Size and molecular parameters of adenosine triphosphatase from Escherichia coli.

In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 99%

Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and Fo sectors of the mitochondrial ATPase complex

Duszyński¹,

Dupuis²,

Lux³

et al. 1988

“…Rotational diffusion measurements suggested similar overall dimensions for the enzyme, 180 X 180 X 70 Á (Wagner & Junge, 1982), and recent measurements using time-resolved laser spectroscopy gave 170 X 170 X 85 Á (Wagner et al, 1985). These results are strikingly different from the smaller volumes obtained by X-ray and neutron scattering (Paradles & Schmidt, 1979;Satre & Zaccai, 1979). The discrepancy likely arises from the hydration brought about by glycerol, which may change the composition of the solvent around the molecule, affecting its diffusion properties (Fredericq & Houssier, 1973).…”

Section: Discussionmentioning

confidence: 72%

“…However, even in this simple case where a single rotary relaxation time is observed, a model has to be assumed to estimate the molecular dimensions. Although F, was first visualized as spherical particles of about 90-Á diameter (Kagawa & Racker, 1966;Howell & Moudrianakis, 1967;Munoz et al, 1968;Catterall & Pedersen, 1974;Garber & Steponkus, 1974;Yamato et al, 1978), it now appears that the F,-ATPase is an oblate ellipsoid (Süss et al, 1978;Paradles & Schmidt, 1979;Satre & Zaccai, 1979;Amzel et al, 1982;Wagner & Junge, 1982;Ackey et al, 1983;Tiedge et al, 1983;Wagner et al, 1985). Therefore, the complete hydrodynamical characterization of such a particle is possible only if at least a second parameter is available.…”

Section: Discussionmentioning

confidence: 99%

“…The F, sector isolated from various sources consists of five different subunits, the stoichiometry of which is o3The molecular weight of this hydrophilic sector ranges from 300000 to 400000. Structural studies obtained by hydrodynamic methods (Wagner & Junge, 1982;Wagner et al, 1985) as well as X-ray (Süss et al, 1978;Paradles & Schmidt, 1979) and neutron scattering (Satre & Zaccai, 1979) and electron microscopy of single crystals (Ackey et al, 1983) led to the conclusion that Ft is rather eccentric with an axial ratio of 1.5-2. Fj has generally been described as an ellipsoid with a maximal dimension of 110-120 Á and a thickness of 70-80 Á.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structural aspects and orientation mechanism of mitochondrial F1-adenosine triphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

Deléage¹,

Roux²,

Marion³

1986

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.

“…(Paradles & Schmidt, 1979;Suss et al, 1978;Akey et al, 1983) ellipsoids have been used to describe the shape of coupling factors, usually with an axial ratio of about 2. At the present time, an unequivocal distinction between the two types of ellipsoids cannot be made.…”

mentioning

confidence: 99%

Chloroplast coupling factor 1: dependence of rotational correlation time on polypeptide composition

Schinkel¹,

Hammes²

1986

Time-resolved fluorescence depolarization measurements were made on chloroplast coupling factor 1 (CF1) labeled with pyrenylmaleimide. Rotational correlation times were determined for native CF1, for CF1 lacking epsilon and/or delta polypeptides, and for activated enzyme. The rotational correlation time measured is characteristic of the rotation of the entire enzyme. Removal of the delta polypeptide resulted in a 25% smaller rotational correlation time, although the delta polypeptide contributes less than 5% of the mass of CF1. Removal of the epsilon polypeptide was without effect. Simultaneous removal of delta and epsilon polypeptides produced a 30% smaller rotational correlation time. Activation of CF1 ATPase by incubation with dithiothreitol reduced the rotational correlation time by 15% relative to that of the latent enzyme. The rotational correlation time of CF1 with delta and epsilon polypeptides removed is essentially that expected for a spherical molecule, whereas the other forms of the enzyme can be approximated as ellipsoids of revolution; the axial ratio of the latent enzyme is estimated from the rotational correlation time and the intrinsic viscosity. These data indicate that the delta polypeptide significantly alters the shape of the enzyme and that a conformational change accompanies dithiothreitol activation of the enzyme.