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P C Hinkle

Publications and source records attributed to P C Hinkle.

At least 19 recordsLinked to original sources
PubMed1991

Mechanistic stoichiometry of mitochondrial oxidative phosphorylation.

P/O ratios of rat liver mitochondria were measured with particular attention to systematic errors. Corrections for energy loss during oxidative phosphorylation were made by measurement of respiration as a function of mitochondrial membrane potential. The corrected values were close to 1, 0.5, and 1 at the three coupling sites, respectively. These values are consistent with recent measurements of mitochondrial proton transport.

Adenosine Diphosphate↗
PubMed1990

Information content of amino acid residues in putative helix VIII of the lac permease from Escherichia coli.

Mutants in putative helix VIII of lactose permease that retain the ability to accumulate lactose were created by cassette mutagenesis. A mutagenic insert encoding amino acid residues 259-278 was synthesized chemically by using reagents contaminated with 1% each of the other three bases and ligated into a KpnI/BclI site in the lacY gene in plasmid pGEM-4. Mutants that retain transport activity were selected by transforming a strain of Escherichia coli containing a wild-type lacZ gene, but deleted in lacY, with the mutant library and identifying colonies that transport lactose on indicator plates. Sequencing of the mutated region in lacY in 129 positive colonies reveals 43 single amino acid mutations at 26 sites and 26 multiple mutations. The variable amino acid positions are largely on one side of the putative alpha-helix, a stripe opposite Glu269. This mutable stripe of low information content is probably in contact with the membrane phospholipids.

Amino Acid Sequence↗
PubMed1988

Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase.

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1986

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent.

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.

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↗
PubMed1979

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.

Adenosine Diphosphate↗
PubMed1978

Characterization of the glucose transporter from human erythrocytes.

The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394--7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight. The KD (0.14 micrometer) and extent (11 nmoles/mg protein) for binding of 3H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.

Biological Transport↗
PubMed1978

How cells make ATP.

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Adenosine Triphosphate↗
PubMed1977

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.

Adenine Nucleotides↗
PubMed1977

Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. I. Symmetrical facilitated diffusion and proton gradient-coupled transport.

Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.

Biological Transport↗
PubMed1976

Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.

A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes rather than binding to the reconstituted liposomes.

Biological Transport↗
PubMed1975

Reconstitution of ion transport and respiratory control in vesicles formed from reduced coenzyme Q-cytochrome c reductase and phospholipids.

Reduced coenzyme Q-cytochrome c reductase from bovine heart mitochondria (complex III) was incorporated into phospholipid vesicles by the cholate dialysis procedure. Soybean phospholipids or mixtures of purified phosphatidylcholine, phosphatidylethanolamine, and cardiolipin could be used. Oxidation of reduced coenzyme Q2 by the reconstituted vesicles with cytochrome c as oxidant showed the following energy-coupling phenomena. 1. Protons were translocated outward with a coupling ratio, H+/2e, of 1.9 +/- 0.2. Measurements with mitochondria under similar conditions showed an H+/2e ratio of 1.8. Proton translocation was not seen in the presence of uncoupling agents and was in addition to the net acidification of the medium from the over-all oxidation reaction. 2. Potassium ions were taken up by the reconstituted vesicles in the presence of valinomycin in a reaction coupled to electron transfer. The coupling ratio for K+ uptake, K+/2e, was 2.0 in the vesicles and approximately 1.5 in mitochondria. 3. The rate of oxidation of reduced coenzyme Q2 by the reconstituted vesicles was stimulated up to 10-fold by uncouplers or by valinomycin plus nigericin and K+ ions. Addition of valinomycin alone in a K+ medium caused a transient stimulation of electron transfer. The results indicate that energy coupling can be observed with isolated reduced coenzyme Q-cytochrome c reductase if the enzyme complex is properly incorporated into a phospholipid vesicle.

Animals↗
PubMed1975

Ion transport and respiratory control in vesicles formed from reduced nicotinamide adenine dinucleotide coenzyme Q reductase and phospholipids.

NADH-coenzyme Q reductase from bovine heart mitochondria (complex I) was incorporated into phospholipid vesicles by the cholate dialysis procedure. Mixtures of purified phosphatidylcholine and phosphatidylethanolamine were required. Oxidation of NADH by coenzyme Q1 catalyzed by the reconstituted vesicles was coupled to proton translocation, directed inward, with an H+/2e ratio greater than 1.4. Similar experiments measuring proton translocation in submitochondrial particles gave an H+/2e ratio of 1.8. The proton translocation in both systems was not seen in the presence of uncoupling agents and was in addition to the net proton uptake from the reduction of coenzyme Q1 by NADH. Electron transfer in the reconstituted vesicles also caused the uptake of the permeant anion tetraphenylboron. The rate of electron transfer by the reconstituted vesicles was stimulated about 3-fold by uncouplers or by valinomycin plus nigericin and K+ ions. The results indicate that energy coupling can be observed with isolated NADH-coenzyme Q reductase if the enzyme complex is properly incorporated into a phospholipid vesicle.

Animals↗