ATPase activity and transport by a cGMP transporter in human erythrocyte ghosts and proteoliposome-reconstituted membrane extracts.
We previously described the [(3)H]cGMP-binding characteristics of a CHAPS-solubilized protein that we proposed to be a cGMP transporter. We now report the ATPase activity of the membrane-bound, solubilized and reconstituted form of a cGMP transporter. The membrane-bound protein of unsealed ghosts had a linear ATPase activity over a 120 min incubation period with optimal activity of about 400 pmol/mg/min. The apparent K(m) and V(max) for ATP were about 0.5 mM and 300 pmol/mg/min, respectively. When solubilized with CHAPS the specific activity of the protein was reduced to about 70 pmol/mg/min. Reconstitution of the CHAPS preparation into phospholipid bilayer using rapid detergent removal by Extracti-gel column resulted in proteoliposomes which had ATPase activity similar to that found in the erythrocyte membranes. The proteoliposomes displayed a linear ATP-dependent uptake of [(3)H]cGMP with an apparent K(m) value of 1. 0 microM. This low K(m)-uptake of [(3)H]cGMP in proteoliposomes was not affected by 10 microM of AMP, cAMP and GMP, but was completely abolished in the presence of the non-hydrolyzable ATP analogue, ATP-gamma-S. Some ATPase activation was also observed in the presence of 2 microM cAMP, but it is unclear whether this activity was coupled to the cGMP transporter. Our results show that the membrane protein responsible for cGMP transport has an ATPase activity and transports the cyclic nucleotide in the presence of ATP.
Boadu
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Department of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037, Tromsø, Norway.
Sager
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D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002793:Cholic Acids; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D002793:Cholic Acids; D011510:Proteolipids; C020485:proteoliposomes; D014316:Tritium; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000251:Adenosine Triphosphatases; D006152:Cyclic GMP; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate