Hermann Koepsell

Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: Renaturation of antigenic sites and reduction of nonspecific antibody binding☆

The immunochemical reaction of monoclonal antibodies directed against native membrane proteins was investigated after their separation in sodium dodecyl sulfate polyacrylamide gels and electrotransfer to nitrocellulose. Nonspecific binding of antibodies to membrane proteins, which was increased by beta-mercaptoethanol treatment or heat denaturation of the antibodies, could be significantly reduced if 1 M D-glucose plus 10% (v/v) glycerol was added during the incubation with the antibodies. It was found that specific antibody binding was drastically reduced by SDS treatment of the membrane proteins. During the electrotransfer to nitrocellulose and the simultaneous removal of SDS, some increase in antibody binding was observed. Considerable renaturation of antigenic sites in the blotted proteins could be induced if the nitrocellulose blots were incubated for 16 h at 37 degrees C in phosphate-buffered saline. With the introduction of both modifications, the renaturation step, and the addition of D-glucose and glycerol to reduce nonspecific antibody binding, the immunoblot technique may be successfully applied to detect conformational antibodies against membrane proteins.

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

SummaryTime courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na+ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na+) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na+) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na+ on both membrane sides and with a clamped membrane potential,KD values of 0.4 and 7.9 μM were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na+(out>in). Low and high affinity transport could be fitted with identicalKm values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentKm ofhigh affinity transport whereas the apparentKm of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na+-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

SummaryAntibodies which were raised against highly purified membrane-bound (Na+−K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+−K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na+−K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+−K+)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na+−K+)-ATPase activity (n=0.77). By measuring the Na+ dependence of the (Na+−K+-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na+−K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+−K+)-ATPase reaction and on the ATP binding of the enzyme.Vmax of the (Na+−K+)-ATPase reaction and the number of ATP binding sites were reduced whileK0.5 ATP for the (Na+−K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK0.5 value for the Na+ stimulation of the (Na+−K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+−K+)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na+−K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+−K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+−K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.

Comparison of a Na+/d-glucose contransporter from rat intestine expressed in oocytes of Xenopus laevis with the endogenous cotransporter

Epithelial Na+/D-glucose cotransport was incorporated into the plasma membrane of Xenopus oocytes after microinjection of poly(A)(+)-mRNA from rat intestine tissue and was detected by measurements of uptake of [14C]AMG (methyl alpha-D-glucopyranoside). In mRNA-injected oocytes, the rate of AMG uptake exceeds the rate of endogenous Na+/AMG cotransport by a factor of up to 30. It is demonstrated that the additionally expressed transport differs qualitatively from the endogenous transport with respect to several parameters which is a prerequisite for the demonstration of expression of a foreign transporter: (1) The expressed system is more sensitive to external glucose or AMG and to the specific inhibitor phlorizin, (2) it is less sensitive to external Na+ and to changes in membrane potential, and (3) it is susceptible to inhibition by monoclonal antibodies, known to bind specifically to Na+/glucose cotransporters and to modulate the cotransport in kidney and intestine. The use of the antibodies allows one to distinguish between endogenous Na+/AMG cotransport and foreign cotransport expressed by injection of foreign mRNA. The expression of the foreign transport leads to transport rates that are high enough to detect the electrical current generated by the Na+/glucose cotransport. This allows future characterization of the cotransport system under voltage-clamp conditions by analyzing membrane current.

A simple liposomal system to reconstitute and assay highly efficient Na+/d-glucose cotransport from kidney brush-border membranes

A simple procedure to reconstitute highly efficient Na+/D-glucose cotransport from solubilized brush-border membranes of proximal kidney tubules is described. Reconstitution of transport activity was possible with various phospholipid and cholesterol combinations; the presence, however, of cholesterol and at least one phospholipid was essential. When liposomes were synthesized from only one phospholipid and cholesterol, the highest uptake rats were observed with phosphatidylserine; phosphatidylcholine was less effective and phosphatidylethanolamine showed insignificant uptake of D-glucose in the presence of Na+. The rate at which an inward-directed Na+ gradient dissipated across the liposomal membranes was reduced if the cholesterol concentration of liposomes was increased. In the optimized system, proteoliposomes were formed from cholesterol and phosphatidylserine by a heat-sonication-freeze-thaw procedure. A Na+-gradient persisted for hours across these proteoliposomal membranes and a Na+/D-glucose cotransport with the following characteristics could be demonstrated: (1) dependency on the Na+ gradient; (2) a transient (3) rheogenicity; (4) stereospecificity; and (5) high-affinity phlorizin inhibition. Since the Na+-gradient-stimulated D-glucose uptake is linear for minutes, the initial uptake rates can be measured and the Na+/D-glucose cotransport activity of different protein fractions can be compared.

Endogenous L-glutamate transport in oocytes of Xenopus laevis

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.

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

Affinity labeling D‐Glucose analog D‐Glucose binding site Na±D‐glucose cotransporter Brush‐border membrane Kidney

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

SummaryTo distinguish ligand-induced structural states of the (Na+−K+)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na+, K+, Mg++ and ATP. It was found that first the large and then the small polypeptide chain of the (Na+−K+)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na+−K+)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na+−K+)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg++, Na+ and K+ present during trypsin digestion influenced the time course and degree of degradation of the (Na+−K+)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na+−K+)-ATPase was the same, whether Na+ or K+ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na+ or K+ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na+ and K+ also influenced the degradation pattern of the (Na+−K+)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na+ and K+ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg++, Na+ and K+ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na+−K+)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na+−K+)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na+−K+)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na+−K+)-ATPase is regulated by binding of Na+ or K+ to the enzyme-ATP complex. The effects of Na+ and K+ on the (Na+−K+)-ATPase structure are modulated by the ATP binding to “high affinity” and to “low affinity” ATP binding sites.

Conformational changes of membrane-bound (Na+-K+)-ATPase as revealed by antibody inhibition.

SummaryAs different structural states of the (Na+−K+)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na+, K+, Mg++, Pi and ATP on the reaction between highly purified (Na+−K+)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na+−K+)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na+, K+, Mg++ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg++, (ii) Mg++ and Na+ or (iii) Mg++ and K+ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na+, Mg++ and ATP were present together. (c) It was inhibited even more if only (i) Na+, (ii) K+, (iii) ATP or both (iv) ATP and Na+, (v) ATP and K+, (vi) ATP and Mg++, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg++, ATP and K+ were present together.In the presence of Mg++ plus ADP and in the presence of Mg++ plus the ATP analog adenylyl (β-γ-methylene) diphosphonate, Na+ and K+ did not influence the degree of antibody inhibition as they did in the presence of Mg++ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg++, ATP and K+ was affected by the sequence in which K+ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na+−K+)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na+ and K+ in the presence of Mg++ plus ATP are proposed to occur during the Na+−K+ transport.

Identification of an Mr 75 000 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.