Decreased Na+-gradient-dependent d-glucose transport in brush-border membrane vesicles from rabbits with experimental fanconi syndrome

The effect of anhydro-4-epitetracycline on sodium gradient-dependent d-glucose transport of rabbit renal brush-border membrane vesicles was studied. The purity of isolated brush-border membrane vesicles as judged by enzyme activities was not different between normal control and anhydro-4-epitetracycline-administered rabbits. There was no difference in estimate of intravesicular volume, either. When NaCl was used for sodium gradient, the overshoot of d-glucose uptake into brush-border membrane vesicles isolated from anhydro-4-epitetracycline-treated rabbits was significantly smaller than that of normal control rabbits. In the cases of NaSCN or Na₂SO₄, the former was also smaller than the latter, but not significantly so. To avoid the possible effect of membrane potential on d-glucose uptake, the voltage-clamp method was applied. Even in the voltage-clamped condition, the overshoot of d-glucose uptake into vesicles from anhydro-4-epitetracycline-treated rabbits was decreased compared to that of normal rabbits. In vitro incubation of brush-border membrane vesicles with 20 mM anhydro-4-epitetracycline caused no alteration in sodium gradient-dependent d-glucose uptake. Our results demonstrate that there exists a disorder in sodium gradient-dependent d-glucose uptake of renal brush-border membrane in anhydro-4-epitetracycline-treated rabbits, and suggest that this disorder is one of the underlying mechanisms of experimental Fanconi syndrome.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Characterization of an essential disulfide bond associated with the active site of the renal brush-border membrane d-glucose transporter

In a previous report (J. Biol. Chem. 258 (1983) 3565–3570) we have demonstrated that the disulfide-reducing agent dithiothreitol has two effects on the sodium-dependent outer cortical brush border membrane d-glucose transporter; the first results in a reversible increase in the affinity of the transporter for the non-transported competitive inhibitor phlorizin, while the second results in a partially reversible loss of phlorizin binding and glucose-transport activity. Evidence was presented that both of these effects are the result of the reduction of disulfide bonds on the transport molecule. In the present paper we extend our observations on the inactivation of the transporter by dithiothreitol. We provide evidence here (i) that the inactivation of the transporter by dithiothreitol is independent of the effect of the reducing agent on the affinity of the transporter, (ii) that this inactivation process is first-order in dithiothreitol and thus presumably due to the reduction of a single disulfide bond essential to the functioning of the transporter. (iii) that it is the reduction of this disulfide bond and not some subsequent conformational or other change in the transporter which results in its inactivation, (iv) that phlorizin and substrates of the transporter provide protection against inactivation by dithiothreitol and that the degree of protection provided correlates well with the known specificity and phlorizin-binding properties of the transporter, and (iv) that the reactivity of the transporter with dithiothreitol is pH-dependent, decreasing with increasing pH over the pH range 6.5–8.5. We conclude that this site of action of dithiothreitol is a single essential disulfide bond intimately associated with the glucose-binding site on the transport molecule.     

Peculiarities of the Na+/d-glucose cotransport system in necturus renal tubules

The effects of d-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d-glucose produces $\text{10.5}\text{mV}\text{± 1.1}$ (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to $\text{2.1 ± 0.1}\text{mV}$. (3) The glucose-evoked depolarization, $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$, does not alter the intracellular K⁺ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na⁺ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li⁺ substituting for Na⁺) does not change the magnitude of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$; complete removal of sodium from the lumen lowers the value of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$ ($\text{3.2 ± 0.2}\text{mV}$) but the response is not abolished. This observation suggests that the d-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.     

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Kinetic analysis of l-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system

Three parallel pathways of l-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternately to the outside and the inside of the membrane.     

Sensitivity of Na-coupled d-glucose uptake, Mg-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of n-aliphatic alcohols and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations (C) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / C and log P, P being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.