Influence of nucleotides on the cold stability of chloroplast coupling factor 1.
Chloroplast coupling factor 1 (CF1), a cold-labile enzyme, contains six nucleotide binding sites. These sites are located at the alpha/beta interfaces of the alpha 3 beta 3 heterohexamer. The cold lability of CF1 is decreased by the presence of nucleotides in the medium. We have studied the influence of both different nucleotides and different binding sites on the cold dissociation of CF1. To monitor the dissociation of CF1 during cold treatment, 8-anilino-1-naphthalenesulfonic acid (ANS) was employed. The increase in ANS fluorescence during cold treatment is the result of increased accessibility of intersubunit hydrophobic regions as the complex dissociates. Mg(2+)-adenosine triphosphates, tightly bound to CF1, markedly stabilize the enzyme in the cold. ADP only protects CF1 from dissociating in the cold when it is bound to the loose sites or when it is bound in conjunction with Mg2+. CF1 that contained 2 mol of ADP/mol and little bound Mg2+ was nearly as cold labile as CF1 that contained just 0.2 mol of ADP/mol. When about one of the two bound ADPs was replaced with adenylyl beta, gamma-imidodiphosphate (AMP-PNP), some protection from cold dissociation was observed. These results show that the site(s) occupied, as well as the nucleotides they contain, strongly influence(s) the structural stability of CF1.
Hightower
K E
KE
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
McCarty
R E
RE
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D003080:Cold Temperature; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium