Some Unique Characteristics of Thylakoid Unisite ATPase

Zhang, Shiying; Jagendorf, André T.

doi:10.1074/jbc.270.12.6607

Cited by 10 publications

(1 citation statement)

References 42 publications

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“…Azide was earlier found to inhibit the CF 1 -ATPase by increasing the binding of inhibitory MgADP (Murataliev et al, 1991). Zhang and Jagendorf (1995) have shown that azide inhibits even unisite ATP hydrolysis by spinach thylakoid-bound CF 1 as long as this CF 1 contains some bound ADP. It seemed therefore possible that in the presence of ADP azide will inhibit also the MgATPase activity of CF 1 -R 3 β 3 .…”

Section: Resultsmentioning

confidence: 96%

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Sokolov

Gromet-Elhanan

1996

Biochemistry

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A minimal chloroplast coupling factor CF1 core complex, containing only alpha and beta subunits, has been isolated from spinach thylakoids [Avital, S., & Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072]. This CF1(alpha beta) exhibited a low MgATPase activity, which was stimulated but not inhibited by low concentrations of the species-specific CF1 effector tentoxin. As is reported here, the structure of CF1(alpha beta) could not be determined due to its instability. However, its pretreatment with high tentoxin concentrations resulted in a remarkable 50-fold stimulation of the MgATPase activity as well as stabilization of its hexameric structure, thus enabling the isolation of a more active CF1-alpha 3 beta 3 complex by size-exclusion chromatography. A detailed characterization of the MgATPase activity of this tentoxin-stabilized CF1-alpha 3 beta 3 hexamer, as compared to the activity of a CF1 complex lacking the epsilon subunit, revealed similar apparent Km values and a similar stimulation by the presence of 100 microM tentoxin in the assay medium, but drastic differences in all other tested assays. Most pronounced were their different temperature profiles and different responses to all added inhibitors and stimulators of the CF1 MgATPase activity and to excess free Mg2+ ions. The specific properties of the stable CF1-alpha 3 beta 3 hexamer are identical to those earlier reported for its parent-unstable CF1(alpha beta). These results indicate that, although the CF1 gamma subunit is not required for the low CF1(alpha beta) ATPase activity nor for the higher activity of the tentoxin-stabilized CF1-alpha 3 beta 3, it plays a central role in obtaining the typical functional properties of the CF1-ATPase. Kinetic cooperativity could not be critically tested as yet with any F1-alpha 3 beta 3. However, tentoxin, as azide, has been shown to inhibit multisite but not unisite catalysis. Therefore, the observation that CF1-alpha 3 beta 3 is only stimulated by tentoxin suggests that the required presence of CF1-gamma for obtaining inhibition by tentoxin reflects the role of this subunit in cooperative interactions between the catalytic sites.

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Section: Resultsmentioning

confidence: 96%

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Sokolov

Gromet-Elhanan

1996

Biochemistry

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Mutational Analysis of ATP Synthase An Approach to Catalysis and Energy Coupling

Futai

Omote

1999

Frontiers of Cellular Bioenergetics

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Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

Covián

Chess

Balaban

2012

Analytical Biochemistry

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Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed.

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Some Unique Characteristics of Thylakoid Unisite ATPase

Cited by 10 publications

References 42 publications

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Mutational Analysis of ATP Synthase An Approach to Catalysis and Energy Coupling

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

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Some Unique Characteristics of Thylakoid Unisite ATPase

Cited by 10 publications

References 42 publications

Spinach Chloroplast Coupling Factor CF1-α3β3 Core Complex: Structure, Stability, and Catalytic Properties

Spinach Chloroplast Coupling Factor CF1-α3β3 Core Complex: Structure, Stability, and Catalytic Properties

Mutational Analysis of ATP Synthase An Approach to Catalysis and Energy Coupling

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

Contact Info

Product

Resources

About

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties