Search

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong. It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.

FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.

Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in beta-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

Steady state fluorescence anisotropy (rs) of TMA-DPH was measured to study the effect of respiratory burst activation with PMA, FMLP, and PAF on the physico-chemical structure of PMNs plasma membrane. Our results show a significant increase in rs during the respiratory burst activation. In the presence of NADPH-oxidase inhibitor DPI, only PAF induces changes in rs values. This suggests a non-specific effect of PAF on plasma membrane. Azide, which induces a supranormal release of H2O2, fails to increase the basal rs value after activation. Moreover, the catalase does not abolish the increase in rs induced upon activation. This rules out the possibility that changes of rs during the respiratory burst activation are attributed mainly to H2O2 release. We conclude that multiple processes accompanying the respiratory burst activation are responsible for the changes in the physico-chemical properties of PMNs plasma membrane.

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.

Extraction of endogenous ubiquinone from lyophilized beef heart mitochondria results in increases of both the order parameter of the spin label 5-NS and of the rotational correlation time of 16-NS; reconstitution with the pentane extract results in restoration of the original spectral parameters. On the other hand, addition of purified ubiquinone homologs restores the original spectra only in the case of 16-NS, whereas the order parameter of 5-NS is restored by addition of mixed phospholipids. The same amounts of ubiquinone homologs incorporated in mixed phospholipid vesicles induce much lower effects. It is suggested that ubiquinone in mitochondria is intercalated with the lipid chains of the membrane in such a way to perturb the fluidity of the hydrophobic core.

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.