Purification and Partial Characterization of Cardiac Plasma Membranes Rich in Beta-Adrenoreceptors

Abstract

A procedure was developed to purify plasma membranes from cardiac tissue. Canine ventricular tissue was homogenized relatively gently in hypotonic buffer, extracted briefly with 0.15 M sodium perchlorate at 4°C, and membranes were collected in an intermediate pellet by centrifugation at 27,500 × g_max. At this stage, most contractile proteins were removed (although Z-band structures were apparent), and the recoveries of Na⁺, K⁺-ATPase, beta-adrenoreceptors and 5′ -nucleotidase were 40-509%. Treatment of these particles with 0.5% dextran sulfate and discontinous density gradient centrifugation resulted in the separation of membranes having 111 and 3.6 pmol/mg protein specific binding sites for [³H] ouabain and [³H] dihydroalprenolol, respectively. Electron micrographs showed medium-sized vesicles. 5′-Nucleotidase was also markedly purified but could be partially separated from the other plasma membrane markers in continuous sucrose gradients. Enzymes associated with mitochondria, lysosomes, and endoplasmic reticulum were efficiently removed. SDS-polyacrylamide gels showed six major and many minor bands. Adenylate cyclase remained responsive to hormones and Gpp(NH)p, but little purification was apparent. The membrane preparation has proved useful for studies of a nucleotidebinding protein associated with cyclase activation.

Key Words:

• cardiac plasma membranes
• dextran sulfate
• beta-adrenoreceptors
• Na⁺
• K⁺-ATPase
• adenylate cyclase