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Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 415 records · Page 23Linked to original sources
PubMed1983

Polymorphism of alpha-actinin. Electrophoretic and immunological studies of rabbit skeletal muscle alpha-actinins.

Heterogeneity of alpha-actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related alpha-actinins from rabbit fast, white muscle. One isoprotein (designated type I alpha-actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II alpha-actinin) appears to be preferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast-twitch muscles. A standard polyacrylamide gel--sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different alpha-actinin dimers and to determine their subunit compositions. By this technique, both type I and type II alpha-actinins appeared to be homodimers. No heterodimeric species of alpha-actinin were detected. alpha-Actinin of red, slow-twitch muscles was similar to type II alpha-actinin of fast, white muscle on one-dimensional and two dimensional gels. However, slow, red muscle alpha-actinin was significantly different from fast, white muscle alpha-actinins in terms of one-dimensional peptide mapping and immunological cross-reactivity.

Actinin↗
PubMed1983

Purification and characterization of an alpha-actinin-like protein from porcine kidney.

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.

Actinin↗
PubMed1983

Microtiter latex antiglobulin test for the detection of antibodies to DNP, digoxin, HCG and similar antigens.

A microtiter latex antiglobulin test (LAGT) with heavy-and-stable latex particles was developed. Antibodies of weak avidity, such as rabbit antisera to DNP or digoxin were much enhanced by the LAGT, whereas the method only slightly promoted strong avidity antibody, such as that found in rabbit antisera to human chorionic gonadotrophin. A microtiter latex agglutination-inhibition test (LAGIT) proved to be an effective tool for detection of weak antigenic substances such as digoxin and DNP.

Animals↗
PubMed1983

Quantitation of DNP, digoxin, HCG and anti-DNP antibody by electronic data processing of latex antiglobulin reaction results.

Volumes of agglutinates produced by the microtiter latex antiglobulin test (LAGT) were measured by an electronic data processing (EDP) method using a particle volume distribution analyzer. This method proved highly sensitive for quantitating weak antibodies, such as anti-digoxin and anti-DNP. EDP measurement of the microtiter latex antiglobulin-inhibition test (LAGIT) is also a suitable method for quantitating HCG, digoxin and DNP.

Animals↗
PubMed1983

Human hypoxanthine-guanine phosphoribosyltransferase.

A mutant form of human hypoxanthine-guanine phosphoribosyltransferase (HPRTToronto) was isolated from erythrocytes of a male patient with gout due to a partial deficiency of enzyme activity. The tryptic peptides of HPRTToronto were mapped by reverse-phase high pressure liquid chromatography in an attempt to define the precise abnormality in its primary structure. Sequence analysis of the single aberrant peptide in HPRTToronto revealed an arginine to glycine amino acid substitution at position 50. A single nucleotide change in the codon for arginine 50 (CGA leads to GGA) could explain this substitution.

Amino Acid Sequence↗
PubMed1983

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid.

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.

Actins↗
PubMed1983

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes.

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KC1, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KC1 and 2 mM MgCl2 but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.

Actins↗
PubMed1983

Purification and some properties of porcine kidney tropomyosin.

A tropomyosin has been purified from porcine kidney and its properties compared with those of rabbit skeletal muscle tropomyosin. Kidney tropomyosin was separated from contaminating vascular smooth muscle tropomyosin by hydroxylapatite chromatography. Kidney tropomyosin resembles tropomyosin from other non-muscle cells in regard to subunit size, mobility on SDS-polyacrylamide gels in the presence and absence of 6 M urea, amino acid composition and morphology. The binding of tropomyosin to F-actin is strongly dependent on the Mg2+ concentration. With kidney tropomyosin, binding begins at 1 mM Mg2+ and is complete at about 4-5 mM, while with muscle tropomyosin, binding is initiated at 1 mM Mg2+ and reaches saturation at 2-3 mM Mg2+. Both kidney and muscle tropomyosins bind to actin in a similar ratio of 1 tropomyosin/6-7 actin monomers at saturation. Both kidney and skeletal muscle tropomyosins prevent "severing" of F-actin filaments induced by gelsolin/villin-like protein purified from kidney. These results suggest that, in non-muscle cells, tropomyosin may protect microfilaments from Ca2+-dependent solation at the site where they may interact with myosin.

Actins↗
PubMed1983

Partial purification and characterization of myosin light chain kinase from bovine thyroid gland.

Myosin light chain kinase has been purified approximately 1700-fold from bovine thyroid gland, using Affigel blue column chromatography, ammonium sulfate fractionation (0-60% saturation), Sepharose 6B gel filtration, and calmodulin-Sepharose affinity column chromatography. This partially purified kinase was Ca2+ and calmodulin dependent for its activity and specifically phosphorylated the 20,000-dalton light chain of chicken gizzard myosin. At higher CaCl2 concentration, thyroid myosin light chain kinase demonstrated a novel inhibition in the presence of calmodulin. The mechanism of this Ca2+-dependent inhibition is not clear at present. The existence of myosin light chain kinase in thyroid gland provides additional support for the role of the contractile system in the secretion of thyroid hormones.

Animals↗
PubMed1982

Human hypoxanthine-guanine phosphoribosyltransferase. Tryptic peptides and post-translational modification of the erythrocyte enzyme.

We describe the isolation and characterization of tryptic peptides of human erythrocyte hypoxanthine-guanine phosphoribosyltransferase. The digest was separated by reverse-phase high pressure liquid chromatography into 30 peaks, 26 of which contained purified peptides. The four complex peaks were resolved by high pressure liquid chromatography with a different reverse-phase column. Each peptide predicted from the recently described amino acid sequence of human erythrocyte hypoxanthine-guanine phosphoribosyltransferase was isolated from this peptide map. Sequence analysis of the purified peptides identified two peptides, 14 and 14d, that differed by an Asn/Asp heterogeneity at the position corresponding to residue 106 of the intact protein. Additional studies indicate that this heterogeneity is due to deamidation of the protein in vivo. This post-translational modification appears to be responsible for some of the electrophoretic heterogeneity observed in the normal erythrocyte enzyme.

Amino Acid Sequence↗
PubMed1982

Actin in Triton-insoluble cytoskeleton of thyroid.

Treatment of bovine thyroid with the non-ionic detergent Triton X-100 extracts most of the cell protein and leaves insoluble residue. This Triton-insoluble cytoskeleton consists of five major polypeptides on sodium dodecyl sulfate polyacrylamide gels. One of these polypeptides is actin. Based on DNase inhibition assay, 30% of the total actin is associated with the cytoskeleton as the filamentous form. Thyroid actin from the cytoskeleton has been solubilized by dialysis against a low ionic strength buffer at pH 8.0 and purified to homogeneity by a polymerizing-depolymerizing cycle. The overall purification was about 144-fold with a yield of 10%. Bovine thyroid actin is very similar to actins from other tissues on the basis of: (1) comigration with rabbit skeletal muscle actin during gel electrophoresis in sodium dodecyl sulfate, (2) its amino acid composition, which includes about 1 mole of 3-methylhistidine per 42,000 g, (3) its ability to bind and inhibit pancreatic deoxyribonuclease I, and (4) its ability to form 7-8 nm microfilaments which is similar to that of skeletal filamentous actin. Thyroid actin contains beta- and gamma-isoactins, with isoelectric points more alkaline than the alpha-actin of rabbit skeletal muscle.

Actins↗
PubMed1982

Paraquat poisoning--a review of four cases.

Paraquat poisoning has been associated with very high mortality. The toxic mechanism is considered to be related to the production of superoxide ions. Review of the literatures reveals that with vigorous treatment recovery from poisoning is not impossible. Clinical experience in four cases of paraquat poisoning and the principles of management of this intoxication are presented.

Adult↗