Changes in chemical properties of mitochondrial adenosinetriphosphatase upon removal of tightly bound nucleotides.
The removal of tightly bound nucleotides from mitochondrial F1-ATPase was found to affect the inhibition by ADP and chemical reactivity toward 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1) and sulfhydryl reagents. Preincubation of nucleotide-depleted F1 with 40 microM ADP in the presence of ethylenediaminetetraacetic acid (EDTA) resulted in a 51% inhibition of the steady-state level of ATPase activity whereas only a 25% inhibition was observed for native F1. Both partially inhibited states of the enzyme could be reversed by the subsequent addition of ATP. Measurement of [14C]ADP binding to nucleotide-depleted F1 in the presence of EDTA reveals three equivalent ADP binding sites with a Kd of 0.45 microM, and a fourth site of lower affinity. The sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were found to inhibit the ATPase activity of nucleotide-depleted F1 but not native F1 or nucleotide-depleted F1 in the presence of ADP or ATP. Polyacrylamide gel electrophoresis of nucleotide-depleted F1 labeled with [14C]NEM gave a 2-fold increase in incorporation into the (alpha + beta) subunits and a 7-fold increase in label in the gamma subunit after 90 min compared to when ADP was present during the reaction. ADP binding to the noncatalytic sites enhanced the rate of inhibition of nucleotide-depleted F1 by NBD-C1 about 2-fold while retarding the subsequent intramolecular transfer from an essential phenol group to an amino group about 2.8-fold. The results suggest a conformational change in F1 caused by changes in nucleotide--protein interaction at the noncatalytic sites.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide