Adenosine triphosphatase and nucleotide binding activity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase.
Adenosine triphosphatase activity and nucleotide binding affinity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase were studied. The aim was to find out whether isolated beta-subunit would provide an experimental model in which effects of mutations on catalysis per se, unencumbered by complications due to their effects on positive catalytic cooperativity, could be studied. Three types of purified, isolated beta-subunit preparations were studied. Type I-beta was from a strain lacking all F1F0 subunits except beta and epsilon. Type II-beta was from F1 carrying the alpha S375F mutation which blocks positive catalytic cooperativity. Type III-beta was from normal F1. Type I- and II-beta had very low ATPase activity (less than 10(-4) s-1) which was azide-insensitive, aurovertin-insensitive, and unaffected by anti-beta antibody. Type I-beta activity was EDTA-insensitive. We conclude that isolated beta-subunit from E. coli F1F0 has zero or at most very low intrinsic ATPase activity. Type III-beta had low ATPase activity (8.4 x 10(-5) s-1 to 1.1 x 10(-3) s-1 in seven different preparations). This activity was aurovertin-sensitive, but varied in azide sensitivity from 0 to 34% inhibited. The azide-sensitive component, like F1 and alpha 3 beta 3 gamma oligomer, was inhibited by anti-beta and anti-alpha antibodies. The azide-insensitive component was stimulated by anti-beta and unaffected by anti-alpha. We show here that (alpha beta)-oligomer has ATPase activity which is azide-insensitive, aurovertin-sensitive, stimulated by anti-beta, and unaffected by anti-alpha. The intrinsic ATPase activity of Type III-beta could be due to contaminating (alpha beta)-oligomer plus alpha 3 beta 3 gamma-oligomer. Isolated beta had very low affinity for nucleotide as compared to the first catalytic site on F1. Taken together with the very low ATPase activity of isolated beta (even if real), the work shows that isolated beta is not a good experimental model of F1 catalysis.
al-Shawi
M K
MK
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Parsonage
D
D
Senior
A E
AE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000494:pharmacology; D001313:Aurovertins / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D001313:Aurovertins; D001386:Azides; D046911:Macromolecular Substances; D009711:Nucleotides; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; C039026:aurovertin D; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium