Reconstitution of ATP-dependent cGMP transport into proteoliposomes by membrane proteins from human erythrocytes.
The cellular efflux of cGMP from human erythrocytes has previously been characterized in functional studies. The purpose of the present study was to find membrane proteins with the ability to restore ATP-dependent uptake of cGMP into proteoliposomes. Human erythrocyte membranes were solubilized with CHAPS (3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate) and gel filtration gave three protein fractions with the ability to restore active transport. Only two of these fractions were retained on a lentil lectin column. By using these two purification steps, active transport was 11 times higher in the first fraction compared to the original material and SDS-PAGE showed the presence of proteins with sizes of 145 kDa and 165 kDa. The second fraction gave 20 times higher active transport after purification and comprised proteins with sizes of 145 kDa and 180 kDa. At present three members of the MRP (multi-resistance associated protein) family have been detected in human erythrocytes: MRPI, MRP4 and MRP5. The last two proteins have been shown to transport cyclic nucleotides. The present findings are compatible with MRP4 as the 145 kDa protein, MRP5 as the 165 kDa protein and MRP1 as the 180 kDa protein. However, the 145 kDa protein could also be SMRP (short multi-resistance protein), the gene splice variant of MRP5. Immunoprecipitation of MRP5 from CHAPS-solubilized extract reduced active transport and specific binding by about 45% and 40%, respectively. This shows that MRP5 is an important cGMP-transporting protein in human erythrocytes.
Boadu
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Department of Pharmacology, Institute of Medical Biology, Medical Faculty, University of Tromsø, Norway.
Sager
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D001693:Biological Transport, Active; D006152:Cyclic GMP / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D011233:Precipitin Tests; D011510:Proteolipids / Q000378:metabolism*
D000255:Adenosine Triphosphate; D006152:Cyclic GMP; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011510:Proteolipids; C120809:ABCC5 protein, human; C020485:proteoliposomes