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G Rudnick

Publications and source records attributed to G Rudnick.

80 records · Page 5Linked to original sources
PubMed1978

Reconstitution of 5-hydroxytryptamine transport from cholate-disrupted platelet plasma membrane vesicles.

The bile acid cholate disrupts platelet plasma membrane vesicles and inactivates 5-hydroxytryptamine transport activity. The transporter appears not to be solubilized by this procedure, but remains associated with a high molecular weight aggregate. Merely removing cholate does not restore the vesicular structure or transport activity, but in the presence of added soybean phospholipids, cholate removal generates proteoliposomes which accumulate 5-hydroxytryptamine. This reconstituted transport activity is similar in its ionic requirements and inhibitor sensitivity to transport in native membrane vesicles and intact platelets.

Biological Transport↗
PubMed1978

Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium.

Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine.

Animals↗
PubMed1977

Active transport of 5-hydroxytryptamine by plasma membrane vesicles isolated from human blood platelets.

Plasma membrane vesicles isolated from human platelets accumulate 5-hydroxytryptamine when an electrical potential (interior negative) or an Na+ gradient (out greater than in) is imposed across the vesicle membrane. Kinetic studies reveal a Km of 0.5 micronM for the transport process. Uptake is inhibited strongly by tricyclic antidepressants and by ionophores such as gramicidin which catalyze transmembrane exchange of Na+ for K+. Transport is absolutely dependent upon external Na+ and Cl- and is only mildly, if at all, inhibited by reserpine, cinanserin, ouabain, or arsenate. Experiments are presented which suggest that a single positive charge crosses the vesicle membrane with each molecule of 5-hydroxytryptamine. The results provide direct evidence for Na+-coupled active 5-hydroxytryptamine transport by the platelet plasma membrane.

Biological Transport, Active↗
PubMed1976

Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli.

p-Nitrophenyl alpha-D-galactopyranoside is a competitive inhibitor of lactose transport in membrane vesicles prepared from Escherichia coli ML 308-225 (Ki congruent to 6.6 muM) but is not accumulated by the vesicles. Binding of p-nitrophenyl alpha-D-[6-3H]galactopyranoside to membrane vesicles has been measured by flow dialysis. In the presence of D-lactate, ligand binds to the vesicles with a KD of about 6 muM, and a total of 2.3 nmol per mg of membrane protein is bound at saturation. In the absence of D-lactate, a small amount of binding can be detected (approximately 0.2 nmol per mg of membrane protein) with a similar affinity constant (KD congruent to 9 muM). Binding inthe presence or absence of D-lactate is dependent upon a functional lac y gene product and upon the structural integrity of the vesicle membrane and is reversed by p-hydroxymercuribenzenesulfonate. Agents such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and valinomycin, alone or in combination, abolish D-lactate-dependent binding but do not affect binding in the absence of electron donors. The results confirm previous observations that the bulk of the lac carrier protein is unable to bind ligand unless the membrane is energized. and they also corroborate observations that a small amount of binding occurs in the absence of energy coupling. The findings are discussed in terms of a model in which the lac carrier protein exists in a state of dynamic equilibrium between two forms: (i) a low affinity, cryptic form which predominates in the absence of energy coupling; and (ii) a high affinity form, accessible from the external surface of the membrane, which predominates in the presence of an electrochemical gradient of protons (interior negative and alkaline).

Bacterial Proteins↗
PubMed1975

Reaction mechanism and structure of the active site of proline racemase.

Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.

Amino Acid Isomerases↗
PubMed1975

Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with an impermeant azidophenylgalactoside.

2'-N-(2-Nitro-4-azidophenyl) aminoethyl-1-thio-beta-D-galactopyranoside (APG2) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 30 to 40 muM, but is not transported. When irradiated with visible light in the presence of D-lactate, APG2 irreversibly inactivates the lac transport system. Imposition of a membrane potential (positive outside) by valinomycin-induced potassium efflux also causes APG2 photoinactivation. Strikingly, photoinactivation is not observed in the absence of D-lactate or a potassium diffusion gradient. Kinetic studies of the inactivation process yield a KD of 35 muM. Since lactose protects against the inactivation, it is apparent that these effects are specific for the lac transport system. The results show that APG2 inactivates from the outer surface of the vesicle membrane and support the previous hypothesis that the lac carrier protein is unable to bind external substrate in the absence of energy coupling.

Amino Acids↗
PubMed1975

Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside.

2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM. The initial rate and steady state level of [3H]azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM. Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system. Strikingly, photolytic inactivation is not observed in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 77 muM. Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system. The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling.

Amino Acids↗