcine liver acylamino acid-releasing enzyme deduced from cDNA sequences.

Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] have been isolated from porcine liver cDNA lambda gt10 cDNA libraries and sequenced. Sequence analyses of the cDNA and several Achromobacter protease I-digested peptides of the purified protein revealed that porcine liver AARE consists of four identical subunits, and each comprising a single chain of 732 amino acids with acetylmethionine at the N-terminus.

Animals↗
PubMed1989

Vapor-phase hydrazinolysis for microdetermination of carboxyl-terminal amino acids of proteins.

The "vapor-phase" hydrazinolysis method was devised for the microdetermination of the carboxyl-terminal residue of a protein. With this method, a polypeptide sample is degraded with vaporized hydrazine. The optimum conditions for hen egg-white lysozyme were established to be 2 to 4 h at 90 or 100 degrees C, the recovery of the carboxyl-terminal leucine being about 70%. With this vapor-phase method, side reactions are reduced and the time of hydrazinolysis is shortened. The limit of quantitation for the carboxyl-terminus of a protein is about 50 pmol, as judged so far with hen egg-white lysozyme. The carboxyl-termini of several proteins were determined using this novel procedure.

Amino Acids↗
PubMed1989

Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.

Amino Acid Sequence↗
PubMed1988

Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae.

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.

Amino Acid Sequence↗
PubMed1988

Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation.

Three clones of full-length cDNA encoding human myeloperoxidase were isolated from a human leukemia HL-60 cell cDNA library in lambda gt10 and characterized. Analysis of the nucleotide sequence of one of the cDNA clones, lambda MP-H17, indicated that the cDNA contained 3207 bp with an open reading frame of 2238 bp, a 5' noncoding region of 159 bp, a 3' noncoding region of 800 bp, and a poly(A) tail of 10 bp. cDNA of the two other clones, lambda MP-H7 and lambda MP-H14, each contained insertions with shorter sequences of 96 and 82 bp, respectively, on the open reading frame of lambda MP-H17 cDNA. A myeloperoxidase genomic clone was isolated, and the structure of its 5' region was determined and compared with the structures of these cDNAs. The comparison revealed that the three cDNAs were derived from myeloperoxidase mRNAs produced by alternative splicing from a transcript of the single gene. Nucleotide sequence analysis of the 3' region of the cDNAs of several clones indicated that the mRNAs were polyadenylated at five different sites. Amino acid sequence determination of the amino-terminal and carboxy-terminal portions of the myeloperoxidase light and heavy chains revealed that, during processing of a precursor polypeptide into the mature protein, the amino-terminal polypeptide, the small peptide between the light and heavy chains, and the carboxy-terminal amino acid were excised.

Amino Acid Sequence↗
PubMed1988

Renotropic activity in ovine luteinizing hormone isoform(s).

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

Amino Acid Sequence↗
PubMed1988

Purification, characterization and amino-acid sequencing of two pancreatic secretory trypsin inhibitors in rat pancreatic juice.

We purified two pancreatic secretory trypsin inhibitors (PSTI-I and PSTI-II) from rat pancreatic juice. PSTI-I consisted of 61 and PSTI-II of 56 amino-acid residues. Their amino-acid sequences were similar (40 out of 56 amino acid residues of PSTI-II being identical with those of PSTI-I), but PSTI-I and PSTI-II appeared to be translation products of different genes. There was no difference in inhibitory properties between PSTI-I and PSTI-II.

Amino Acid Sequence↗
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

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↗