Biomedical subjects

H Sinohara

Publications and source records attributed to H Sinohara.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1988

Amidolytic activity of porcine trypsin bound to human plasma alpha-1-antiproteinase.

Human plasma alpha-1-antiproteinase interacted with porcine trypsin in two different manners. One was a well known interaction, which resulted in inhibition of the proteolytic activity of the trypsin. The other has not been described to date, and resulted in retention of the amidolytic activity of the trypsin towards benzoyl-L-arginine p-nitroanilide in the presence of soybean trypsin inhibitor. The latter, so-called trypsin-protein amidase, activity is essentially the same as that observed with vertebrate alpha-macroglobulin and rodent murinoglobulin under similar conditions. All attempts to separate the two different activities as well as to abolish either activity by means of chemical or physical modifications were unsuccessful. The proteolysis-inhibiting interaction, which was virtually completed within 5 min, was predominant over the amidolysis-retaining interaction, when the inhibitor/trypsin molar ratio was less than 1. On the other hand, the amidolysis-retaining interaction, which proceeded much more slowly, became evident when the molar ratio was greater than 1.

Amidohydrolases↗

PubMed1988

Acute phase response of plasma proteins in analbuminemic rats.

In healthy Nagase analbuminemic rats (NAR), the highest degree of relative increase in serum protein concentration was found for alpha-2-macroglobulin, the most prominent acute phase protein in rats. Its levels were about 30- and 60-fold higher in males and females, respectively, than those in the control Sprague-Dawley (SD) rats. In terms of absolute concentration, however, alpha-1-inhibitor 3 (also called alpha-X-protein or murinoglobulin) showed the most conspicuous change, its levels being higher by about 7 mg/ml than those in SD. When the acute phase reaction was induced by subcutaneous injection of turpentine, the levels of alpha-1- and alpha-2-macroglobulins, alpha-1-cysteine proteinase inhibitor, alpha-1-antiproteinase, and alpha-1-inhibitor 3 in NAR changed in essentially the same way as in SD: alpha-1-inhibitor 3 decreased markedly while the rest increased further. These results suggest that mechanisms responsible for the elevation of serum globulins in healthy NAR are not directly related to those involved in the acute phase response. On the other hand, the antithrombin III levels in healthy NAR were about twice the control values and changed little during the inflammation. In contrast, this protein in SD doubled during the acute phase, its maximal levels being close to those in healthy or inflamed NAR. This suggests that the antithrombin III level in healthy NAR is regulated by a mechanism similar to that in SD maximally reacting to the acute phase stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Proteins↗

PubMed1988

Synthesis of contrapsin and alpha-1-antiproteinase in inflamed and tumor-bearing mice.

In mice bearing ascites tumors, such as Ehrlich, L1210, Meth-A, and P-815, the plasma levels of contrapsin and alpha-1-antiproteinase remained virtually unchanged. However, the total body pools of these proteins as well as their hepatic mRNA levels increased severalfold, and isoelectrofocusing patterns of these proteins shifted to the lower pH side. Under the same conditions, plasma albumin level decreased by 25%, but its total body pool and hepatic mRNA level increased severalfold. On the other hand, induction of acute phase reaction by injection of bacterial lipopolysaccharide caused differential effects on the two proteinase inhibitors: hepatic translatable mRNA for contrapsin was doubled while that for alpha-1-antiproteinase remained unchanged.

Animals↗

PubMed1987

Sex-steroid-binding plasma protein (SBP), testosterone, oestradiol and dehydroepiandrosterone (DHEA) in prepuberty and puberty.

Measurement of sex-steroid-binding plasma protein in serum of healthy individuals in prepuberty and puberty (74 males and 94 females) was performed using a radioimmunoassay procedure. An age-related decrease of serum SBP was demonstrated during these ages in both sexes. In parallel studies, the serum level of testosterone, oestradiol and dehydroepiandrosterone was evaluated in subjects under 20 years of age. A rise of serum dehydroepiandrosterone levels in both sexes was observed to occur at approximately 8 years of age, being a little bit earlier than the ages for testosterone to rise in males and for oestradiol to rise in females, both of them being at about 10 years of age, respectively. When the testosterone/sex-steroid-binding plasma protein ratio was evaluated as an index of free testosterone concentration in serum, a sharp increase of the ratio was found to occur at 10 years of age and to continue during puberty in both sexes with more marked increment in males than in females. It was suggested that the decrease of SBP in puberty might be largely influenced by alteration in the hormonal balance of testosterone, oestradiol and dehydroepiandrosterone.

Adolescent↗

PubMed1986

Isolation and characterization of alpha-macroglobulin from guinea pig plasma.

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.

Animals↗

PubMed1986

Guinea pig plasma murinoglobulin. Purification and some properties.

Murinoglobulin, a newly identified mouse plasma protein resembling alpha-macroglobulins [Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781], was also found in guinea pig plasma, and purified to homogeneity. Guinea pig murinoglobulin consisted of a single 180-kDa polypeptide chain containing about 18% carbohydrate. It inhibited the proteolytic activities of trypsin and thermolysin towards Remazol brilliant blue hide powder, but stimulated the amidolytic activities of trypsin and Staphylococcus aureus V8 protease towards small synthetic substrates. Heat treatment of murinoglobulin completely abolished the former activities, but partially retained the latter activities. The ability of guinea pig murinoglobulin to inhibit the proteolysis was much weaker than that of the mouse homologue. On interaction with trypsin, murinoglobulin underwent cleavage of one susceptible bond with concomitant unmasking of one thiol group. Methylamine treatment also released one thiol group per molecule.

Amino Acids↗

PubMed1986

Studies of sex-steroid-binding plasma protein (SBP) in Graves' disease before and under antithyroid drug therapy.

The serum concentration of sex-steroid-binding plasma protein (SBP) was measured with radioimmunoassay for 92 patients with Graves' disease, 36 males and 56 females, as well as 92 age- and sex-matched controls. In 24 male patients with hyperthyroidism, the mean SBP concentration was 6.4 +/- 0.2 (mean +/- SD) microgram/ml, whereas it was 2.3 +/- 0.2 micrograms/ml for the controls. The mean SBP concentration in 28 female patients with hyperthyroidism was 7.4 +/- 0.6 micrograms/ml, whereas it was 3.2 +/- 0.6 micrograms/ml for the controls. In Graves patients who were euthyroid following treatment, SBP levels were decreased near normal in both sexes. There was a significant correlation between T4 and SBP concentrations as well as between T3 and SBP concentrations in patients with Graves' disease in both sexes, with a better correlation in females. Increased SBP in untreated Graves' disease initiated to decline within a month following administration of antithyroid drugs, taking over 3 months to decline to the normal range.

Adolescent↗

PubMed1986

Effects of alpha-macroglobulins and murinoglobulins on the hemagglutination by influenza C virus.

Rat alpha-1- and alpha-2-macroglobulins as well as rat murinoglobulins I and II were shown to inhibit hemagglutination by influenza C virus. In marked contrast, neither alpha-macroglobulins nor murinoglobulins from mouse or guinea pig plasma had the inhibitory activity. These results suggest that the hemagglutination-inhibiting activity of rat alpha-macroglobulins or murinoglobulins is not related to their protease-binding capacity.

Animals↗