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Biomedical subjects

Y Hamaguchi

Publications and source records attributed to Y Hamaguchi.

At least 163 records · Page 9Linked to original sources
PubMed1978

The isolation and characterization of a trypsin inhibitor from Kintoki bean (Phaseolus vulgaris).

A trypsin inhibitor was isolated from beans of Phaseolus vulgaris, cultivar. Kintoki, and the specific activity increased 200 times as high as that of the crude extract. It was homogeneous on several electrophoreses and the molecular weight was about 13,000. The amino acid composition was characterized by high ratios of cystine, aspartic acid, and serine. It inhibited trypsin in a molar ratio of 1 : 1 and alpha-chymotrypsin in a molar ratio of 2 : 1. It, however, inhibited neither pepsin nor pronase. It was relatively stable to heat treatment in the acidic medium, but not in the alkaline medium. Neither pepsin nor pronase destroyed the inhibitory function.

Amino Acids↗
PubMed1977

Use of rabbit antiboty IgG bound onto plain and aminoalkylsilyl glass surface for the enzyme-linked sandwich immunoassay.

Rabbit antibody IgG was bound onto aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, rabbit antibody IgG was also bound onto glutaraldehyde-activated aminoalkylsilyl glass rods. These antibody-glass rods were tested by the sandwich procedure using Fab' fragments of rabbit antibody conjugated with beta-D-galactosidase from Escherichia coli. The glutaraldehyde-activated aminoalkylsilyl glass showed the largest capacity to bind antigen and the plain glass showed the smallest. However, the antibody-glass rods prepared by simple adsorption were as useful for the sandwich immunoassay of macromolecular antigens as those prepared with glutaraldehyde. With all the antibody-glass rods prepared, 0.1 to 10 fmol of ornithine delta-aminotransferase from rat liver and 2,4-dinitrophenyl human IgG were measurable. More than 10 fmol of the antigens may be measurable with larger amounts of the antibody-beta-D-galactosidase complexes, although the non-specific binding of the complexes to the solid phase increases to limit the sensitivity of the immunoassay.

Animals↗
PubMed1977

[Pharmacological studies on diuretics (8). Diuretic activity and mechanism of action of a new hypotensive diuretic, SE-1520 (author's transl)].

Effects of SE-1520 were compared with those of thiazide diuretics, hydrochlorothiazide (HCT) and trichlormethiazide (TCM) in rats and dogs. SE-1520 in an oral dose of 0.1 approximately 10.0 mg/kg resulted in a strong and dose-dependent increase in urine volume as well as in the urinary excretion of Na+ and Cl-, while that of K+ significantly increased with an oral dose over 3.0 mg/kg. In experimental acidotic, alkalotic, nephritic and spontaneously hypertensive rats, almost the same strong natriuretic activity seen in normal rats was observed with SE-1520. The diuretic potency of these three diuretics in rats was MCT greater than or equal to SE-1520 greater than HCT. SE-1520 also exhibited a strong diuretic activity in dogs. In the renal clearance test of dogs, SE-1520 0.1 approximately 1.0 mg/kg i.v. did not influence the renal plasma flow (RPF) or glomerular filtration rate (GFR). The renal tubular Na+ reabsorption rate decreased by a maximum of 2.11% and the urinary Na+ excretion increased by 15.3 times the normal level 20 min after the injection of SE-1520 in a dose of 1.0 mg/kg i.v. From the results of clearance test and stop flow test in dogs, the site of action of SE-1520 was observed to be mainly in the distal tubules.

Acidosis↗
PubMed1977

The isolation and characterization of a lethal protein from Kintoki beans (Phaseolus vulgaris).

A lethal protein with hemagglutinating activity but without trypsin inhibitory activity was isolated from beans of Phaseolus vulgaris, cultiva, and Kintoki and proved homogeneous by ultracentrifugation, disc polyacrylamide gel electrophoresis, sodium dodesyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight was estimated to be 104, 000 by ultracentrifugal analysis and gel filtration on Sephadex G-200. The molecule dissociates into three identical subunits in the presence of 8 M urea or 0.1% sodium dodesyl sulfate. The amino acid composition was characterized by the high content of aspartic acid and the complete absence of methionine and cystine. The carbohydrate content was 8.1%; 5.0% mannose and 3.1% glucosamine. The addition of the lethal protein to a basal diet (0.4%) resulted in the intensive depression of the growth and finally in the death of rats. The intraperitoneal injection of 250 microgram per g body weight of mouse brought about an acute toxicity which caused death of all the injected mice.

Amino Acids↗
PubMed1976

Enzyme-linked sandwich immunoassay of macromolecular antigens using the rabbit antibody-coupled glass rod as a solid phase.

A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab' - beta-D-galactosidase complex and the rabbit antibody immunoglobulin-G-coupled glass rod as a solid phase is described. The Fab' fragments of rabbit antibody IgG are conjugated with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide. Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde. A wide range of the concentrations of rabbit IgG fraction (20-2000 mug/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled. The smallest amounts of ornithine delta-aminotransferase from rat liver, human immunoglobulin G and 2,4-dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes. The assay with the rabbit antibody immunoglobulin-G-coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin-G-coupled Sepharose 4B.

Adsorption↗
PubMed1976

Enzyme-linked immunoassay. Conjugation of rabbit anti-(human immunoglobulin G) antibody with beta-D-galactosidase from Escherichia coli and its use for human immunoglobulin G assay.

Rabbit immunoglobulin G (IgG) was reduced by incubating with 10 mM 2-mercaptoethylamine and then treated with excess amounts of N,N'-o-phenylenedimaleimide. As a result, maleimide residues were introduced into rabbit IgG molecules. Rabbit IgG containing maleimide residues could be coupled to beta-D-galactosidase from Escherichia coli which has sulfhydryl groups in the molecule. The resulting rabbit IgG (antibody)-enzyme complex may be useful for immunoassay of antigens. As an example, human IgG was assayed by the sandwich method using the rabbit anti-(human IgG) IgG-beta-D-galactosidase complex and amounts of human IgG as small as 3 fmoles were measurable.

Animals↗
PubMed1976

Enzyme-linked sandwich immunoassay of ornithine delta-aminotransferase from rat liver using antibody-coupled glass rods as solid phase.

A macromolecular antigen, ornithine delta-aminotransferase [EC 2.6.1.13] from rat liver (OAT) was assayed by the sandwich procedure using rabbit (anti-OAT) Fab'-beta-D-galactosidase complex and rabbit (anti-OAT) IgG-coupled glass rods as a solid phase. The Fab' fragments of the rabbit (anti-OAT) IgG were conjugated with beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N, N'-o-phenylenedimaleimide. The rabbit (anti-OAT) IgG was coupled to the aminoalkylsilyl glass rods using glutaraldehyde. The rabbit (anti-OAT) IgG-coupled glass rods were incubated with OAT and then with the rabbit (anti-OAT) Fab'-beta-D-galactosidase complex. The amount of OAT was determined from the activity of beta-D-galactosidase bound to the glass rods. A minimum of 0.03 fmoles of OAT could be determined by this method and use of the glass rods gave greater reproducibility, and was more sensitive and simpler than use of Sepharose 4B.

Animals↗
PubMed1976

Enzyme-linked immunoassay: conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from E. coli and its use for immunoassay.

1. A method for the conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from Escherichia coli is described. The method consists of two main steps: treatment of the Fab' fragments containing sulfhydryl groups with excess N,N'-o-phenylenedimaleimide, to introduce maleimide residues into the fragments, and then incubation of the dimaleimide-treated Fab' fragments with beta-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab'-beta-D-galactosidase complex. More than 90% of the enzyme used can be converted to the Fab'-enzyme complex, and the complex is readily separated from free Fab' fragments by chromatography on a Sepharose 6B column. 2. The application of the rabbit Fab'-beta-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG by the sandwich method. The rabbit (anti-human IgG) IgG-coupled Sepharose 4B is incubated with human IgG and then with the rabbit (anti-human IgG) Fab'-enzyme complex, and the enzyme activity bound to the Sepharose is measured. In this way it is possible to determine as little as 0.3 fmoles of human IgG.

Animals↗
PubMed1975

Studies on new intracellular proteases in various organs of rat. 2. Mode of limited proteolysis.

1. The mechanism of proteolysis of ornithine transaminase apoenzyme II by group-specific protease and the relation between the confirmations of ornithine transaminase and its susceptibility to group-specific protease were studied to elucidate the mode of action of the protease. 2. Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group-specific protease, while the holoenzyme was not. 3. Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group-specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme II was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively. 4. The initial step of intracellular proteins degradation is discussed on the basis of these results.

Amino Acids↗
PubMed1975

Enzyme-linked immunoassay. II. A simple method for synthesis of the rabbit antibody-beta-D-galactosidase complex and its general applicability.

Rabbit anti-human IgG antibody was mildly reduced in the presence of 10 mM 2-mercaptoethylamine and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. Human IgG and the anti-human IgG antibody-beta-D-galactosidase complex were successively adsorbed on Sepharose 4B binding rabbit anti-human IgG antibody. In this way amounts of human IgG as small as 5X10(-15) moles could be measured by determining the activity of beta-D-galactosidase bound to the Sepharose.

Animals↗
PubMed1975

Enzyme-linked immunoassay. I. Novel method for synthesis of the insulin-beta-D-galactosidase conjugate and its applicability for insulin assay.

Pork insulin was subjected to mercaptosuccinylation and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. The competitive binding of the conjugate and insulin to anti-insulin antibody was tested. Results showed that formation of an insulin-beta-D-galactosidase conjugate could be used for immunoassay of insulin.

Binding, Competitive↗