Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.
The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Martins
O B
OB
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Salgado-Martins
I
I
Grieco
M A
MA
Gómez-Puyou
A
A
de Gómez-Puyou
M T
MT
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D000227:Adenine Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007293:Inosine Triphosphate; D011506:Proteins; D000097795:ATPase Inhibitory Protein