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Biomedical subjects

S Ohtaki

Publications and source records attributed to S Ohtaki.

At least 145 records · Page 8Linked to original sources
PubMed1981

Highly sensitive sandwich enzyme immunoassay of human IgE with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human IgE was developed. Polystyrene balls were coated with goat anti-human IgE immunoglobulin (IgG) by physical adsorption. Goat anti-human IgE Fab' was purified by affinity chromatography and conjugated with beta-D-galactosidase from Escherichia coli. Using thus prepared anti-IgE-coated polystyrene balls and anti-IgE-beta-D-galactosidase conjugate, 0.2 mU (2 amol)--1 U of IgE per assay could be determined. When 0.1 microliter of serum per assay was used, the range of IgE levels in serum that could be determined was 2--10000 U/ml, and even 0.01 U/ml was measurable by using 20 microliters of serum. The regression equation and coefficient for correlation to radioimmunoassay were gamma (RIA) = 0.94 chi (EIA) + 18.2 and 0.96 (n = 81), respectively. The coefficients of within- and between-assay variations ranged from 5.4 to 8.5%. The mean levels of serum IgE determined by the present assay were 103 U/ml in 70 normal children and 1064 U/ml in 38 children with bronchial asthma.

Antibody Specificity↗
PubMed1981

Analyses of catalytic intermediates of hog thyroid peroxidase during its iodinating reaction.

The formation of Compounds I, II, and III of hog thyroid peroxidase was demonstrated in its reaction with H2O2 by spectrophotometric and kinetic methods combined with a stopped flow technique. Hog thyroid peroxidase reacted with H2O2 to form Compound I with a rate constant of 5.2 X 10(6) M-1 s-1. Compound I was then spontaneously converted to Compound II with a rate constant of 8 s-1. In the presence of 200 microM H2O2, Compound II ws further converted to Compound III (or oxyform) with a half-life of 0.8 s. During the peroxidative oxidation of tyrosine, thyroid peroxidase was present as Compound I, and the rate constant for the reaction of Compound I with tyrosine was measured at 1.4 X 10(4) M-1 s-1. The addition of iodide to the above reaction system markedly decreased the steady state concentration of Compound I. From the kinetic data, it was concluded that the reaction between Compound I and iodide occurred by way of a 2-electron transfer and the rate constant was roughly estimated at 2.1 X 10(7) M-1 s-1. This value was much higher than that obtained from overall kinetic data. Similar reactions were carried out with bovine lactoperoxidase and the kinetic data for the two enzymes were compared. The primary oxidation product of iodide was suggested to be iodinium cation (I+) in either enzyme reaction.

Animals↗
PubMed1981

Stimulation of protein-bound iodine formation by lipid extracts from hog thyroid microsomes.

The lipid materials extracted from hog thyroid microsomes were found to contain factors which stimulated both the NADPH oxidation by NADPH-cytochrome c reductase and PBI formation by a reconstituted system containing the reductase as a hydrogen peroxide generator and thyroid peroxidase. For both reactions, the stimulating activity in the extracts from the other subcellular fractions was less than that observed in the extracts from microsomal fraction. Lipids extracted from hog liver microsomes did not show any NADPH oxidation stimulating activity. Thin-layer chromatography of the lipid extracts from thyroid microsomes revealed the presence of at least two stimulating factors differing in chromatographic behaviour. At least one of the stimulating factors separated on TLC stimulated the oxygen consumption accompanied by generation of hydrogen peroxide due to the NADPH oxidation by NADPH-cytochrome c reductase. From these results it was suggested that in thyroid microsomes, but not in liver microsomes, there were stimulating factors extractable by chloroform-methanol. The factors could stimulate NADPH oxidation by NADPH-cytochrome c reductase followed by generation of hydrogen peroxide, resulting in the enhancement of PBI formation coupled with thyroid peroxidase.

Animals↗
PubMed1981

Iodide transport of thyroid plasma membranes.

Plasma membranes consisting of closed vesicles were isolated from hog thyroid homogenate. The membrane vesicles showed uphill transport of iodide from an external medium containing monovalent cations, of which K+ induced iodide transport more potently than Na+. The activity of the iodide transport expressed as T/M[I-] was as little as 3 to 11 in the presence of K+, but was invariably present. The ratio reached a maximum within about 10 min and then decreased fairly rapidly to unity. The addition of SCN- or CIO-4 to the external medium inhibited iodide transport. The transport activity was found to be maximum at pH 7.0 to 7.5 in the external medium. A kinetic study showed that the transport rate was saturated with respect to the iodide concentration. These observations suggested the presence of a carrier-mediated iodide transport system which was coupled with K+ flux across the plasma membrane.

Animals↗
PubMed1981

A new protease in hog thyroid lysosomes. I. The presence of a leupeptin-sensitive protease in the soluble fraction of thyroid lysosomes.

The presence of protease activity at pH 7.5 and at acidic pH was demonstrated in the soluble fraction of lysosomes which were prepared from hog thyroid homogenates by subcellular fractionation. The ratios of specific activity measured at pH 7.5 to that at pH 3.5 were 0.07 to 0.08 for casein (at pH 7.5) or haemoglobin (at pH 3.5) as substrate and 0.13 for iodoamino acid-releasing activity from thyroglobulin as substrate. The protease activity measured at pH 7.5 was inhibited by inhibitors for neutral type protease, such as leupeptin, and by sulfhydryl inhibitors, such as iodoacetamide, indicating that the protease was a leupeptin-sensitive protease and contained a sulfhydryl group for its active site. Insensitiveness of the protease activity to di-isopropyl fluorophosphate excluded the involvement of a serine group at the active site. At pH 5.5 the proteolytic and iodoamino acid-releasing activities using casein and thyroglobulin, respectively, were also investigated. Several lines of evidence suggested that both activities measured at pH 5.5 were due to a mixture of acidic and leupeptin-sensitive protease activities.

Animals↗
PubMed1981

A new protease in hog thyroid lysosomes. II. A partial purification and characterization of a leupeptin-sensitive protease.

A leupeptin-sensitive new protease was partially purified from hog thyroid lysosomes. The purification procedure included solubilization by hypotonic treatment of lysosomes, and Sephacryl S-300 and Sephadex G-100 gel chromatography, and the purification ratio was 10-fold from lysosomes. The pH optimum of the protease activity was around 5.5 and its molecular weight was estimated to be 22 000 by gel filtration. 2-Mercaptoethanol activated the hydrolysis of protein substrates and its effect was most pronounced in the case of thyroglobulin as substrate. Among the inhibitors used, leupeptin, antipain, toluenesulfonyl-lysine chloromethyl ketone and, to a lesser degree, chymostatin and toluene-sulfonyl-phenylalanine chloromethyl ketone effectively inhibited the hydrolysis of casein by the enzyme at pH 5.5, whereas pepstatin did not inhibit the activity significantly. The enzyme activity was also inhibited by sulfhydryl inhibitors such as iodoacetamide, p-chloromercuribenzoate, and N-ethylmaleimide. The release of iodoamino acids from thyroglobulin by the enzyme was inhibited in the same manner by the inhibitors used in the hydrolysis of casein. The physiological role of the new protease is discussed in comparison with cathepsin B and L found in liver lysosomes.

Animals↗
PubMed1981

Amyloid fibril protein in type I familial amyloidotic polyneuropathy in Japanese.

Amyloid fibrils were isolated from the kidneys of two Japanese patients with type I FAP and fractionated on Sephadex G-100 gel column. The elution pattern showed three peaks. Molecular weight of the third peak protein was about 14,000, which was absent in the chromatogram obtained from the tissue of a nonamyloidotic patient. Antisera raised against whole denatured amyloid fibril components reacted with this protein, giving rise to a precipitin arc on immunoelectrophoretic analysis in the area almost corresponding to prealbumin but slightly cathodal to it. The amino acid composition of this protein was different from those of AA protein. AL protein, or normal human PreA subunit. The major subunit GAM III of amyloid fibril protein (AFj) appears immunologically related to PreA.

Amino Acids↗
PubMed1980

An enzyme immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum.

An enzyme-linked sandwich immunoassay using human thyroglobulin conjugated with beta-D-galactosidase and silicone rods coated with human thyroglobulin was developed for the measurement of circulating anti-thyroglobulin autoantibody. The volume of serum needed for the assay was as little as 5 microliter. The sensitivity of the assay was approximately 7 x 10(-15) mol/tube of antithyroglobulin immunoglobulin G corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by (1) parallelism of the standard curve with dilution of sample sera of patients with thyroid diseases, and (2) non-detectability of anti-thyroglobulin in autoantibody in the sera of normal subjects. The precision of the assay was proven by (1) sufficient recovery of antithyroglobulin immunoglobulin G added to serum, and (2) coefficients of variance within and between assays were 6.5 to 10.3% and 4.9 to 14.1%, respectively. No effect of thyroglobulin on the present assay was observed when the ratio of the amount of thyroglobulin to that of anti-thyroglobulin immunoglobulin G was lower than 1 : 10. Furthermore, a significant correlation was observed between anti-thyroglobulin autoantibody concentrations measured by our enzyme immunoassay and those by tanned red cell hemagglutination (r = 0.78), and between those by the enzyme immunoassay and those by radioimmunoassay (r = 0.80). The application to clinical samples ensured the high sensitivity and the adequate validity of the present assay.

Autoantibodies↗