Mitochondrial F(1)F(O)-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F(1) sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0-9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5.
3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.
The activation of the Ca2+‐ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half‐maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin‐Sepharose resin and in dependence on calmodulin concentrations when the pH is increased.
In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin‐binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.
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