ATPase oligomycin-sensitive

Decreased membrane-bound ATPase Oligomycin sensitivity not modified... 

Mitochondrial oligomycin-sensitive mg2+ATPase is thought to play a major role in oxidative phosphorylation (Boyer et al. 1977). It has been suggested that impairment of mitochondrial energy metabolism by chlordecone may contribute to the decreases in body weight observed following exposure to this chemical (Desaiah 1981). 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

One striking characteristic of the coupling ATPase of energy-transducing membranes, apart from the extraordinarily large number of different polypeptide subunits, is the existence of two different polypeptides involved in the response of the enzyme to the inhibitor oligomycin. One binds the inhibitor, the other, separated in space from the former by possibly as much as 10 to 15 A, confers oligomycin sensitivity to the entire enzyme complex. How could the transfer of information between these two polypeptide subunits and their concerted interaction with the ATPase proper be visualized ... 

Figure 4.1.1 (a) % Inhibition of beef heart mitochondrial Ca -ATPase ( ----- ) and oligomycin-sensitive... 

Sciortino, F., Poole, P. H., Stanley, H. E., Havlin, S. (1990). Phys. Rev. Letters, 64,1686-1689. Solaini, G., Baracea, A., Parenti Castilla, G., Lenarz, G. (1984). Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase. J. Bioenerg. Biomembr. 16, 391-406. 

Mitochondrial ATP synthase of yeast contains at least 13 different kinds of subunits and that of ani-mals 16, twice as many as in E. coli. Subunits a, p, y, a, b, and c of the mitochondrial synthase correspond to those of E. coli. However, the mitochondrial homolog of E. coli 5 is called the oligomycin-sensitivity-conferring protein (OSCP). It makes the ATPase activity sensitive to oligomycin. The mitochondrial 8 subunit corresponds to e of E. coli or of chloroplasts. Mitochondrial e has no coimterpart in bacteria. ... 

The fact that ATP can be synthesized by establishing a pH differential of some 3.5 units across the lamellae of spinach chloroplasts has been interpreted as evidence in favour of the reversibility of the ATPase system in chloroplasts and calculations based on the pH differential required to dehydrate ADP-f-Pj and the stoichiometry of synthesis of ATP per proton estimated to pass through the membrane suggest that the system may be a 2H -translocator-ATPase, as in mitochondria . The reversibility of the mitochondrial ATPase system has similarly been inferred from the recent observations of Reid et on the oligomycin-sensitive synthesis of ATP by rat-liver mitochondria subjected to a pH differential. 

In the FoFi-ATPase of mammalian and yeast mitochondria, the oligomy-cin sensitivity conferring protein (OSCP) is one of the subunits of the enzyme complex, and is needed for inhibition of ATP hydrolysis by oligomycin (1). The amino acid sequence of OSCP from beef heart mitochondria is homologous to the amino acid sequence of the S-subunit of the Fi-ATPase from E. coli, 26.4% (2), chloroplasts, 25.3% (3), and Rhodospirill urn rubrum, 28.9% (2). However, only R. rubrum is sensitive to oligomycin (4). It has also been shown that R. rubrum Fj-ATPase, with the B-subunits substituted with B from E. coli, is not oligomycin sensitive when reconstituted to depleted membranes (5). 

I admit that when I first read his proposition, I was not impressed. In 1965 I published a book on Mechanisms in Bioenergetics and did not even mention the chemiosmotic hypothesis. Phil Handler who wrote a generous review of my book in Science objected to my failure to discuss Mitchell s hypothesis. By the time his review appeared I knew that his criticism was justified because Peter Mitchell had visited me in New York in 1965. This was another important event in my scientific life. Not that I really understood most of what Mitchell said during these days of intensive discussions, but I opened my mind to a new way of thinking. I am now convinced that the basic formulations of his chemiosmotic hypothesis are correct, namely that the function of the respiratory chain is to translocate protons and that the return of those protons via the oligomycin-sensitive ATPase is responsible for ATP formation. Thus the problem of the mecham sm of coupling of oxidation and phosphorylation is basically solved. 

The oligomycin-sensitive ATPase complex, a major enzyme of the mitochondrial inner membrane, can serve to illustrate some of these principles. The ATPase complex is a water-insoluble enzyme which spontaneously associates into vesicular membranes in the presence of phospholipids. The ultrastructural appearance of the reconstituted ATPase membranes is similar to that of the native inner mitochondrial membrane. The complex consists of at least 10 different subunit polypeptides which have been resolved into 3 components, each with a measurable function (1) Five of the polypeptides are part of a catalytic unit called Fi, a water-soluble ATPase which neither requires phospholipids for activity nor has any detectable capacity for binding phospholipids. (2) Four other polypeptides form a unit which has no presently known enzymatic function, but when combined with Fi modifies its physical and catalytic properties. The proteins of this unit are extremely insoluble in water and can combine with phospholipids to form membranes. One of the proteins of the membrane unit is characterized by an unusually large proportion of nonpolar amino acids and a high affinity for phospholipids. (3) The third component (OSCP) is a single polypeptide whose function is to link F to the hydrophobic membrane unit. In the purified state, this protein is completely water soluble. ... 

Fig. 2. Identification of fMet on subunit 9 of oligomycin-sensitive ATPase. Sac-charomyces cerevisiae, strain 4D, was grown on SSA 3% lactate, labeled with PH]formate for 6 h, and mitochondria prepared as previously described. Preparation of chloroform-methanol (CM) fractions was done as described by Sierra and Tzagoloff. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis was done by the method of Weber and Osborn, with 8% acrylamide for 11 h at 5 mA per gel. The 12-cm gels were cut into 1-mm slices and counted. Different patterns were normalized thru mobility of protein standards and tracking dye. (A) Final purified CM fraction after several ether...

When dealing with pleiotropic respiratory-deficient mutants of yeast, three practical difficulties constantly arise. The major inconvenience is the general genetic instability of the chromosomal pleiotropic respiratory-deficient mutants in S. cerevisiae where an additional p mutation spontaneously arises with very high frequency Since both the nuclear and the mitochondrial mutations are often expressed by deficient cytochromes aas and b, as well as by the loss of oligomycin sensitivity of the ATPase, the biochemical study of these double mutants is very difficult. An easy way out of this difficulty is to use chromosomal respiratory-deficient mutants of a petite-negative yeast species like 5. pombe, where no viable p mutation can be induced. 

Subik et al. discovered S. cerevisiae mutants in which multiple deficiencies in cytochromes aa and b were accompanied by loss of the oligomycin sensitivity of the mitochondrial ATPase. Mutants with similar properties were reported for S. pombe. It has been suggested that in S. cerevisiae,these pleiotropic effects were the result of a deficient mitochondrial protein synthesis. Total absence of mitochondrial ATPase activity has also been reported to accompany multiple cytochrome deficiencies in S. pombe and S. cerevisiae. In the latter case, mitochondrial protein synthesis was shown to be functional. ... 

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