ATPase activity and transport by a cGMP transporter in human erythrocyte ghosts and proteoliposome-reconstituted membrane extracts

We previously described the [H-3]cGMP-binding characteristics of a CHAPS-so lubilized protein that we proposed to be a cGMP transporter. We now report the ATPase activity of the membrane-bound, solubilized and reconstituted fo rm of a cGMP transporter. The membrane-bound protein of unsealed ghosts had a linear ATPase activity over a 120 min incubation period with optimal act ivity of about 400 pmol/mg/min. The apparent K-m and V-max for ATP were abo ut 0.5 mt and 300 pmol/mg/min, respectively. When solubilized with CHAPS th e specific activity of the protein was reduced to about 70 pmol/mg/min. Rec onstitution of the CHAPS preparation into phospholipid bilayer using rapid detergent removal by Extracti-gel(R) column resulted in proteoliposomes whi ch had ATPase activity similar to that found in the erythrocyte membranes. The proteoliposomes displayed a linear ATP-dependent uptake of [H-3]cGMP wi th an apparent K-m value of 1.0 muM. This low K mu -uptake of [H-3]cGMP in proteoliposomes was not affected by 10 muM of AMP, cAMP and GMP, but was co mpletely abolished in the presence of the non-hydrolyzable ATP analogue, AT P-gamma -S. Some ATPase activation was also observed in the presence of 2 m uM cAMP, but it is unclear whether this activity was coupled to the cGMP tr ansporter. Our results show that the membrane protein responsible for cGMP transport has an ATPase activity and transports the cyclic nucleotide in th e presence of ATP. (C) 2000 Elsevier Science B.V. All rights reserved.