Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

Abstract

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [ adenine-2,8- 3H]NAD +, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20°C, the equilibrium binding was obtained at 15 min, while NAD binding at 0°C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20°C. Brush border membrane inactivated by heating at 95°C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both α-NAD and β-NAD stereoisomers displaced bound [ 3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD +. Adenine, nicotinamide, pyrophosphate, of 5′-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10 −4 M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site ( K d = 1.9·10 −5 M) and a low-affinity binding site ( K d = 2.2·10 −3 M). (4) Unlike concentrative uptake of d-[ 14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na o + > Na i +], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike d-[ 14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.