naphthoquinones and oxophenylarsine

The role of thiol groups in H+-gradient-dependent dipeptide transport in rabbit renal brush-border membrane vesicles was investigated using glycylsarcosine as the substrate. Treatment of the membrane vesicles with a thiol-group-reducing agent, dimercaptopropanol, stimulated Gly-Sar transport. On the other hand, treatment with thiol group oxidants such as 5,5'-dithiobis(2-nitrobenzoic acid), plumbagin and phenazine methosulfate inhibited Gly-Sar transport. These effects were irreversible, because washing the membranes after treatment failed to reverse the effects. Incubation of the membrane vesicles with phenylarsine oxide, a reagent which interacts specifically with vicinal dithiols, significantly inhibited Gly-Sar transport. In all cases, the stimulation or the inhibition of the dipeptide transport was primarily due to changes in the maximal velocity of the transport system, the apparent affinity constant remaining unaltered. These results demonstrate the involvement of one or more vicinal dithiol groups in the function of the renal dipeptide transport system and that these thiol groups must exist in reduced form to maintain maximal transport activity. In addition, these data indirectly suggest that a dithiol-disulfide interchange may play a role in the function of the renal dipeptide transport system.

Topics: Animals; Arsenicals; Biological Transport; Carrier Proteins; Dimercaprol; Dipeptides; Dithionitrobenzoic Acid; Kidney Cortex; Kinetics; Methylphenazonium Methosulfate; Microvilli; Naphthoquinones; Oxidation-Reduction; Protons; Rabbits; Structure-Activity Relationship; Sulfhydryl Compounds

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p-nitrophenyl alpha-D-galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification. In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H.R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221-6225; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480-5484] must be re-interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.

Topics: Arsenicals; Binding Sites; Biological Transport; Chemical Phenomena; Chemistry; Diethyl Pyrocarbonate; Escherichia coli; Escherichia coli Proteins; Formates; Galactosides; Glycosides; Membrane Transport Proteins; Monosaccharide Transport Proteins; Naphthoquinones; Protein Binding; Symporters

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

Topics: Arsenicals; Biological Transport, Active; Dithiothreitol; Escherichia coli; Escherichia coli Proteins; Kinetics; Lactose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Naphthoquinones; Oxidation-Reduction; Proline; Sulfhydryl Reagents; Symporters