Structure-function studies of canine cardiac sarcolemmal membranes. I. Estimation of receptor site densities

A novel method for the estimation of receptor site densities in purified canine cardiac sarcolemmal vesicles is described. Canine sarcolemmal vesicles, purified by the method of Jones et al. (Jones, L.R., Maddock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980) had high (Na+ + K+)-ATPase specific activity (127 +/- 1.9 mumol Pi/mg per h). Total phospholipid content, estimated by measurements of total phosphorus and total fatty acid contents, was 3.09 mumol/mg. Saturation isotherms for several receptor ligands gave the following values for Kd and Bmax: ouabain 32.6 +/- 2.7 nM, 365 +/- 59 pmol/mg; quinuclidinyl benzilate 0.055 +/- 0.010 nM, 5.8 +/- 0.7 pmol/mg; dihydroalprenolol 4.6 +/- 1.0 nM, 2.2 +/- 0.2 pmol/mg; and nitrendipine 0.21 +/- 0.04 nM, 0.93 +/- 1.04 pmol/mg. Membrane phospholipid surface area per ligand-binding sites was estimated from the Bmax values for each receptor ligand utilizing 3.09 mumol phospholipid/mg and 60 A2 as the average surface area occupied by each phospholipid molecule. The following receptor site densities per micrometer 2 phospholipid surface were obtained: ouabain, 400; quinuclidinyl benzilate, 6; dihydroalprenolol, 2; and nitrendipine, 1. As the surface area contributed by protein was estimated to be less than 20% of the lipid surface area, these values must be reduced by approx. 20% to estimate site densities per micrometer 2 membrane surface. These data demonstrate much lower beta-adrenergic and muscarinic receptor density compared to that of Na+ pump sites.