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The Journal of Biological Chemistry Jul 1998Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism,...
Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.
Topics: 3-O-Methylglucose; Animals; Biological Transport; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Humans; Insulin; Mice; Mice, Transgenic; Monosaccharide Transport Proteins; Muscle Proteins; Muscle, Skeletal
PubMed: 9660777
DOI: 10.1074/jbc.273.29.18173 -
The Journal of Biological Chemistry Jan 1990A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer...
A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.
Topics: Animals; Brain; Digitonin; Glucose; Glucosephosphates; Hexokinase; L-Lactate Dehydrogenase; Male; Mitochondria; Rats
PubMed: 2295599
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1991von Willebrand factor (vWf) is a multimeric plasma glycoprotein that functions in hemostasis as the initiator of platelet adhesion to damaged blood vessels and as the...
von Willebrand factor (vWf) is a multimeric plasma glycoprotein that functions in hemostasis as the initiator of platelet adhesion to damaged blood vessels and as the carrier of Factor VIII (FVIII). Montgomery et al. (Montgomery, R.R., Hathaway, W.E., Johnson, J., Jacobsen, L., and Muntean, W. (1982) Blood 60, 201-207) reported a variant of von Willebrand disease characterized by the abnormal interaction between FVIII and a defective vWf. To identify the molecular basis of this abnormal interaction, we isolated platelet RNA from members of one of the affected families and determined the nucleotide sequence of the FVIII-binding domain encoded by the vWf mRNA. A single G to A transition at nucleotide 2561 was linked with disease expression and results in the substitution of Gln for Arg91 in mature vWf. A restriction fragment containing this mutation was introduced into a full-length vWf expression vector, and both wild type and mutant vWf were expressed in COS-7 cells. In a solid-phase binding assay, expressed vWf was captured with anti-vWf monoclonal antibody AVW1 and then incubated with 6.25-400 milliunits of recombinant FVIII. After washing, vWf-bound FVIII activity was determined with a chromogenic assay. Mutant vWf showed reduced binding of FVIII compared with wild type, suggesting that the substitution of Gln for Arg91 is the likely basis for the abnormal vWf/FVIII interaction in this von Willebrand disease variant.
Topics: Arginine; Autoradiography; Base Sequence; Cell Line; Electrophoresis, Polyacrylamide Gel; Factor VIII; Gene Expression; Glutamine; Humans; Molecular Sequence Data; Mutation; Polymorphism, Genetic; RNA, Messenger; von Willebrand Diseases; von Willebrand Factor
PubMed: 1918030
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1995The isolated working rat heart was adapted for simultaneous determination of glycogen synthesis and degradation using a dual isotope technique. After prelabeling of...
The isolated working rat heart was adapted for simultaneous determination of glycogen synthesis and degradation using a dual isotope technique. After prelabeling of glycogen with [U-14C]glucose, glycogenolysis was determined continuously from the washout of 14CO2 plus [14C]lactate. Glycogen synthesis was determined during the same period from incorporation of [5-3H]glucose. In the absence of added hormones, hearts were predominantly glycogenolytic (1.5 mumol/min/g, dry weight), and there was simultaneous synthesis (11% of the rate of glycogenolysis). The percentage of glucose taken up by the heart that could traverse the glycogen pool as a consequence of glycogen turnover was minor (5%). Insulin (10 milliunits/ml) predictably stimulated glycogen synthesis (3.6-fold) and nearly abolished glycogenolysis. Addition of glucagon (1 microgram/ml) increased contractile performance and initially stimulated glycogenolysis (3.8-fold) until glycogen was largely depleted. Net tritium incorporation was unaffected by glucagon. Both hormones stimulated glycolytic flux from exogenous glucose (3H2O from [5-3H]glucose) as well as total glycolytic flux (3H2O plus glycogenolysis). The initial stimulation in total glycolytic flux with glucagon was largely from glycogen, explaining the lag in stimulation from exogenous glucose. The relationship between the specific radioactivity and amount of glycogen remaining after different degrees of glycogenolysis suggests that the preference of glycogenolysis for newly synthesized glycogen is only partial.
Topics: Animals; Carbon Dioxide; Glucose; Glycogen; Lactates; Lactic Acid; Male; Myocardium; Perfusion; Rats; Rats, Sprague-Dawley
PubMed: 7721842
DOI: 10.1074/jbc.270.16.9234 -
The Journal of Biological Chemistry Nov 2002The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty... (Comparative Study)
Comparative Study
The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis. PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established. Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His(6)-PhaG, gave K(0.5) values of 28 microm (ACP) and 65 microm (3-hydroxyacyl-CoA) considering V(max) values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz). In addition, the alignment with the alpha/beta-hydrolase fold region indicated that PhaG belongs to alpha/beta-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% alpha-helix, 22% beta-sheet, 18% beta-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme. Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.
Topics: Acyltransferases; Amino Acid Sequence; Amino Acid Substitution; Animals; Chromatography, Gel; Circular Dichroism; Epoxide Hydrolases; Fatty Acids; Kinetics; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Pseudomonas putida; Recombinant Proteins; Sequence Alignment; Subcellular Fractions
PubMed: 12200450
DOI: 10.1074/jbc.M207821200 -
The Journal of Biological Chemistry Nov 1986Adenylate cyclase activity in purified rat adipocyte membranes is stimulated by the calcium- and phospholipid-dependent enzyme protein kinase C. Over the concentration...
Adenylate cyclase activity in purified rat adipocyte membranes is stimulated by the calcium- and phospholipid-dependent enzyme protein kinase C. Over the concentration range of 100-1000 milliunits/ml, both highly purified (approximately 3000 units/mg of protein) protein kinase C from rat brain and partially purified (14 units/mg of protein) protein kinase C from guinea pig pancreas stimulate cyclase activity. The actions of both protein kinase C preparations on adenylate cyclase activity are dependent on added calcium, which is effective at concentrations less than 10 microM. Exogenous phospholipids are not required for stimulation of adenylate cyclase by protein kinase C; but, under typical cyclase assay conditions, the adipocyte membranes satisfy the lipid requirement for protein kinase C phosphorylation of histone. The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate enhances the kinase action on cyclase, and the phorbol ester is effective at concentrations equimolar with the kinase (less than 10 nM). With the brain protein kinase C, 12-O-tetradecanoylphorbol-13-acetate effects are especially evident at limiting calcium concentrations. Inhibitors of protein kinase C activity, such as chlorpromazine, palmitoylcarnitine, and polymyxin B, inhibit selectively that adenylate cyclase activity which is stimulated by protein kinase C plus calcium. It is concluded that protein kinase C acts directly on the adipocyte adenylate cyclase system.
Topics: Adenylyl Cyclases; Adipose Tissue; Animals; Brain; Calcium; Cell Membrane; Chlorpromazine; Enzyme Activation; Guinea Pigs; Kinetics; Male; Palmitoylcarnitine; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate
PubMed: 3771540
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Jan 1993Glucose oxidase and peroxidase (lactoperoxidase or myeloperoxidase) are virucidal to human immunodeficiency virus type 1 (HIV-1) in the presence of sodium iodide, as...
Glucose oxidase and peroxidase (lactoperoxidase or myeloperoxidase) are virucidal to human immunodeficiency virus type 1 (HIV-1) in the presence of sodium iodide, as assessed by the loss of viral replication in a syncytium-forming assay or by the inhibition of cytopathic effects on infected cells. In the presence of low concentrations of sodium iodide, five HIV-1 isolates were equally susceptible to this virucidal system at enzyme concentrations of a few milliunits. The loss of viral replication was linearly related to the time of incubation in the enzyme solutions, with an inactivation rate of 1 log unit every 30 min. These enzymes and this halide were also cytotoxic to chronically infected, but not to uninfected, cultured CEM cells. Protein conjugates were prepared by using the enzymes and murine antibody 105.34, which recognized the V3 loop of HIV-1 LAI isolate surface glycoprotein, or recombinant human CD4. The protein conjugates inactivated free virus at rates similar to those of the free enzymes and were more effective than antibody or recombinant CD4 alone. These in vitro findings demonstrate that the peroxidase-H2O2-halide system provides potent virucidal activity against HIV-1.
Topics: Antibodies, Monoclonal; Antiviral Agents; CD4 Antigens; Cell Line; Enzyme-Linked Immunosorbent Assay; Glucose Oxidase; HIV-1; Humans; Lactoperoxidase; Peroxidase; Peroxidases; Proteins; Virus Replication
PubMed: 8381638
DOI: 10.1128/AAC.37.1.26 -
The Journal of Biological Chemistry Feb 1999Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 micrograms/ml) are found circulating in plasma during diverse inflammatory events. The saturable,...
Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 micrograms/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (Kd = 6 nM) at 4 degreesC to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by approximately 50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome c reduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both protease-reversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.
Topics: Animals; Cattle; Cell Compartmentation; Cells, Cultured; Endothelium, Vascular; Hot Temperature; Humans; Kinetics; Nitric Oxide; Oxidation-Reduction; Rabbits; Rats; Signal Transduction; Substrate Specificity; Xanthine Oxidase
PubMed: 9988743
DOI: 10.1074/jbc.274.8.4985 -
Journal of Bacteriology May 1978A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of...
A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.
Topics: Chromosome Mapping; Chromosomes, Bacterial; Escherichia coli; Genes; Genetic Linkage; Glutamate-Ammonia Ligase; Glutamine; Mutation; Transduction, Genetic; UDPglucose-Hexose-1-Phosphate Uridylyltransferase
PubMed: 26660
DOI: 10.1128/jb.134.2.569-577.1978 -
Proceedings of the National Academy of... Sep 2001Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was...
Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an "edible vaccine" provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication.
Topics: Animals; Genetic Vectors; Hepatitis B; Hepatitis B Surface Antigens; Mice; Plants, Genetically Modified; Plasmids; Solanum tuberosum
PubMed: 11553782
DOI: 10.1073/pnas.191617598