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J Robbins

Publications and source records attributed to J Robbins.

At least 19 recordsLinked to original sources
PubMed1979

Effect of deglycosylation on the binding and immunoreactivity of human thyroxine-binding globulin.

Thyroxine-binding globulin (TBG), prepared from human serum by an improved purification method, was treated with a mixture of neuraminidase, beta-galactosidase, alpha-mannosidase, and beta-N-aectylglucosaminidase, which resulted in the removal of approximately 86% of saccharides. Purification by thyroxine-Sepharose affinity chromatography gave a homogeneous protein as shown by equilibrium sedimentation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Amino acid and NH2-terminal sequence analysis indicated that the protein moiety was intact. Deglycosylation had no effect on the stoichiometry of the binding of L-thyroxine as shown by tryptophanyl fluorescence quenching and equilibrium dialysis at pH 8.6 and 25 degrees C. However, the affinity constant for L-thyroxine was reduced from 1.6 X 10(9) M-1 to 0.58 X 10(9) M-1. Analysis of radioimmunoassay data revealed that deglycosylation resulted in a slight decrease of the affinity constant for anti-TBG antibody from 3.9 X 10(10) M-1 to 1.8 X 10(10) M-1. These results suggest that the polypeptide moiety, rather than the heterosaccharides, contains the antigenic determinants. Removal of the majority of the heterosaccharides of TBG has only a minor effect on its immunoreactivity and on the binding of thyroid hormone.

Amino Acids↗
PubMed1979

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.

Animals↗
PubMed1979

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.

Animals↗
PubMed1978

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol.

Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.

Animals↗
PubMed1978

Quantification of myosin heavy-chain mRNA during myogenesis.

Complementary DNA (cDNA) was synthesized from a 26-S poly(A)-containing RNA fraction consisting predominately of myosin mRNA, and used to quantitate the amounts of myosin mRNA during development. RNA . cDNAmyosin hybridizations were performed with RNA isolated from tertiary cultures of chick fibroblasts, and the proportion of myosin RNA determined. The levels of myosin RNA present in primary chick myoblast cultures undergoing development were also analyzed by RNA . cDNAmyosin hybridizations. Correlation of these values with the rate of myosin heavy chain synthesis indicates that myosin mRNA is subject to post-transcriptional or translational control.

Animals↗
PubMed1978

Thyroxine inactivation by starvation in cultured hepatocarcinoma cells, Formation of reverse triiodothyronine.

The effect of starvation on thyroid hormone metabolism was studied in monkey hepatocarcinoma monolayer cultures. Nonphenolic ring monodeiodination of thyroxine, 3, 5, 3'-triiodothyronine and 3, 3'-diiodothyronine was accelerated. Since phenolic ring deiodination of 3, 3',5'-triiodothyronine (reverse T3) was unaffected, this metabolite accumulated in the medium during thyroxine metabolism. This suggests that increased serum reverse T3 in malnourished humans may be caused by enhanced deiodination of thyroxine rather than decreased rT3 catabolism.

Animals↗
PubMed1978

[Hormonal control of thyroxine-binding globulin synthesis and metabolism (author's transl)].

Serum thyroxine-binding globulin (TBG), the major plasma transport protein for thyroid hormones in man, was shown to be altered under the influence of estrogen and in hypothyroidism. In order to study these alterations, we used an animal model. Synthesis of TBG was demonstrated in hepatocytes isolated from adult Rhesus monkeys, and in a monkey hepatocarcinoma continuous cell culture line (NCLP-6-E). When the hepatocytes were obtained from monkeys pretreated with beta-estradiol (E2), a specific 2.5-2.9 fold increase of TBG synthesis and secretion was shown; similar data were obtained with the tumor line. Furthermore, an increased TBG production was shown with these cells when T4 (from 10(-14) to 10(-11) M) was added to the culture medium. The in vitro results were correlated with in vivo data obtained by investigating the metabolism of TBG after iv injection of tracer doses of purified, radiolabelled TBG to normal, E2-treated and thyroidectomized monkeys. The main effect of estrogen administration was a marked increase of the production rate. During hypothyroidism, the catabolism and the production rate of TBG were decreased. In both conditions, there were significant changes in the distribution volume of TBG, the physiologic relevance of which remains to be investigated.

Animals↗
PubMed1977

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.

Amino Acids↗
PubMed1977

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.

Circular Dichroism↗