Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

Monoclonal antibodies against the renal Na+-D-glucose cotransporter. Identification of antigenic polypeptides and demonstration of functional coupling of different Na+-cotransport systems

Eight monoclonal antibodies are described which are directed against the renal Na+-D-glucose cotransporter. In porcine renal brush-border membranes, the antibodies either bind to one or to three polypeptides which have been identified as components of the Na+-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H., (1987) J. Biol. Chem. 262, 10718-10727). Their molecular weights and isoelectric points are 75,000 and pH 5.5, 60,000 and pH 5.2, and 47,000 and pH 5.4. Six antibodies were able to influence Na+-dependent D-glucose uptake and/or Na+-dependent high affinity phlorizin binding. In the presence of Na+, the binding of all antibodies to native membrane proteins was altered by D-glucose but not by D-mannose. Since this effect was observed with D-glucose concentrations less than 1 x 10(-8) M, a high affinity D-glucose-binding site on the D-glucose transporter has been implied. Some of the antibodies probably interact also with other Na+-coupled transporters since their binding was altered by micromolar concentrations of L-lactate, L-alanine, or L-glutamate but not by the nontransported control substances D-alanine and D-glutamate. L-lactate increased the binding of one antibody in the absence but not in the presence of D-glucose. Effects of L-lactate and L-alanine on the binding of another antibody were only observed when D-glucose was present. Thus, some epitopes on the Na+-D-glucose cotransporter are altered by D-glucose and also by substrates of other Na+ cotransporters. This finding suggests functional coupling of different Na+-cotransport systems.     

Identification of an Mr 75000 component of the H+/D-glucose cotransporter from Zea mays with monoclonal antibodies directed against the mammalian Na+/D-glucose cotransporter

Monoclonal antibodies which interact with the mammalian Na+/D-glucose cotransporter and bind to Mr 75,000 and Mr 47,000 polypeptide components of this transporter have been described (Koepsell, H., Korn, K., Raszeja-Specht, A., Bernotat-Danielowski, S. and Ollig, D. (1988) J. Biol. Chem., 263, 18419-18429). The interaction of these antibodies with plasma membranes from Zea mays L. coleoptiles containing an H+/D-glucose cotransporter was studied. Four monoclonal antibodies cross-reacted with Mr 75,000 and Mr 33,000 polypeptides. One of these antibodies, which inhibits Na+/D-glucose cotransport in the kidney and stimulates Na+/D-glucose cotransport in intestine, stimulates electrogenic uptake of 3-O-methyl-D-[14C]glucose in plant membrane vesicles. The data indicate common epitopes in the mammalian Na+/D-glucose cotransporter and the H+/D-glucose cotransporter of plants and suggest that both transporters contain an Mr 75000 polypeptide component.     

Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter

The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+.     

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Chemical modification of the small intestinal Na+/d-glucose cotransporter by amino group reagents: Evidence for a role of amino group(s) in the binding of the sugar

Some amino group reagents inactivate the small-intestinal Na⁺/d-glucose cotransporter, as measured either as a catalyst of Na⁺-dependent d-glucose transport or as a Na⁺-dependent phlorizin ligand. The amino group(s) studied in this paper are not identical with those investigated previously (Biber, J., Weber, J. and Semenza, G. (1983) Biochim. Biochim. Acta 728, 429–437): these are protected from inactivation by the simultaneous presence of Na⁺ plus sugar substrates. They are thus likely to be located within the substrate-binding site.     

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.     

Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., B?nziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge.     

Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus oocytes, and transmembrane currents associated with this transporter were monitored using a two-electrode voltage clamp. Addition of D-glucose to a Na(+)-containing solution bathing these oocytes generated a current which was blocked by phlorizin. Water-injected control oocytes did not exhibit any currents under these conditions. The magnitude and shape of the currents were dependent on the extracellular glucose and Na+ concentrations and the membrane potential. At Vhold = -50 mV, the Km values for glucose and Na+ were 14 +/- 2 (N = 4) microM and 17 +/- 1 (N = 3) mM, respectively. These Km values and imax exhibited voltage dependence: increasing the membrane potential from -30 to -150 mV increased KGlcm and imax threefold and decreased KNam eightfold. The reversal potential (VR) of the phlorizin-sensitive, glucose-dependent current varied with log Nao+ (slope 46 +/- 6 [N = 9] mV). In the absence of sugar, a Na(+)-dependent, phlorizin-sensitive (Ki = 3 +/- 0.5 microM) current was detected only in RNA-injected oocytes. The amplitude of this current at -50 mV was 6 +/- 1% (N = 13) of the maximum current measured in the presence of D-glucose. The VR of this sugar-independent current varied with log Nao+ (slope 63 +/- 1 [N = 4] mV), indicating that the cotransporter may carry Na+ in the absence of sugar. We conclude that the Na+/glucose cotransporter is electrogenic and that investigations of currents associated with its operation can yield valuable insights into the mechanisms of solute translocation.     

Characterization and histochemical localization of the rat intestinal Na(+)-D-glucose cotransporter by monoclonal antibodies

The localization of the Na(+)-D-glucose cotransporter in rat small intestine was investigated with four monoclonal antibodies which were raised against porcine renal brush-border membrane proteins. The antibodies alter high affinity phlorizin binding or Na+ gradient-dependent D-glucose uptake in kidney and intestine. In both organs, the antibodies react with polypeptides with apparent molecular weights of 75,000 and 47,000. In pig kidney, these polypeptides were identified as components of the Na(+)-D-glucose cotransporter (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18419-18429 (1988)). The electron microscopic localization of antibody binding was investigated by immunogold labeling of ultrathin plastic sections. In villi and crypts of duodenum, jejunum and ileum the antibodies bound specifically to brush-border membranes of enterocytes and did not react with the basolateral membranes. The density of antigenic sites in brush-border membranes was highest in jejunum, intermediate in ileum and lowest in duodenum. On the tip, the middle and the basis of the villi the density of antigenic sites was similar. The data demonstrate homologous Na(+)-D-glucose cotransporters in kidney and intestine. They suggest that during maturation of the enterocytes when the total area of brush-border membrane increases, the concentration of the Na(+)-D-glucose cotransporter in the brush-border membrane remains constant. However, we found that different segments of small intestine not only contain different surface areas of the transporter-containing brush-border membrane per intestinal length but also different densities of the transporter within the brush-border membrane.     

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled D-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and D-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of D-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and D-glucose to the cotransporter.     

Effects of amiloride analogues on Na+ transport in toad bladder membrane vesicles. Evidence for two electrogenic transporters with different affinities toward pyrazinecarboxamides

Most of the electrical potential-driven 22Na+ uptake in toad bladder membrane vesicles can be blocked by the diuretic amiloride. Analysis of the amiloride inhibition curve indicates the presence of two pathways with low and high affinities to the diuretic (Garty, H. (1984) J. Membr. Biol. 82, 269-279). The selectivity of these pathways to amiloride was explored by comparing the inhibition curve of this diuretic with those of 10 of its structural analogues. The relative potencies of various amiloride-like compounds as blockers of the flux component with high affinity to amiloride were in good agreement with the structure-activity relationships elucidated from transepithelial short-circuit current measurements. Thus, this pathway is most probably the apical Na+-specific channel. The other pathway with lower affinity to the diuretic was relatively insensitive to modifications of the amiloride molecule, and the structure-activity relationships measured for the inhibition of this pathway were different from those reported for any other amiloride-blockable process. Other experiments have established that the Na+ flux with low affinity to amiloride is electrogenic and is not mediated by a Na+/H+ or Na+/Ca2+ exchanger, Na+-hexose cotransporter, or the Na+/K+-ATPase. The data indicate that tracer flux measurements in toad bladder membrane vesicles monitor, in addition to the well-characterized apical Na+ channels, another amiloride-blockable electrogenic Na+ transporter. This pathway could be responsible for the basolateral amiloride-blockable Na+ conductance recently observed in nystatin-treated bladders (Garty, H., Warncke, J., and Lindemann, B. (1987) J. Membr. Biol. 95, 91-103).     

Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives.

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.     

Evidence of an essential carboxyl residue in membrane-bound pyrophosphatase ofRhodospirillum rubrum

Chemical modifications with water-soluble carbodiimides (EDC and CMC) were performed to elucidate whether some carboxyl residues are involved in the catalytic activity of membrane-bound pyrophosphatase ofRhodospirillum rubrum. EDC and CMC cause a loss of hydrolytic activity following pseudo-first-order kinetics up to 10 min of reaction. The enzyme was completely protected against EDC inhibition by PPi or Mg²⁺, whereas PPi or Mg²⁺ gave partial protection against CMC inactivation. Mg-PPi protected completely against the inhibition caused by both carbodiimides. These data suggest that the carboxyl moiety modified by EDC is at the active site. At longer times of inactivation with both carbodiimides, we could not observe a linear relationship in semilogarithmic plots of residual activity versus time, indicating that at least two carboxyls are involved in the inactivation, which correlates with the partial protection against CMC inactivation by PPi. We found that the activator site for Mg²⁺ is apparently at or near the active site of the enzyme. This is supported by the fact that PPi protects completely the activator effect of this divalent cation.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Alteration of intracellular [Ca2+] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca2+ is coupled to Na+ entry and increased intracellular pH

The egg peptide speract increases intracellular pH (pHi) and cyclic nucleotides in sperm of the sea urchin Strongylocentrotus purpuratus by a mechanism dependent on seawater Na+ but not Ca2+ (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 2235-2241; Repaske, D. R., and Garbers, D. L. (1983) J. Biol. Chem. 258, 6025-6029). Using the Ca2+ indicators quin2 and indo-1, we show that speract stimulates a transient rise in intracellular [Ca2+] ([a2+]i) when millimolar Ca2+ is present in seawater. The rise is increased and extended by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), which also enhances 22Na+ uptake with or without Ca2+. Without MIX, speract initiates a rise in [Ca2+]i that peaks within approximately 5 s and decreases with a t1/2 of approximately 9 s. Activation of Na+:H+ exchange without speract by either Na+ addition to sperm in Na+-free seawater (NaFASW) or by monensin also increases [Ca2+]i, but neither change is transient. Inhibition of Na+:H+ exchange by increased seawater [K+] prevents the rise in [Ca2+]i initiated by either speract or Na+ addition to sperm in NaFASW. Increasing pHi by adding 10 mM NH4+ or by addition of Li+ to sperm in NaFASW does not increase [Ca2+]i. The data suggest that speract binding leads to rapid activation of Na+:H+ exchange; and, as a consequence, [Ca2+] entry increases transiently through either Na+:Ca2+ exchange or else through a verapamil-insensitive Ca2+ channel. MIX prevents the inactivation of this entry mechanism.     

Covalent modification of the renal Na+/H+ exchanger by N,N'-dicyclohexylcarbodiimide

We studied the effect of the carboxyl group-specific reagent N,N'-dicyclohexylcarbodiimide on the Na+/H+ exchanger present in microvillus membrane vesicles isolated from rabbit renal cortices. Pretreatment of membrane vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of Na+/H+ exchange which was not due to vesicle disruption or collapse of imposed pH gradients. Inhibition by dicyclohexylcarbodiimide followed pseudo-first-order kinetics, resulted primarily from a decrease in binding affinity for substrate, was pH-dependent in a manner consistent with reaction with carboxyl groups, and was greater than inhibition by hydrophilic carbodiimides. Substrates Na+ and Li+ and the competitive inhibitor amiloride protected against inhibition by dicyclohexylcarbodiimide in a pH-dependent fashion. Finally, we demonstrated amiloride-sensitive covalent binding of radiolabeled dicyclohexylcarbodiimide to a 100-kDa protein. In conclusion, a catalytically important carboxyl group is located in a relatively hydrophobic microenvironment at or near the external transport site of the renal Na+/H+ exchanger; and the transporter itself, or a subunit thereof, may be a 100-kDa protein.     

Identification of the D-glucose binding polypeptide of the renal Na+-D-glucose cotransporter with a covalently binding D-glucose analog

The covalently binding D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG) was synthesised and shown to be a high-affinity inhibitor of the renal Na+-D-glucose contransporter. From renal brush-border membranes a protein fraction was isolated, in which the concentration of Na+-dependent phlorizin binding sites per mg protein was enriched 7-fold. In labeling experiments with this protein fraction a polypeptide of Mr approximately 79000 was identified as containing the D-glucose binding site of the renal Na+-D-glucose cotransporter.