The decay of the ATPase activity of light plus thiol-activated thylakoid membranes in the dark.
Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the gamma subunit of the ATP synthase in the light enhances the rate of Mg2+-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH4Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.
McCarty
Richard E
RE
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA. rem1@jhu.edu
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000609:Aminoacridines; D000643:Ammonium Chloride / Q000494:pharmacology; D002021:Buffers; D025222:Chloroplast Proton-Translocating ATPases / Q000502:physiology*; D003624:Darkness; D004983:Ethanolamines / Q000494:pharmacology; D005456:Fluorescent Dyes; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006868:Hydrolysis; D007093:Imidazoles / Q000494:pharmacology; D008027:Light; D018724:Spinacia oleracea / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D020524:Thylakoids / Q000502:physiology*; D014325:Tromethamine / Q000494:pharmacology
D000609:Aminoacridines; D002021:Buffers; D004983:Ethanolamines; D005456:Fluorescent Dyes; D007093:Imidazoles; D013438:Sulfhydryl Compounds; D000643:Ammonium Chloride; D014325:Tromethamine; C010847:9-amino-6-chloro-2-methoxyacridine; D000244:Adenosine Diphosphate; C029899:imidazole; D000255:Adenosine Triphosphate; C020283:diethanolamine; D025222:Chloroplast Proton-Translocating ATPases; D005998:Glycine; C100184:tricine