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H Sinohara

Publications and source records attributed to H Sinohara.

At least 19 recordsLinked to original sources
PubMed1995

Molecular cloning and sequence analysis of cDNA encoding plasma alpha-1-antiproteinase from Syrian hamster: implications for the evolution of Rodentia.

Complementary DNA clones encoding plasma alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) were isolated from Syrian hamster liver cDNA library and sequenced. The deduced amino acid sequence of putative reactive site (P3-P'3) was Ile-Pro-Met-Ser-Val-Pro, characteristic of alpha-1-antiproteinase of orthodox type (Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932). A molecular phylogenetic tree of all known orthologous proteins was constructed based on the synonymous substitution rate. The result shows that the hamster has branched off first before the divergence among mice, rats, and gerbils, and that the rabbit is the closest relative of the guinea pig which is separated from the rodents. Although this tree differs largely from the classical phylogeny based on the morphology (hamsters and gerbils belong to the same family, Cricetidae, and the guinea pig belongs to the order Rodentia), it lends support to recent concepts that the hamster and guinea pig differ, in a number of biochemical features, not only from each other but also from mice and rats, and that the guinea pig may belong to an order distinct from Rodentia.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of mouse countertrypin, a new trypsin inhibitor belonging to the mammalian fetuin family.

A novel trypsin inhibitor, tentatively named countertrypin, was isolated from mouse plasma in an apparently homogeneous state. Countertrypin is a 53-kDa glycoprotein having about 30% carbohydrate, and did not cross-react immunologically with either mouse alpha 1-antiproteinase (also called alpha 1-proteinase inhibitor or alpha 1-antitrypsin) or contrapsin. Countertrypin had no inhibitory activity against chymotrypsin, pancreatic elastase, neutrophil elastase, thrombin, plasmin, plasma kallikrein, pancreatic kallikrein, clotting factor Xa, or papain. This inhibitory spectrum does not correspond to any of the known plasma proteinase inhibitors that have been well characterized in human or other mammals. NH2-terminal amino acid sequence analysis of the intact molecule and three peptides obtained by CNBr digestion revealed that a total of 93 amino acid residues could be aligned with stretches in human alpha 2-HS glycoprotein, bovine fetuin, and rat pp63 (rat fetuin). Human alpha 2-HS glycoprotein and bovine fetuin prepared without use of ethanol inhibited trypsin and pancreatic and neutrophil elastases. These results indicate that mouse countertrypin is a new member of the mammalian fetuin family, which possibly has the trypsin-inhibiting activity in common.

Amino Acid Sequence↗
PubMed1993

Hamster alpha-macroglobulin and murinoglobulin: comparison of chemical and biological properties with homologs from other mammals.

alpha-Macroglobulin and murinoglobulin were purified to homogeneity from Syrian hamster plasma and their properties were compared with those of their respective homologs from other mammals. The trypsin-inhibiting capacity of hamster murinoglobulin was much weaker than those of rat and mouse murinoglobulins. Hamster alpha-macroglobulin was cleaved by trypsin at a number of sites whereas the human homolog was split essentially only in a "bait" region into two fragments of similar size. Hamster alpha-macroglobulin treated with methylamine differed from that treated with trypsin in the electrophoretic mobility, intensity of fluorescence induced by binding of bis(8-anilino-1-naphthalenesulfonate), and plasma clearance pattern, whereas virtually no difference was observed between the human homologs treated in the same manner. The reaction of hamster alpha-macroglobulin with methylamine, as measured by the generation of thiol groups and the decrease in trypsin-protein amidase activity, was much slower than that of the human homolog. Trypsin in a complex with hamster alpha-macroglobulin retained its fibrinolytic activity, but this was not the case for human or rabbit alpha-2-macroglobulin. These results suggest that, compared with the human homolog, hamster alpha-macroglobulin is more loosely packed in the native state, undergoes conformational change more slowly on treatment with methylamine, and less efficiently hinders the access of proteinaceous substrates to trapped proteinase. The serum concentration of hamster alpha-macroglobulin was 6.9 mg/ml, or about 3-fold higher than that of the human type, and showed little change during the acute-phase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗
PubMed1992

Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin.

We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin (bone sialoprotein) (nucleotides 265-886 and 1183-1424). Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that osteopontin is presumably involved in stone formation as stone matrix.

Base Sequence↗
PubMed1991

Cloning, structure and expression of cDNA for mouse contrapsin and a related protein.

A cDNA clone (lambda MC-2) for contrapsin, a serine-proteinase inhibitor, was isolated from a lambda ZAP mouse liver cDNA library. The 1.6 kb cDNA insert of lambda MC-2 contained an open reading frame that encodes a 418-residue polypeptide (46,970 Da), in which a signal peptide of 21 residues was identified by comparison with the N-terminal sequence of the purified protein. The predicted structure (MC-2) also contained other peptide sequences determined by Edman degradation. Four potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (63 kDa). Further screening of the cDNA library with an EcoRI-EcoRI fragment (510 bp) of lambda MC-2 as a probe yielded another cDNA clone (lambda MC-7), which encodes a 418-residue polypeptide (MC-7) with a calculated mass of 47,010 Da. MC-2 showed 83% similarity at the amino acid level to MC-7, in contrast with 44% similarity to alpha 1-proteinase inhibitor. The possible reactive site (P1-P'1) for serine proteinase is suggested to be Lys-Ala for MC-2 and Ser-Arg for MC-7. Northern-blot analysis revealed that both MC-2 and MC-7 mRNAs have the same size of 1.8 kb and are markedly induced in response to acute inflammation. Construction of the expression plasmids pSVMC-2 and pSVMC-7 and their transfection into COS-1 cells demonstrated that pSVMC-2 directs the synthesis of a 63 kDa form whereas pSVMC-7 expresses a 56 kDa form. The difference in molecular mass between the two may be explained by the fact that the MC-7 sequence contains three potential sites for N-glycosylation, one site less than that of MC-2.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and sequence analysis of cDNAs coding for guinea pig alpha 1-antiproteinases S and F and contrapsin.

The cDNAs encoding two isoforms, S (slow) and F (fast), of alpha 1-antiproteinase (also referred to as alpha 1-antitrypsin or alpha 1-proteinase inhibitor) as well as contrapsin were obtained by screening lambda gt11 cDNA library prepared fro inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of alpha 1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own alpha 1-antiproteinases F and S, rat alpha 1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of alpha 1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of alpha 1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P'3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple alpha 1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin superfamily.

Amino Acid Sequence↗
PubMed1991

Purification, characterization, and acute phase response of plasma alpha-1-antiproteinase in the hamster, Mesacricetus auratus.

1. alpha-1-Antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) with a molecular mass of 60 kDa was purified to apparent homogeneity from hamster plasma. 2. It inhibited elastase, chymotrypsin and trypsin, but did not significantly affect pancreatic kallikrein, plasma kallikrein or plasmin. 3. It has the same N-terminal heptapeptide sequence as that of rat alpha-1-antiproteinase. 4. Its plasma level decreased after injection of bacterial lipopolysaccharide.

Acute-Phase Proteins↗
PubMed1991

Cloning and sequencing of cDNA coding for rabbit alpha-1-antiproteinase F: amino acid sequence comparison of alpha-1-antiproteinases of six mammals.

Rabbit liver cDNA coding for alpha-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit alpha-1-antiproteinase F showed 74 and 64% homology to human alpha-1-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of alpha-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.

Amino Acid Sequence↗
PubMed1990

Electron microscopic demonstration of a common structural motif in human complement factor C3 and rat alpha 1-inhibitor 3 (murinoglobulin).

Electron microscopy of two homologous giant proteins revealed that complement factor C3 and alpha 1-inhibitor 3 have a common structural motif of a semicircularly bent string 18-20 nm long with two or three bumps indicating globular domains. C3 had a structure similar to the letter C with a small but distinct hole in the center. alpha 1-Inhibitor 3 was a more complete ring sometimes ajar at one corner. When the latter was treated with a proteinase, it became slightly flattened and adopted a squarish C-shape.

Acute-Phase Proteins↗
PubMed1990

Trypsin inhibitors in guinea pig plasma: isolation and characterization of contrapsin and two isoforms of alpha-1-antiproteinase and acute phase response of four major trypsin inhibitors.

Contrapsin and two isoforms, F (fast) and S (slow), of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of alpha-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of alpha-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and alpha-macroglobulin showed moderate (1.5 to 2.3-fold) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different alpha-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of alpha-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i.e., contrapsin, alpha-1-antiproteinase, alpha-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.

Acute-Phase Reaction↗
PubMed1990

Amino acid sequence at the reactive site of rabbit alpha-1-antiproteinases.

Rabbit alpha-1-antiproteinases S and F were treated with trypsin, chymotrypsin, Staphylococcus aureus protease V8, and thermolysin, and the liberated peptides encompassing the reactive region of the respective inhibitors were separated and sequenced. The reactive center of the F form was methionine, and the residues from P3 to P'1 (Ile-Pro-Met-Ser) were the same as those of human alpha-1-antiproteinase. The S form, on the other hand, was found to be a mixture of two distinct proteins (S-1 and S-2), and their reactive centers (P1-P'1) were Ser-Ser and Tyr-Ser, respectively. Seven out of 17 amino acids in the F form and 7 out of 16 in the S-1 form were the same as the corresponding residues of human alpha-1-antiproteinase, while 5 of 10 residues in the S-2 form were the same as those of the human inhibitor. Ten out of 16 residues were the same between the F and the S-1 forms, whereas the sequence P1 to P'3 of the S-2 form (Tyr-Ser-Met-Pro) was the same as the corresponding residues of mouse alpha-1-antiproteinase.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin.

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.

Amino Acid Sequence↗
PubMed1990

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.

Animals↗
PubMed1989

Changes in trypsin-binding properties and conformation of rabbit alpha-2-macroglobulin on reaction with methylamine.

Reactions of rabbit alpha-2-macroglobulin with methylamine and trypsin were studied and the results were compared with those obtained for previously described 2-macroglobulins from other species. Rabbit alpha-2-macroglobulin was cleaved by trypsin at a number of sites, whereas the human homologue was split essentially only in the "bait" region into two fragments of similar sizes. Reaction of native or methylamine-treated rabbit alpha-2-macroglobulin with trypsin resulted in a substantial decrease in the intensity of fluorescence induced by binding of 6-(p-toluidino)-2-naphthalenesulfonate or bis(8-anilino-1-naphthalenesulfonate). Under the same conditions, the fluorescence of the human protein increased. The time course of the reaction of rabbit alpha-2-macroglobulin with methylamine was studied by measuring (i) the generation of thiol groups, (ii) the decrease in trypsin-inhibiting activity with remazol brilliant blue hide powder as the substrate, and (iii) the decrease in trypsin-protein amidase activity. The thiol appearance reaction exhibited a multiphasic time course. The initial phase was found to follow second-order kinetics with an apparent rate constant of 1.2 M-1.s-1. Under the same conditions, the human protein showed monophasic kinetics with a rate constant of 12 M-1.s-1. Both the trypsin-inhibiting activity and the trypsin-protein amidase activity concurrently decreased at a slower rate than the thiol appearance. These results indicate that rabbit alpha-2-macroglobulin is more stable to nucleophilic attack by methylamine but less resistant to proteolysis by trypsin than the human homologue, and that the final conformation induced by methylamine differs considerably from that induced by trypsin.

Animals↗
PubMed1989

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin.

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.

Animals↗
PubMed1989

Isolation and partial characterization of a novel form of low molecular weight kininogen from guinea pig plasma.

Two kinds of low molecular weight kininogen (termed LK1 and LK2) were isolated from pooled plasma of guinea pigs. When polyclonal antisera raised against the individual proteins were used, immunological cross-reactions were observed between LK1 and high molecular weight kininogen (HK), but not either between LK1 and LK2 or between LK2 and HK. After tissue injury, plasma level of LK1 doubled while those of LK2 and HK remained relatively unchanged.

Animals↗
PubMed1988

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin.

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.

Amino Acid Sequence↗