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The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins, N-acetyl-S-all-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, wereused to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-S-all-trans and trans-farnesyl-L-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, whereas Ras, which is farnesylated, was not. Furthermore, N-acetyl-S-all-trans-geranylgeranyl-L-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaP(i)-2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration of N-acetyl-S-all-trans-geranylgeranyl-L-cysteine. Membrane treatments with 0.7 m KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine likely occurs via different mechanisms.

The effect of phenylglyoxylation on brush-border-membrane functions was studied with membrane vesicles from rat kidney cortex. Na+-gradient-dependent uptake of phosphate, glucose and alanine was inhibited by 65, 88 and 70% by pre-incubation of vesicles with 50 mM-phenylglyoxal for 2 min. The inhibition showed a dependency for alkaline pH. Borate co-operativity in butanedione inactivation was used to prove that inhibition was caused by arginine modification. Intravesicular volumes, alkaline phosphatase, aminopeptidase M and Na+-H+ exchange were not affected by phenylglyoxal treatment. Inhibition of phosphate uptake was studied in more detail and showed that the chemical modification introduced by phenylglyoxal inhibited the overshoot of phosphate uptake caused by the Na+ gradient, and decreased the apparent maximal velocity of the phosphate-transport system in its interaction with Na+. Phosphate uptake measured in the absence of Na+ was not affected by phenylglyoxal. Shunting of the transmembrane electrical potential with K+ and valinomycin had no effect on phenylglyoxal inhibition, proving that the alteration of transmembrane electrical potential could not be responsible for this effect. Phenylglyoxal had no ionophoric effect on the Na+ gradients studied (1-100 mM). Na+ efflux was also unaffected by phenylglyoxal treatment. Na+, harmaline and amiloride were ineffective in protecting against phenylglyoxal inhibition, suggesting that the site modified was not an Na+-binding site. These results indicate the involvement of highly reactive arginine residues in phosphate, glucose and alanine uptake.

The interaction between P-glycoprotein (140-180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs. Two monoclonal antibodies, which are able to discriminate between two major domains of cyclosporin A (the cyclophilin and calcineurin binding domains), were used to detect the photolabeled proteins. A protein of 155 kDa corresponding to P-glycoprotein was much more strongly photolabeled in membranes of CHRC5 cells than in membranes of their drug-sensitive parent cell line AuxB1. The antitumor drug vinblastine and the reversal agents verapamil and cyclosporin A inhibited the photolabeling, and the nonimmunosuppressive derivative PSC-833 caused a stronger inhibition than cyclosporin A. P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized specifically by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein.

Phosphate transport across brush border membranes from kidney cortex is very sensitive to inhibition by phenylglyoxal, an arginine modifier. Sodium-dependent phosphate influx into brush border membrane vesicles was inhibited by 60%. In contrast, phenylglyoxal had no effect on passive influx or on sodium-dependent efflux of phosphate. Preincubation of the vesicles with sodium prior to the addition of phenylglyoxal demonstrated a strong protective effect of intravesicular sodium (73% protection). Phosphate also protected the transporter from inhibition, but from the extravesicular side only (63%). Substitution of phosphate by sulfate offered no protection at all, indicating the specificity of protection. Addition of both substrates (sodium and phosphate) offered an additional protection from the extravesicular side compared to that offered by phosphate alone (92 versus 55%). There was no additional protection when both substrates were added to the intravesicular side. Phosphate influx measured in the presence of sodium but in the absence of a sodium gradient was totally unaffected by phenylglyoxal modification. There was no inhibition on phosphate influx measured in equilibrium exchange conditions. We propose a model for the phosphate carrier in which the sodium gradient induces a conformational change and an arginine residue is essential for the coupled flux of sodium and phosphate.

Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5′-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.