Functional lac carrier proteins in cytoplasmic membrane vesicles isolated from Escherichia coli. 1. Temperature dependence of dansyl galactoside binding and beta-galactoside transport

Therisod, H.; Letellier, L.; Weil, R.; Shechter, E.

Biochemistry 16(17): 3772-3776

1977


ISSN/ISBN: 0006-2960
PMID: 332221
DOI: 10.1021/bi00636a007
Accession: 068526437

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Abstract
Dansyl galactoside (6'-N-dansyl)aminohexyl 1-thio-.beta.-D-galactopyranoside) binds in an energy-dependent way to the lac carrier proteins of membrane vesicles isolated from E. coli cells. The binding is not followed by transport but is accompanied by a large increase in fluorescence and a shift of the emission maximum to a lower wavelength. These properties make it possible to titrate the amount of lac carrier proteins which become accessible upon energizing the membrane. The temperature dependence of the binding of dansyl galactoside was determined in membrane vesicles of E. coli K 1059 and ML 308225. The number of binding sites of dansyl galactoside decreases with decreasing temperature. The range over which this occurs overlaps that at which the conformational order-disorder transition of the membrane lipids takes place. The Kd is temperature independent. The initial rates of binding are graphed according to the Arrhenius equation. This is represented as nmol of dansyl galactoside bound per min either per mg of protein or per nmol of accessible lac carrier proteins. In the former case, the best fit to the data requires 2 lines. The line in the low-temperature range has a steeper slope. In the latter case, the data can be fit either by only 1 line (K 1059 membranes) or by 2 lines but with a much less pronounced difference in slope (ML 308225 membranes). There is a striking parallel between the temperature dependence of the initial rates of dansyl galactoside binding to a given membrane and the temperature dependence of the initial rates of .beta.-galactoside transport by the same membrane. This parallelism indicates that the carriers which participate in the transport (functional carriers) are the ones which become accessible to the external medium upon energizing. Apparently, the change in slope usually observed in the Arrhenius plot of transport in the temperature range extending over the order-disorder transition is mainly a result of a change in the number of functional lac carriers.