Fluorescence properties of 2′ (or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and its use in the study of binding to heavy meromyosin ATPase

Hiratsuka, Toshiaki

doi:10.1016/0005-2795(76)90277-4

Cited by 50 publications

(31 citation statements)

References 14 publications

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“…F obs for unbound TNP-ATP in buffer without protein was low and increased in a nonlinear, concentrationdependent manner (Fig. 2, A and B), consistent with the intrinsic fluorescence of this ATP analogue and the inner filter effect (29,30,31). All of the buffer data were well fit using a second order polynomial that accounts for this inner filter effect (see "Materials and Methods"; r 2 Ն 0.99).…”

Section: -Azido-[␥-mentioning

confidence: 48%

“…TNP-ATP Binding-To assess the binding of ATP to these recombinant fusion proteins, we used fluorescent TNP-ATP (Molecular Probes, Inc.) (29,30), which has been widely employed to study nucleotide binding to enzymes and other proteins (31)(32)(33)(34). The binding of TNP-ATP to recombinant proteins was performed generally as described by Faller (32).…”

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

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The Carboxyl Termini of KATP Channels Bind Nucleotides

Vanoye¹,

MacGregor²,

Dong³

et al. 2002

Journal of Biological Chemistry

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Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K ATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer. ATP-sensitive or ATP-regulated potassium (K ATP )1 channels couple metabolism to either cell excitability (Kir6.x) (1-6) or potassium secretion (Kir1.1 in kidney) (7,8) and provide therapeutic targets for diseases including tissue ischemia, diabetes, hypertension, and disorders of potassium homeostasis. K ATP channels are formed by an octameric complex of four pore-forming subunits (Kir6.x or Kir1.1) and four sulfonylurea receptors, SUR1 or SUR2 for Kir6.x (2) or the cystic fibrosis transmembrane conductance regulator or SUR2b for Kir1.1 (9, 10).Although the SUR/fibrosis transmembrane conductance regulator subunits contain nucleotide-binding folds (11, 12), this subunit is not required for ATP-mediated inhibition of K ϩ channel activity. For example, deletion of the last 36 amino acids from the COOH terminus of Kir6.2 (Kir6.2⌬C36) produces functional K ϩ channels in the absence of coexpressed SURs that are sensitive to ATP (13). Nevertheless, SUR subunits are required for ADP-mediated activation of K ATP channels (14 -16). Thus, ATP inhibition of K ATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[␥-32 P]ATP (17, 18) also supports the direct binding of ATP with the pore-forming subunit of K ATP channels. In addition, mutations in both the NH 2 -and COOH-terminal regions of the Kir6.2 (13, 19 -23) and Kir1.1 (24) subunits alter the EC 50 for ATP-mediated channel gating. Because ATP-mediated inhibition of channel activity must be a complex process involving residues that form an ATP-binding pocket and others that may be required for linking ATP binding to channel closure, those mutational studies of channel gating by nucleotides do not provide unequivocal evidence for direct involvement of those residues in ATP binding.In the present study, we assessed the direct binding of fluorescent 2Ј,3Ј-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate (TNP-ATP) to purified maltose-binding fusion proteins of the cytosolic NH 2 and COOH termini of the three known K ATP channels and the COOH terminus of a ATP-insensitive inward rectifier K ϩ channel, Kir2.1 (25). We provide herein what we believe to be the first evidence of direct binding of ATP to cytosolic domains of the pore-forming subunits of K ATP channels and show that the COOH termini, but not the NH 2 termini, of Kir subunits of K ATP channels bind TNP-ATP. The kinetic analyses of TNP-ATP binding suggest that the COOH termini have a single nucleotide-binding site. Based on glutaraldehyde cross-linking...

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Section: -Azido-[␥-mentioning

confidence: 48%

Section: Dna Constructs and Mutagenesis-dnas Encoding The Nhmentioning

confidence: 99%

The Carboxyl Termini of KATP Channels Bind Nucleotides

Vanoye¹,

MacGregor²,

Dong³

et al. 2002

Journal of Biological Chemistry

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“…Binding of TNP-nucleotides causes an increase in fluorescence (enhanced fluorescence), a phenomenon that has been widely used to characterize the nucleotidebinding abilities of proteins (20). Fluorescence enhancement emerges from the changes in polarity in the near environment of the TNP moiety upon binding (14,26). TraG⌬2 and TrwB⌬1 produced significant fluorescence enhancement of TNP-ATP and TNP-ADP (Fig.…”

Section: Resultsmentioning

confidence: 99%

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Schröder

Lanka

2003

J Bacteriol

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TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We charac- Type IV secretion is an energy-driven mechanism for delivery of effector molecules from bacterial donors to recipient cells. It mediates the transfer of toxic components into eukaryotic hosts by many pathogenic bacteria such as Helicobacter pylori, Legionella pneumophila, Brucella spp., and Bartonella henselae and enables DNA transfer in bacterial conjugation systems such as that encoded by plasmid RP4.

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“…The general structural formula for a TNP nucleotide, the ribosemodified chromophoric fluorescent analogue of ATP, ADP and AMP at neutral pH (Hiratsuka & Uchida, 1973). The anionic structure of the trinitrophenyl moiety is responsible for the fluorescence, pKa (−log of the acid dissociation constant) being 5·0 (Hiratsuka, 1976).…”

Section: Chemicals and Solutionsmentioning

confidence: 99%

“…2' (or 3')-O-(2,4,6,-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) was obtained from Molecular Probes and purified on a Sephadex LH_20 column packed in water (Hiratsuka, 1976). N_Methylanthraniloyl-ATP (Mant_ATP) was synthesized by reaction of ATP with N-methylisatoic anhydride as described by Hiratsuka (1983).…”

Section: Chemicals and Solutionsmentioning

confidence: 99%

Fluorescence changes of a label attached near the myosin active site on nucleotide binding in rat skeletal muscle fibres

Fujita

Nawata

Yamada

1999

The Journal of Physiology

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1. Trinitrophenyl AMP (TNP_AMP) in the concentration range 10-300 ìÒ induced an increase in fluorescence intensity at around 530 nm in skinned skeletal muscle fibres freshly obtained from rat psoas muscle. 2. The fluorescence intensity of the fibres depended on TNP_AMP concentration up to•200 ìÒ. The Kd of TNP_AMP binding to the muscle fibres was 38·0 ± 8·4 ìÒ (mean ± s.d., n = 4 measurements) in three fibres. TNP_AMP fluorescence was readily washed out. 3. Various nucleotides affected the fluorescence of the fibres incubated in 20 ìÒ TNP_AMP.MgATP (1 mÒ) and caged ATP (5 mÒ) reduced the fluorescence in 20 ìÒ TNP_AMP by more than 40 % of the value measured in the absence of nucleotide. 4. When the fibres were stretched to almost no filament overlap, the extent of the quenching of the TNP_AMP (20 ìÒ) fluorescence due to ATP binding was reduced by 14%. This might be explained by assuming that the association of the thin filament affected the TNP_AMP fluorescence in muscle fibres. 5. The distance between the active site and the specific site for TNP was measured by the fluorescence resonance energy transfer between N-methylanthraniloyl-ATP (Mant_ATP) bound to the active site and the TNP_AMP bound to the TNP_specific site in muscle fibres. The results showed that the distance between the two may be less than 2 nm. 6. It may be concluded that the fluorescence intensity at 530 nm in skinned muscle fibres in low concentrations of TNP_AMP changes directly reflecting the conformational state of the nucleotide-binding region that is determined by the binding of nucleotides.8726

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Fluorescence properties of 2′ (or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and its use in the study of binding to heavy meromyosin ATPase

Cited by 50 publications

References 14 publications

The Carboxyl Termini of KATP Channels Bind Nucleotides

The Carboxyl Termini of KATP Channels Bind Nucleotides

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Fluorescence changes of a label attached near the myosin active site on nucleotide binding in rat skeletal muscle fibres

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