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T J Wheeler

Publications and source records attributed to T J Wheeler.

33 records · Page 2Linked to original sources
PubMed1986

Reconstitution of glucose transport activity from erythrocyte membranes without detergent and its use in studying effects of ATP depletion.

The direct reconstitution of unsolubilized membrane proteins by the freeze-thaw procedure avoids possible changes in properties produced by detergent solubilization and fractionation. Glucose transport activity was reconstituted using human erythrocyte membranes, with about 2/3 of the glucose uptake being stereo-specific. The highest specific activity occurred at low ratios of protein to lipid in the reconstitution, where most transport was due to liposomes containing single transporter molecules. Transporters were reconstituted with a scrambling of orientations, indicated by a 50% inactivation by added trypsin. Separation of unreconstituted protein doubled the specific activity. Similar results were obtained using the purified transporter (Wheeler, T.J. and Hinkle, P.C. (1981) J. Biol. Chem. 256, 8907-8914). The same ratio of net uptake to equilibrium exchange was observed for the two preparations. Their relative reconstituted transport activities and cytochalasin B binding activities were equal, indicating that the two were reconstituted with similar efficiencies. The decrease in glucose transport in erythrocytes produced by ATP depletion and the stimulation produced by resealing with ATP (Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378) were confirmed. However, no difference was observed in reconstituted transport activity using ghosts resealed with or without ATP, indicating that ATP produces indirect effects rather than modifications of the transporter.

Adenosine Triphosphate↗
PubMed1985

Reconstitution of the glucose transporter from bovine heart.

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.

Animals↗
PubMed1985

The glucose transporter of mammalian cells.

The glucose transporter is now identified but may have modifications or other subunits that control its activity. The kinetics and inhibitor binding studies are consistent with the carrier model with different degrees of asymmetry and a single binding site that varies in specificity depending on the conformation of the protein. The physical structure could actually be quite different from the usual diagrams (rocking bananas), however, and could function as a monomer or higher oligomer. The binding site, or filter, that gives specificity could be in the middle as usually depicted; alternatively it could be entirely on the cytoplasmic side, where the protein is trypsin sensitive, and hydrophobic helices could span the membrane forming a simple channel. Possible restrictions on structures in the membrane from the hydrophobic nature of transmembrane segments of membrane proteins (62) may favor a globular domain outside the membrane as the binding site. Such speculations will have to await more structural information about the transporter.

Adipose Tissue↗
PubMed1984

Insulin-induced translocation of intracellular glucose transporters in the isolated rat adipose cell.

Three techniques have now been used to demonstrate that insulin stimulates glucose transport in isolated rat adipose cells through the translocation of glucose transporters from a large intracellular pool to the plasma membrane. By using a specific D-glucose-inhibitable cytochalasin B-binding assay, most of the basal cell's transporters are found associated with a low-density microsomal membrane fraction. However, although Golgi marker enzyme activities are also enriched in this fraction, their distributions over all fractions do not parallel that of the transporters. In response to insulin, more than half of the intracellular transporters are translocated to the plasma membranes without a corresponding redistribution of marker enzyme activities. Furthermore, although the Kd of the transporters in the plasma membranes remains constant at approximately 100 nM, that of the intracellular transporters decreases from approximately 140 to approximately 100 nM. Nevertheless, transport activity is reconstitutable from, and an affinity-purified rabbit IgG against the purified human erythrocyte transporter cross-reacts with a 45,000-dalton band in, both plasma membranes and the low-density microsomal membrane fraction in proportion to the number of glucose transporters determined by cytochalasin B binding. Thus, intracellular glucose transporters in the rat adipose cell appear to be 1) localized to a unique membrane species, 2) either compartmentalized in two distinguishable pools or processed during their cycling to the plasma membrane in response to insulin, but fully functional and indistinguishable when reconstituted into liposomes, and 3) immunologically similar to the human erythrocyte glucose transporter.

Adipose Tissue↗
PubMed1981

Kinetic properties of the reconstituted glucose transporter from human erythrocytes.

The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter.

Blood Glucose↗
PubMed1980

Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle.

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.

AMP Deaminase↗
PubMed1979

Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl.

Adenylate deaminase from rat skeletal muscle has been studied with the objective of understanding how the activity of the enzyme is regulated in vivo. ATP and GTP inhibit the enzyme at low concentrations in the presence of 150 mM KCl. The ATP inhibition is reversed as the ATP concentration is raised to physiological levels. The GTP inhibition is reversed as the GTP concentration is raised to unphysiologically high levels. In the presence of physiological concentrations of ATP, the GTP inhibition is also greatly diminished, but inhibition by orthophosphate remains strong. The apparent affinities of the enzyme for GTP, ATP, and orthophosphate are reduced as the pH is decreased from 7.0 to 6.2. ADP also reduces the apparent affinities of the enzyme for the inhibitors. The regulatory effects of GTP, ATP, and ADP are produced primarily by their unchelated forms. Comparison of the kinetic behavior of the enzyme in vitro with metabolite concentrations in vivo indicates that the major variables that regulate the activity of adenylate deaminase of muscle in vivo are the concentrations of AMP, ADP, orthophosphate, and H+.

AMP Deaminase↗
PubMed1979

Creatine phosphate inhibition of adenylate deaminase is mainly due to pyrophosphate.

Inhibition of rat skeletal muscle adenylate deaminase by creatine phosphate reported previously is due to inorganic pyrophosphate present as a contaminant in commercial preparations of creatine phosphate. This conclusion is based on the following evidence: a compound that inhibits adenylate deaminase can be separated from commercially prepared creatine phosphate by ion exchange chromatography; the inhibition by "creatine phosphate" and by the separated inhibitory compound is relieved by treatment with inorganic pyrophosphatase; inhibition by inorganic pyrophosphate is similar to that produced by unpurified creatine phosphate; and pyrophosphate is present in commercially available creatine phosphate in amounts sufficient to account for the inhibition. Some commercial preparations of creatine phosphate contain much less pyrophosphate than others; these preparations are only weakly inhibitory. Inorganic triphosphate is a more powerful inhibitor of the enzyme than pyrophosphate; it may also be present as a contaminant in creatine phosphate.

AMP Deaminase↗
PubMed1975

Acute sedative properties of SKF 525A in rats: implications for its use as a metabolism inhibitor in the study of psychoactive drugs.

SKF 525A was found possess sedative properties in rats at doses of 25 and 50 mg per kg, when injected intraperitoneally. The behavioural effects of the drug were assessed in two ways. Firstly, by "Time Sampling Behavioural Categorisation" of explatory behaviour; and secondly by activity measurements obtained with an ultrasonic motion recorder. The results clearly demonstrate that SKF 525A has sedative properties in rats at doses which are conventionally used to inhibit metabolism of a wide range of drugs. The implications of these results for the use of SKF 5251 in the study of the actions of psychotropic drugs are discussed.

Animals↗