Characterization of the Membrane Protein Component of the Lactose Transport System of Escherichia coli

Jones, Theodore H.D.; Kennedy, Eugene P.

doi:10.1016/s0021-9258(18)63569-0

Cited by 153 publications

(10 citation statements)

References 9 publications

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“…The apparent Kv calculated from the Scatchard plot (Figure 2B) is approximately 6 µ , and the amount of NPG bound at saturation is about 2.3 nmol per mg of membrane protein. Within experimental error, the latter value is indistinguishable from that obtained with Dns2-Gal and Dns6-Gal (Reeves etal., 1973; Schuldiner et al, 1975Schuldiner et al, , 1976a and from the value reported by Jones and Kennedy (1969) for the total amount of M protein (i.e., lac carrier protein) in the membrane. It is also apparent from the data presented in the body of Figure 2 that addition of neither Dlactate nor NPG to uninduced ML 30 vesicles causes a change in the concentration of [6-3H]NPG in the dialysate (open symbols), indicating that binding of NPG is dependent upon the presence of a functional lac y gene product.…”

Section: Resultsmentioning

confidence: 95%

“…Superficially, the results appear to be mutually exclusive, until the data are examined quantitatively. It then becomes apparent that the number of binding sites observed by Kennedy et al (1974) is approximately 10 to 20 times less than the total amount of lac y gene product present in the membrane as determined by Jones and Kennedy (1969) and by titration studies with the dansyl galactosides (Reeves et al, 1973;Schuldiner et al, 1975aSchuldiner et al, , 1976a). Both sets of observations are corroborated in this paper.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

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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“…The amount of protein in this narrow molecular weight range did not increase proportionately to the increase in specific activity, suggesting that several species of protein are included within this size range. The membrane-localized lactose permease protein (Jones and Kennedy, 1969) migrates in this region of SDS gels. Pretreating enzyme fractions for 5 min at 98°in 1 % SDS-1 % 2-mercaptoethanol destroyed the activity but did not alter the migration of proteins in this gel system.…”

Section: Resultsmentioning

confidence: 97%

Membrane-bound phospholipase A1 purified from Escherichia coli

Scandella¹,

Kornberg²

1971

Biochemistry

214

103

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“…The protection by substrates was exploited to label the susceptible transport proteins, AraE, XylE and GalP, with radioactive A-ethylmaleimide; they were identified as proteins that migrated with an apparent relative molecular mass (Mr) of 35000-40000 in SDSpolyacrylamide gels (Macpherson et al 1981(Macpherson et al , 1983Henderson et al 1983;Henderson & Macpherson 1986;Davis 1986). By similar procedures the lactose/H+ transport protein was the first to be identified, with an apparent M r of about 30000 (Jones & Kennedy 1969). Such labelling experiments are very important for the identification of hydrophobic transport proteins of low abundance (0.3-1.0 % of the membrane proteins), which are refractory towards the usual methods of solubilisation and purification.…”

Section: N-ethylmaleimidementioning

confidence: 99%

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Henderson

Maiden

1990

Phil. Trans. R. Soc. Lond. B

152

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Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.

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Characterization of the Membrane Protein Component of the Lactose Transport System of Escherichia coli

Cited by 153 publications

References 9 publications

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

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

Membrane-bound phospholipase A1 purified from Escherichia coli

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

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