8-N3-3′-Biotinyl-ATP, a Novel Monofunctional Reagent: Differences in the F1- and V1-ATPases by Means of the ATP Analogue

Schäfer, Hans‐Jochen; Coskun, Ünal; Eger, Olaf; Godovac‐Zimmermann, Jasminka; Wieczorek, Helmut; Kagawa, Yasuo; Grüber, Gerhard

doi:10.1006/bbrc.2001.5502

Cited by 17 publications

(22 citation statements)

References 52 publications

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“…The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8,9), revealed a molecule with a single, compact stalk.The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule.…”

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confidence: 99%

Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Rizzo¹,

Coskun²,

Radermacher³

et al. 2003

Journal of Biological Chemistry

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The effect of the ATPase activity of Manduca sexta V 1 ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V 1 were studied. The Vacuolar ATPases (V 1 V O ATPases) define an ubiquitous class of proton pumps, which utilize ATP hydrolysis to maintain an acidic pH inside the vacuole (1). The electrochemical ion gradient created across the vacuolar membrane is used for the accumulation of positively charged substrates such as calcium and basic amino acids (2). In addition to this storage function, the vacuolar compartment has secretory and proteolytic functions (3, 4). The V-ATPases, consisting of at least thirteen distinct subunits (A 3 :B 3 :C:D:E:F:G y :H z :a:d:c:cЈ:cЉ) are morphologically subdivided in two components: a membrane-bound domain, V O , that contains the ion channel, and an extrinsic domain, V 1 , in which ATP hydrolysis takes place (4, 5). The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8,9), revealed a molecule with a single, compact stalk.The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule. The x-ray data define a mushroom-shaped V 1 ATPase, which consists of an ϳ145 Å headpiece, joined by an elongated stalk (8). Image processing of electron micrographs of negatively stained V 1 (9, 12, 13) has revealed that the headpiece consists of a pseudo-hexagonal arrangement of six masses surrounding a seventh mass. These barrel-shaped masses of approximately 30 Å in diameter and 80 Å in length, which consist of the major subunits A and B, are arranged in an alternating manner (9). The hexagonal barrel of subunits A and B encloses a cavity of ϳ32 Å in which part of the central stalk is asymmetrically located. The stalk protrudes from the bottom side of the headpiece forming an angle of ϳ7°with the vertical axis of the molecule. At the upper end of the hexagonal barrel extensions can be observed, assumed to belong to the N termini of subunit A (9, 13). Further insights into the topology of the M. sexta V 1 ATPase were obtained by differential protease sensitivity, release by chaotropic agents (13), and cross-linking studies (13,14). These studies resulted in a model in which the subunits H, C, D, G, and F are exposed in the enzyme, whereas subunit E is shielded in the complex (6,13,14).Here we report an investigation of the structure-function relationship of the V 1 stalk...

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confidence: 99%

Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Rizzo¹,

Coskun²,

Radermacher³

et al. 2003

Journal of Biological Chemistry

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“…Any of these lipids was forming a phase-separated domain, as suggested by the appearance of an additional higher temperature thermotropic transition besides that of POPC. Whereas the insertion of b 5 wt in the bilayer affected the POPC component, b 5 ext caused a decrease of the higher temperature, phase-separated component in all mixtures, except POPC/DSPC. These data have also been confirmed using fluorescence spectroscopy.…”

Section: C4-032pmentioning

confidence: 89%

“…In 1994, this interfacial location of all the nucleotide binding sites was confirmed impressively by X-ray analysis of the F1ATPase from beef heart mitochondria [4]. The introduction of an additional biotin residue, yielding 3¢-biotinyl-8-azido-ATP [5], is advantageous for an easy detection of labeled proteins. Irradiation of the F1ATPase from the thermophilic bacterium PS3 (TF1) in the presence of 3-biotinyl-8-azido-ATP resulted in the nucleotide-specific inactivation of the enzyme as well as in the nucleotide-dependent labeling of alpha and/or beta subunits.…”

Section: C1-057pmentioning

confidence: 98%

“…Irradiation of the F1ATPase from the thermophilic bacterium PS3 (TF1) in the presence of 3-biotinyl-8-azido-ATP resulted in the nucleotide-specific inactivation of the enzyme as well as in the nucleotide-dependent labeling of alpha and/or beta subunits. In addition, 3¢-biotinyl-8-azido-ATP could be used successfully to label V1ATPase from Manduca sexta [5]. Dimerization of 3¢-biotinyl-8-azido-ADP resulted in the formation of the bifunctional diadenine dinucleotide 3¢-dibiotinyl-8-diazido-AP4A.…”

Section: C1-057pmentioning

confidence: 99%

“…To investigate this issue, we studied lipid-protein interaction by differential scanning calorimetry (DSC) and fluorescence techniques, using liposomes embedding cytochrome b 5 wild-type (b 5 wt, an ER resident) or mutant (b 5 ext, having five extra non-polar amino acids in the TMD, transported to the plasma membrane). The proteins were incorporated into liposomes of palmitoyl-oleyl-phosphatidylcholine (POPC) plus 5-20% either palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), distearoyl-phosphatidylserine (DSPS), distearoyl-phosphatidic acid (DSPA) or C-16 ceramide (CER).…”

Section: C4-032pmentioning

confidence: 99%

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Membrane Proteins and their Interactions

Planque¹,

Contera²,

Mendes³

et al. 2005

The FEBS Journal

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Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

Hacker

Welter

Marx

2015

CP Nucleic Acid Chemistry

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This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

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8-N3-3′-Biotinyl-ATP, a Novel Monofunctional Reagent: Differences in the F1- and V1-ATPases by Means of the ATP Analogue

Cited by 17 publications

References 52 publications

Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

Membrane Proteins and their Interactions

Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

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