Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein.

Schuldiner, Shimon; Kerwar, Grace K.; Kaback, H. Ronald; Weil, Rudolf

doi:10.1016/s0021-9258(19)41822-x

Cited by 99 publications

(48 citation statements)

References 28 publications

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“…Surprisingly, only transportable substrates of LP were found. Even for the dansyl galactosides (Schuldiner et al, 1974), transport was demonstrated (Overath et al, 1979). Our results show that a peptide galactoside with a bulky aromatic dipeptide binds to LP, but is not significantly transported.…”

Section: Discussionmentioning

confidence: 62%

“…The aglycon may vary considerably, but nonetheless contributes to the affinity (Sandermann, 1977). Especially aromatic aglycons lead to a high affinity, examples being p-nitrophenyl -D-galactoside (NpaGal) with a nitrophenyl group and a dissociation constant K¿ = 22 µ as well as 6'-[(jV-dansyl)amino]hexyl-1 -thio-/3-D-galactoside (DnsHxSGal) with a dansyl group and K¿ -5 µ (Schuldiner et al, 1974;Schuldiner & Kaback, 1977;Wright et al, 1981). In contrast, carbohydrate aglycons usually lead to a low affinity (lactose, for example, has a K¿ = 14 mM), the only known exception being /3-d-galactosyl l-thio-/3-o-galactoside (GalSGal) with a K¿ -70 µ .…”

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confidence: 99%

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A Nontransportable Substrate for Lactose Permease

Seibert

Dörner²,

Jähnig³

1995

Biochemistry

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A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent. This substrate, 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM. Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease. An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose.

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Section: Discussionmentioning

confidence: 62%

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confidence: 99%

A Nontransportable Substrate for Lactose Permease

Seibert

Dörner²,

Jähnig³

1995

Biochemistry

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“…Studies with (jV-dansyl)aminoalkyl (f-D-galactopyranosides (Reeves et al, 1973;Schuldiner et al, 1975aSchuldiner et al, -c, 1976a) and 2-nitro-4-azidophenyl ß-D-galactopyranosides (Rudnick et al, 1975a,b) indicate that the lac carrier protein in membrane vesicles isolated from Escherichia coli does not bind ligand significantly unless the vesicles are "energized" (see Schuldiner et al, 1976b, for a review). As a result of D-lactate or reduced phenazine methosulfate oxidation, an electrochemical gradient of protons develops across the vesicle membrane [interior negative and alkaline (Ramos et al, 1976)], and active transport, an increase in dansyl galactoside binding, and azidophenyl galactoside-dependent photoinactivation are observed.…”

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confidence: 99%

“…As a result of D-lactate or reduced phenazine methosulfate oxidation, an electrochemical gradient of protons develops across the vesicle membrane [interior negative and alkaline (Ramos et al, 1976)], and active transport, an increase in dansyl galactoside binding, and azidophenyl galactoside-dependent photoinactivation are observed. Many of these effects are mimicked by artificially induced ion gradients of appropriate polarity (Schuldiner et al, 1975a;Rudnick et al, 1975b), but dilution-induced carriermediated lactose efflux also causes increased dansylgalactoside binding in a manner which is apparently independent of the electrochemical potential across the membrane (Schuldiner et al, 1975a). In any case, based on these observations, it has been postulated that a membrane potential (interior negative) causes the lac carrier protein to become accessible to the external medium, to increase its affinity for ligand, or both, and it has been suggested that the lac carrier protein or part of it may be negatively charged (Schuldiner et al, 1975a(Schuldiner et al, , 1976b.…”

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confidence: 99%

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

Rudnick¹,

Schildiner²,

Hr³

1976

Biochemistry

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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).

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“…The position of the nitroxyl group along the fatty acid chain also affects the rate; the rate for a nitroxyl group which is five carbon atoms [1(12,3)] away from the carboxyl group is greater than that for a nitroxyl which is 12 carbon atoms [1(5,10)] away, and still greater than that for a nitroxyl which is 16 carbon atoms [1(1,14)] away. Sulfhydryl reagents (diazinedicarboxylic acid bisdimethylamide, Aeth--R^ecent observations by Kaback and coworkers have shown that bacterial cytoplasmic membrane vesicles can be used as an excellent model system to investigate active transport (Kaback, 1972(Kaback, , 1974. In Escherichia coli membrane vesicles, the respiration-linked transport system is coupled primarily to the oxidation of D-lactate to pyruvate, catalyzed by a flavine-linked, membrane-bound D-lactate dehydrogenase (Kaback, 1972).…”

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confidence: 99%