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P C Hinkle

Publications and source records attributed to P C Hinkle.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1975

Kinetics of adenosine triphosphate synthesis in bovine heart submitochondrial particles.

The kinetics of ATP synthesis by submitochondrial particles were studied with a continuous flow mixing technique allowing measurements of ATP synthesis between 15 and 120 ms after mixing for estimation of the initial rate. During the initial 100 ms, ATP synthesis proceeded at a faster rate when driven by an artificially imposed electrochemical proton gradient than when driven by the respiratory substrate NADH. The rate of ATP synthesis driven by the artificial electrochemical proton gradient was dependent upon the magnitude of the gradient, and was affected similarly by variations of either the pH gradient or membrane potential. Initiation of ATP synthesis driven by NADH oxidation with ADP plus Pi resulted in an initial rate of phosphorylation equivalent to that during steady state respiration. However, a lag in phosphorylation was observed when ATP synthesis was initiated by oxygen or NADH. Valinomycin caused transient uncoupling of ATP synthesis driven by respiration since it strongly inhibited ATP synthesis during the initial 100 ms but it had no effect during steady state phosphorylation. The kinetic results strongly suggest that an electrochemical proton gradient is an obligate intermediate between electron transport and ATP synthesis on the main pathway of energy transduction.

Adenosine Triphosphate↗
PubMed1975

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.

Adenosine Triphosphatases↗