Steady-state ATP synthesis by bacteriorhodopsin and chloroplast coupling factor co-reconstituted into asolectin vesicles.
A method was developed for the co-reconstitution of bacteriorhodopsin and chloroplast coupling factor in asolectin vesicles. First, bacteriorhodopsin was reconstituted from a mixture of octyl glucoside, asolectin, and protein in the presence of ethylenediaminetetraacetic acid by passage through a Sephadex G-50 centrifuge column. Then, the purified coupling factor was reconstituted from a mixture of sodium cholate, bacteriorhodopsin vesicles, and coupling factor in the presence of Mg2+ by passage through the centrifuge column. Sucrose density-gradient centrifugation indicated a band of vesicles with slightly different positions in the gradient for maximum vesicle concentration, bacteriorhodopsin vesicle concentration, ATP synthesis, and ATP hydrolysis. The rate of light-driven ATP synthesis reaches a limiting value as the concentration of bacteriorhodopsin and the light intensity are increased. A steady-state rate of ATP synthesis of 1 mumol per mg of coupling factor X min-1 has been achieved. Apparently this rate is limited by the heterogeneity within the vesicle population and by the ability of bacteriorhodopsin to form a sufficiently large pH gradient.
Krupinski
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Hammes
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism*; D002736:Chloroplasts; D008027:Light; D010713:Phosphatidylcholines; D010743:Phospholipids; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D010713:Phosphatidylcholines; D010743:Phospholipids; D002338:Carotenoids; D001436:Bacteriorhodopsins; C024990:asolectin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases