Effects of neuraminidase and deoxyribonuclease on the beta-adrenergic receptors in rat heart.

Langer (1) reported that external to the lipid bilayer or unit membrane at the surface of the myocardial cell is the glycocalyx which is composed of two layers, the surface coat and the external lamina. Both layers contain abundant fixed negatively charged sites which are due in part to the presence of sialic acid in the glycocalyx. Although several investi gators have studied the pharmacological specificity of ,3-adrenergic receptors in myocardial membranes (sarcolemma) (2-8), it appears that the reproducible determination of the density of i3-adrenergic receptors in the sarcolemma of the myocardium is difficult because of the glycocalyx. The object of this study is to examine the effect of the removal of sialic acid on the ,3-adrenergic receptor assay. In addition, deoxyribonuclease (DNase), which was found to be beneficial in the isolation of sarcolemma from cardiac muscle (9), was also used for the comparison of the effects of these enzymatic treatments on the i3-adrenergic receptors. 3H (-)-dihydroalprenolol (3H-DHA) (49 .4 Ci/mmole) was purchased from New England Nuclear Corp. Neuraminidase (Type V, purified from Clostridium perfringens), DNase I (from bovine pancreas) and dl propranolol were purchased from Sigma Chemical Company. Male Wister rats weighing 200-300 g were used in this study. After the atria, great vessels, valves, and epicardial fat were removed, rat ventricles were suspended in 250 mM sucrose, 10 mM Tris-HCI, pH 7.6, and minced with small scissors. The minced ventricles were homo genized using a Polytron homogenizer for 10 sec twice at setting 8 and filtered through 4 layers of cheese cloth. Three M KCI and 250 mM sodium pyrophosphate were added to adjust the final concentrations to 0.3 M KCI and 25 mM sodium pyrophosphate to solubilize contractile and other proteins, shaken vigorously, and this suspension was centrifuged at 177,000 g for 45 min. The pellets obtained were also used to determine the density of ,3-adrenoceptors as a control. The pellets were then treated with neura minidase (0.31 unit/rng protein) in a medium containing 100 mM KCI, 10 mM MgC12, 20 mM Tris-maleate, pH 7.4, at 25°C for 30 min (10) or DNase (60 kunitz unit/mg protein) in a medium containing 250 mM sucrose, 10 mM Tris-HCI, pH 7.6, at 37°C for 60 min (9). The suspension treated with these enzymes was centrifuged at 177,000 g for 45 min, and the pellets were finally resuspended in an "Incubation buffer" (75 mM Tris-HCI, 25 mM MgCl2, pH 8.0). …