Solubilization and functional reconstitution of the DCCD-sensitive Na+/H(+)-antiporter from Halobacterium halobium.
Na+/H+ exchange activity was solubilized from Halobacterium halobium with octyl-beta-D-glucoside (OG) and was reconstituted into the bacterio-rhodopsin incorporated liposomes (BR-liposomes) by the detergent-dialysis method. Light illumination stimulated uphill 22Na+ uptake into the reconstituted conjugate proteoliposomes. The 22Na+ uptake was FCCP-sensitive and was dependent on the amounts of OG-extract applied. On the other hand, the proteoliposomes reconstituted with the membrane fraction pretreated with N,N'-dicyclohexylcarbodiimide (DCCD) did not exhibit the light-dependent 22Na+ uptake, thus, DCCD-sensitive. When the reconstituted proteoliposome was incubated with [14C]DCCD, radio-labels appeared slightly on 50K but mainly on 11K-Dalton component, which are the same components labeled in the intact membrane vesicles. It is concluded that halobacterial DCCD-sensitive Na+/H(+)-antiporter was solubilized and reconstituted in the conjugate BR-liposomes with preserved functional unit.
Konishi
T
T
Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Japan.
Murakami
N
N
D001436:Bacteriorhodopsins / Q000378:metabolism*; D001692:Biological Transport; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005960:Glucosides / Q000494:pharmacology; D006217:Halobacterium / Q000378:metabolism*; D008081:Liposomes; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002234:Carbodiimides; D002352:Carrier Proteins; D003902:Detergents; D005960:Glucosides; D008081:Liposomes; D017923:Sodium-Hydrogen Exchangers; C018619:octyl-beta-D-glucoside; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D012964:Sodium