flavin-adenine-dinucleotide and Hyperthyroidism

Topics: Animals; Diabetes Mellitus; Electron Transport; Female; Flavin-Adenine Dinucleotide; Glucose; Humans; Hydrolases; Hyperinsulinism; Hyperthyroidism; Insulin; Insulin Secretion; Islets of Langerhans; Kynurenine; Membranes; Mitochondria, Liver; Mixed Function Oxygenases; Pregnancy; Quinaldines; Rats; Streptozocin; Thyroxine; Transaminases

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Dactinomycin; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hyperthyroidism; Hypothyroidism; In Vitro Techniques; Iodine Isotopes; Liver; Male; Phosphotransferases; Rats; Riboflavin; Thyroid Hormones; Thyroxine

A full-length 2.4-kb cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was cloned from rat liver using PCR techniques. The cloned gene encodes a protein of 727 amino acids. The calculated molecular mass of 80,898 Da is higher than the apparent molecular mass observed by SDS/PAGE (74,000 Da) of the purified enzyme. This result indicates that the enzyme is synthesized as a precursor with a putative mitochondrial signal sequence. mRNA for this gene was detected in liver, heart, muscle, brain, testes, and pancreas. With the exception of testes, basal expression levels were very low in all tissues examined. However, application of thyroid hormones led to a 10- to 15-fold increase in liver glycerol-3-phosphate dehydrogenase mRNA, whereas hypothyroidism further decreased the mRNA level.

Topics: Amino Acid Sequence; Animals; Bacillus subtilis; Base Sequence; Brain; Cloning, Molecular; DNA, Complementary; Flavin-Adenine Dinucleotide; Gene Expression Regulation, Enzymologic; Glycerolphosphate Dehydrogenase; Hyperthyroidism; Hypothyroidism; Liver; Male; Mitochondria, Liver; Molecular Sequence Data; Molecular Weight; Muscles; Myocardium; Organ Specificity; Pancreas; Polymerase Chain Reaction; Rats; Rats, Wistar; Recombinant Proteins; RNA, Messenger; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Testis; Thyroid Hormones

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.

Topics: Animals; Brain; Enzyme Induction; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Hyperthyroidism; Immunosorbent Techniques; Male; Mitochondria, Liver; Molecular Weight; NADH Dehydrogenase; Protein Biosynthesis; Rats; Rats, Inbred Strains; RNA, Messenger; Swine

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.

Topics: Amino Acids; Animals; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Glycerolphosphate Dehydrogenase; Hyperthyroidism; Kinetics; Mitochondria, Liver; Rats; Thyroid Gland

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.

Topics: Animals; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hyperthyroidism; Hypothyroidism; Liver; Male; Nucleotidases; Nucleotidyltransferases; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Pyrophosphatases; Rats; Rats, Inbred Strains; Thyroid Hormones

Topics: Animals; Erythrocytes; Flavin-Adenine Dinucleotide; Glutathione Reductase; Hyperthyroidism; Hypothyroidism; In Vitro Techniques; Liver; Male; Rats; Thyroxine

Topics: Animals; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hyperthyroidism; Hypothyroidism; Liver; Male; Nucleotidyltransferases; Phosphoric Monoester Hydrolases; Phosphotransferases; Rats; Thyroidectomy; Thyroxine

Topics: Animals; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hyperthyroidism; Liver; Liver Extracts; Rats; Riboflavin