[35S]ATPyS binding sites in the purified heart sarcolemma membrane

heart sarcolemma membranes were found to bind a slowly hydrolyzable analogue of ATP ( 3”S-labeled adenosine 5 ’-(y-thio)tri’phosphate ([““S]ATPrS) 1 in a specific manner and ex-hibited two apparent affinity sites. The high-affinity site had a dissociation constant (Kn) of 4.7-8.3 nM [maximum binding (B,,,,) = 9.5-18.4 pmol/mg protein], whereas the low-affinity site had a KD of 655~1,257 nM (B,,, = 812-2,955 pmol/mg protein). Like ATP, other nucleotides such as GTP, UTP, ITP, and CTP were effective in displacing [35S]ATPyS binding. Although crude membrane preparations from different tissues also exhibited both high- and low-affinity sites for [35S]ATPyS, Ku values for the high-affinity sites were severalfold higher than that for the purified heart membranes. It is proposed that the high-affinity binding site for nucleotides may represent the ATP receptor in the heart cell membrane