Abstract
Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters. The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli. Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E. coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM. Binding was inhibited by D-glucose, but not by L-glucose. UV irradiation of the membranes in the presence of [4-3H]cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein. Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system. The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters. Structural similarity to the latter was revealed by tryptic digestion of [4-3H]cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter.
Highlights
Cytochalasin B is a potent inhibitor of mammalian The galP gene from E. coli has recently been sequenced.’
The sequence of the GalP protein is -26% identical to of photolabeling by different sugars and sugar ana- the human erythrocyte transporter an-2d3% identical to the logues showed that the substrate specificity ofGalP human liver transporter
Structural similarity to the latter sugar transporters includesglucose/H+ transporters in the was revealed by tryptic digestion of [4-3H]cytochalasin cyanobacterium Synechocystis [27] andinthe greenalga B-photolabeled GalP, which yielded a radiolabeled Chlorella [28] and glucose, galactose,maltose, and lactose fragment ofapparent M, 17,000-19,000, similar to transporters in yeasts[29,30,31,32]. that previously reported for the human erythrocyte Cytochalasin B is a potent reversible inhibitor of mammaglucose transporter
Summary
Organisms-E. coli strain JM1576 (GalP‘) wasgrown in liquid with histidine, leucine, and isoleucine Both strains are constitutive culture on minimal salts [4]plus 22 mM glycerol supplemented with for GalP and are MglP- [6, 42]. 7 other sugar substrates on the transport of n-galactose were studied and 44) or from intact cells by explosive decompression in a French using initial rates estimated from the amount of galactose taken up press [45]. The former procedure yields vesicles predominantly of the after 15 s. Centrifugal binding procedure similar to that described by Cushman and Wardzala [46].Assays wereperformed in plastic Eppendorf tubes containing Kaback vesicles(1.7-3.3mg of protein/mlin 20 mM
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