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The Journal of Biological Chemistry Oct 1998In most hepatoma cells, the high-Km GLUT2/glucokinase proteins are replaced by the ubiquitous low-Km GLUT1/hexokinase type I proteins. In the mhAT3F hepatoma cells, the...
In most hepatoma cells, the high-Km GLUT2/glucokinase proteins are replaced by the ubiquitous low-Km GLUT1/hexokinase type I proteins. In the mhAT3F hepatoma cells, the stimulatory effect of glucose on gene expression and glycogen accumulation was not maximal at 5 mmol/liter glucose. This response to high glucose is observed in mhAT3F cells, where GLUT2 was expressed, but not glucokinase (assessed by Northern blotting and reverse transcription-polymerase chain reaction). A low-Km hexokinase activity (19.6 +/- 3.8 milliunits/mg of protein) was present, but a high-Km (40 mmol/liter) hexokinase activity (13.9 +/- 2.5 milliunits/mg) was also detected in mhAT3F cells. The high-Km hexokinase activity was dependent on both ATP (or PPi) and glucose in the assay and was recovered in a 10-50-kDa fraction after filtration. A 30-kDa protein was detected using an anti-glucokinase antibody and localized by confocal microscopy at the same sites as glucokinase in hepatocytes. In FAO cells, the high-Km hexokinase activity and 30-kDa protein were not found. We conclude that a high-Km hexokinase activity is present in mhAT3F cells. This might explain why the effects of glucose on gene expression were not maximal at a glucose concentration of 5 mmol/liter. A 30-kDa protein identified using an anti-glucokinase antibody may be responsible for this activity present in mhAT3F cells.
Topics: Adenosine Triphosphate; Animals; Carcinoma, Hepatocellular; Female; Gene Expression Regulation; Glucose; Glucose Transporter Type 2; Hexokinase; Immunohistochemistry; Kinetics; Mice; Microscopy, Fluorescence; Monosaccharide Transport Proteins; Nucleotides; RNA, Messenger; Rats; Rats, Wistar; Tumor Cells, Cultured
PubMed: 9748301
DOI: 10.1074/jbc.273.40.26187 -
Proceedings of the National Academy of... Apr 1999We have previously shown that surfactant protein A (SP-A) mediates in vitro killing of mycoplasmas by alveolar macrophages (AMs) from resistant C57BL/6 mice through a...
We have previously shown that surfactant protein A (SP-A) mediates in vitro killing of mycoplasmas by alveolar macrophages (AMs) from resistant C57BL/6 mice through a nitric oxide (.NO)-dependent mechanism. Herein, SP-A-deficient [SP-A(-/-)] and inducible.NO synthase-deficient [iNOS(-/-)] mice were infected intranasally with 10(5) or 10(7) colony-forming units of Mycoplasma pulmonis. SP-A(-/-) mice were as susceptible to mycoplasmal infection as highly susceptible C3H/He mice, and far more susceptible than resistant C57BL/6 mice. iNOS(-/-) mice had significantly greater numbers of mycoplasmas and severity of lung lesions than iNOS(+/+) controls. In vitro, AMs isolated from C57BL/6 mice, activated with IFN-gamma, incubated with SP-A (25 micrograms/ml), and infected with 10(10) colony-forming units of M. pulmonis, killed mycoplasmas within 6 h. Mycoplasmal killing was abrogated by 1,000 units/ml of copper-zinc superoxide dismutase. In the absence of AMs, incubation of M. pulmonis with the peroxynitrite generator 3-morpholinosynodiomine.HCl (SIN-1) effected complete killing of mycoplasmas by 90 min in a dose-dependent manner. Addition of copper-zinc superoxide dismutase (3,000 units/ml), which converts SIN-1 to a.NO donor, prevented this killing. Neither of the reactive oxygen species generated by xanthine oxidase (10 milliunits/ml, plus 500 microM xanthine and 100 microM FeCl3), nor.NO generated by 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate) (100 microM) killed mycoplasmas. These data establish that peroxynitrite generation by AMs is necessary for the killing of a pathogen in vitro and in vivo.
Topics: Animals; Cells, Cultured; Macrophage Activation; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Mycoplasma; Mycoplasma Infections; Nitrates; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants
PubMed: 10220400
DOI: 10.1073/pnas.96.9.4953 -
The Journal of Biological Chemistry Aug 1991We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis...
We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cattle; Cloning, Molecular; DNA; Electrophoresis, Polyacrylamide Gel; Kinetics; Liver; Male; Molecular Sequence Data; Multienzyme Complexes; Myocardium; Nucleic Acid Hybridization; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; RNA, Messenger; Rats; Restriction Mapping; Sequence Homology, Nucleic Acid; Testis
PubMed: 1651918
DOI: No ID Found -
Journal of Dairy Science Feb 1978Xanthine oxidase activity in milk was determined by measuring the rate of formation of vanillic acid from vanillin. Raw milk received at a dairy plant had .208 units...
Xanthine oxidase activity in milk was determined by measuring the rate of formation of vanillic acid from vanillin. Raw milk received at a dairy plant had .208 units xanthine oxidase activity per ml and after 24-h storage at 4 C, .228 units per ml. Upon further storage activity decreased. Heating the fresh raw milk in a water bath to 55 C increased xanthine oxidase activity to .236 units per ml. Partial inactivation of the enzyme occurred when milk was heated at 60, 65, or 70 C for 5 min, and destruction was almost complete with heat at 75 C for 5 min. Raw milk heated at 48 C for 5 min and homogenized at pressures between 70.3 and 281.2 kg/cm2 had xanthine oxidase activities which were a linear function of pressure and showed that each additional kg/cm2 pressure resulted in additional xanthine oxidase activity of .16 milliunits per ml of milk.
Topics: Animals; Cattle; Female; Food Handling; Food Preservation; Hot Temperature; Milk; Pressure; Xanthine Oxidase
PubMed: 641238
DOI: 10.3168/jds.S0022-0302(78)83573-5 -
The Journal of Biological Chemistry Feb 1982We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and...
We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.
Topics: ATP Citrate (pro-S)-Lyase; Animals; Cytosol; In Vitro Techniques; Insulin; Kinetics; Liver; Male; Phosphorylation; Protein Kinases; Rats; Rats, Inbred Strains
PubMed: 7035458
DOI: No ID Found -
Proceedings of the National Academy of... May 2004Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn...
Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41-61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.
Topics: Alkaline Phosphatase; Escherichia coli; Hydrogen; Hydrogenase; Hydrolysis; Mutation; Oxidation-Reduction; Phosphates; Phosphites; Protons
PubMed: 15148399
DOI: 10.1073/pnas.0400664101 -
Proceedings of the National Academy of... Jan 1996Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In...
Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.
Topics: Amino Acid Sequence; Cell Membrane; Cyclic AMP; GTP-Binding Proteins; Humans; Molecular Sequence Data; Peptides; Receptors, Thyrotropin; Signal Transduction; Thyroid Gland; Thyrotropin
PubMed: 8552586
DOI: 10.1073/pnas.93.1.116 -
The Journal of Biological Chemistry Jun 1990In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77%... (Comparative Study)
Comparative Study
In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.
Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Amino Acid Sequence; Animals; Blotting, Western; Brain; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Crystallins; Electrophoresis, Polyacrylamide Gel; Humans; Hydroxyprostaglandin Dehydrogenases; Japan; Lens, Crystalline; Liver; Lung; Molecular Sequence Data; Molecular Weight; Rana catesbeiana; Sequence Homology, Nucleic Acid; Substrate Specificity
PubMed: 2190986
DOI: No ID Found -
Proceedings of the National Academy of... Apr 1979Thyroid cells, obtained from both normal human tissue and benign nodular goiter, were cultured and maintained in vitro in 4-18 passages. Cultures with confluent cells...
Thyroid cells, obtained from both normal human tissue and benign nodular goiter, were cultured and maintained in vitro in 4-18 passages. Cultures with confluent cells accumulated cyclic AMP (10-150 times the basal amount) upon addition of bovine thyrotropin (100 milliunits/ml), indicating that the cells in culture maintained a thyrotropin-sensitive adenylate cyclase system. Addition of high doses of thyrotropin also induced a characteristic and reversible change in the morphology of the cells. The effect of thyrotropin on cell growth was studied in short- and long-term experiments. Thyrotropin reduced [(3)H]thymidine incorporation in a dose-dependent fashion in all cultures of thyroid cells. The maximal inhibition over a 24-hr period was about 50%. The thyroid cells were notably sensitive, and the half-maximal effect occurred at about 100 milliunits of thyrotropin per ml. In contrast, the hormone had no effect on [(3)H]-thymidine incorporation into human glial cells. Low doses of thyrotropin also had no effect on human fibroblasts and, at high doses, a stimulation of [(3)H]thymidine incorporation was seen. Thyroid cell cultures grown in the presence of 10 milliunits of thyrotropin per ml for 7-14 days had a slower growth rate and 24-36% lower cell numbers at saturation density than control dishes, indicating that the hormone also had a long-term effect on cell proliferation. The data agree with in vitro studies by others of the effects of corticotropin and lutropin on target cells and suggest that in vivo the primary action of pituitary trophic hormones on endocrine tissues is not stimulation of growth.
Topics: Cell Division; Cells, Cultured; Cyclic AMP; DNA; Goiter; Humans; Kinetics; Thymidine; Thyroid Gland; Thyroid Neoplasms; Thyrotropin
PubMed: 221911
DOI: 10.1073/pnas.76.4.2022 -
The Journal of Biological Chemistry Jul 1975Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and...
Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.
Topics: Animals; Cattle; Cyclic AMP; Enzyme Activation; Fluorides; In Vitro Techniques; Kinetics; Osmolar Concentration; Phosphorus Radioisotopes; Prostaglandins E; Protein Kinases; Sodium Chloride; Swine; Thyroid Gland; Thyrotropin; Tritium
PubMed: 168196
DOI: No ID Found