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Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria

1985; Elsevier BV; Volume: 184; Issue: 4 Linguagem: Inglês

10.1016/0022-2836(85)90313-4

1089-8638

John E. Walker, Ian M. Fearnley, N Gay, Bradford W. Gibson, F.D. Northrop, Stephen Joseph Powell, M J Runswick, M Saraste, Victor L. J. Tybulewicz,

Metabolism and Genetic Disorders

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits α, β, γ, δ and ε that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the γ-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The α, β, γ, δ and ε subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the α-subunit and one in each of the γ- and ε-chains; they are absent from the β- and δ-subunits. The stoichiometry of subunits in the complex is estimated to be α3β3γ1δ1ε1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the α- and β-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the α- and β-chains are ragged. In 65% of α-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the β-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The δ-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the α- and β-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine δ-subunit of F1-ATPase shows that it is the counterpart of the bacterial ε-subunit. The bovine ε-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial δ-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane. Comparison of the bovine F1-ATPase sequences with sequences in F1-ATPases determined from other species demonstrates that the subunits have evolved at different rates. The α-, and particularly the β-subunits, which together probably contain the catalytic sites of the enzyme, are highly conserved. Overall the minor subunits are rather poorly conserved, but nonetheless contain well-conserved elements. These are probably the segments that interact with the well-conserved α- and β-polypeptides.