Adenosine triphosphate. A constituent of cholinergic synaptic vesicles

Dowdall, M. J.; Boyne, Alan F.; Whittaker, V. P.

doi:10.1042/bj1400001

Cited by 274 publications

(101 citation statements)

References 17 publications

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“…As in the case of acetylcholine, we therefore conclude that ATP is indeed stored inside the vesicles in agreement with earlier results [14] and is the only major phosphate-containing dissolved vesicle component. This does not exclude the presence of small amounts of G T P as detected in synaptic vesicles from Torpedo californica [15], the resonances of which would be indistinguishable from those of ATP under our conditions.…”

Section: Spectra Of Isolated Vesiclessupporting

confidence: 93%

31P-NMR Analysis of Synaptic Vesicles. Status of ATP and Internal pH

Füldner

Stadler

1982

Eur J Biochem

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The status of ATP in cholinergic synaptic vesicles from the electric organ of Torpedo marmoraia has been studied by 31P-NMR. In isolated vesicles, ATP was found to be the only major phosphate-containing constituent. The chemical shifts and the linewidths of the P, and Pp resonances are different from those of uncompartmented ATP added to the suspension. The spectrum of intact electric tissue contains two sets of ATP resonances; one of them belongs to vesicular ATP as identified by its linewidths and chemical shifts.Both spectral characteristics of vesicular ATP, linewidths and chemical shifts, could be explained by an exchange mechanism, which also occurs in model solutions containing acetylcholine, ATP and Mg2+ at a pH around 5.5.We therefore conclude that the cholinergic synaptic vesicles store ATP together with acetylcholine and Mg2 ' essentially in free solution at an acidic pH. Since the chemical shift of the P, resonance of ATP is largely determined by factors other than pH, the concept of its use as an indicator for the internal pH has to be modified in the case of these organelles.The application of NMR to secretory organelles has yielded a wealth of information regarding the internal organization of these structures [I -41. The secretory vesicles of nerve terminals, the synaptic vesicles, store and release transmitter and therefore play a central role in synaptic transmission. For an understanding of their function it is necessary to know the organization of the core and the physical status of the components. The only source of synaptic vesicles homogeneous with respect to transmitter content and available in sufficient quantity are the purely cholinergic electromotor nerve terminals innervating the electric organs of Torpedinidae. The chemical composition of highly purified vesicle fractions from this source has been determined and the membrane proteins have been analysed [S -71. The vesicles behave like highly hydrated structures in which up to 65% of the water is osmotically active and a further 7 % is bound to readily diffusible solutes [8]. The vesicular cores do not contain soluble proteins like catecholamine-storing chromaffine granules [5]. The main storage products are acetylcholine and ATP. They are present at concentrations which, if they behaved as ideal solutes would generate an osmotic pressure considerably in excess of that of the surrounding cytoplasm [7,9] ; therefore some immobilization or complex formation may be expected.Recently, we have shown by 'H-NMR spectroscopy that the transmitter in these vesicles, acetylcholine, is stored in a dissolved form. In addition, 'H-NMR is able to distinguish compartmented from uncompartmented acetylcholine and to follow its release from the compartmented state [lo].Here, we apply 31P-NMR to isolated vesicles and to intact electric tissue. From the chemical shifts and the linewidths of the P,, Po and P, resonances of intravesicular ATP, the physical state of the stored ATP and the intravesicular pH are deduced. The interpretation is based on the sim...

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Section: Spectra Of Isolated Vesiclessupporting

confidence: 93%

31P-NMR Analysis of Synaptic Vesicles. Status of ATP and Internal pH

Füldner

Stadler

1982

Eur J Biochem

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“…Under pathological conditions, when ATPlevels are persistently low and electrochemical gradients over plasma-and vesicle membrane decrease, the terminal may loose its capacity to prepare for exocytosis and terminals may become deficient. In many systems, both the CNS and periphery, ATP appears to be stored together in single synaptic vesicles with different transmitters like acetylcholine (Dowdall et al, 1974) and norepinephrine (Winkler et al, 1981).…”

Section: Differential Transmitter Release: Co-transmission and Changementioning

confidence: 99%

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

Verhage

Ghijsen

Silva

1994

Progress in Neurobiology

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“…ATP was measured loo r ., by the luciferin/luciferase method as modified by Dowdall et al [21]. Acetylcholine was determined by bioassay [22].…”

Section: Analytical Methou'smentioning

confidence: 99%

The role of thiols in nucleotide uptake into synaptic vesicles from Torpedo marmorata

Lee¹,

Witzemann²

1987

European Journal of Biochemistry

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We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N-ethylmaleimide; radiolabelled N-ethylmaleimide was used to locate the sulfhydryl group to the translocase-associated molecule previously identified as a polypeptide of MI 34000 [Lee and Witzemann (1983) Biochemistry 22,6123-61301. The nucleotide uptake was 75% inhibited by the mercurials p-hydroxymercuribenzoate and p-chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.Reactive sulfhydryl groups often play an important role in the transport of molecules across biological membranes. The ADP/ATP carrier from beef heart mitochondria [l], the glucose transporter of the intestinal brush border membrane [2, 31 and Ca2+ transport by the Ca2+, Mg2+-adenosine triphosphate of the sarcoplasmic reticulum [4], for example, all show high sensitivity to sulfhydryl-blocking reagents. In many cases, the sensitive sulfhydryl groups are involved in a dithiol-disulfide interconversion which in turn affects the affinity of the carrier for the substrate. This has been best demonstrated in the case of hexose and lactose transport in bacteria [5]. The importance of such redox-sensitive steps in the diverse transport systems for Ca2+ (61, Pi [7], y-aminobutyric acid in brain synaptosomes [8], and glucose [9, 101 as well as for hexose and lactose mentioned above, has led Robillard and Konings [ll] to propose that these dithioldisulfide interconversions play a general role in regulating membrane transport.Cholinergic synaptic vesicles from the electromotor tissue of Torpedo marmorata contain an adenine nucleotide translocase with general specificity for transporting ATP, AMP, and ADP [12]. This translocase has been identified with a membrane polypeptide of MI 34000 [13,14].For both synaptic vesicles from electric organ as well as chromaffin granules, loading with ATP occurs prior to the import of neurotransmitter [15, 161. This fact has led to the hypothesis that the presence of ATP is essential for the uptake of the neurotransmitter [12, 151. Since acetylcholine uptake and vesicle recycling may be regulated by vesicular ATP concentrations it is important to understand the molecular mechanism of ATP uptake into vesicles. We have initiated experiments to determine whether sensitive thiol or disulfide groups, located on the translocase, would affect ATP transport into synaptic vesicles.From the experiments reported here, free sulfhydryl groups can be shown to be involved in ATP translocation and to play a critical rol...

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Adenosine triphosphate. A constituent of cholinergic synaptic vesicles

Cited by 274 publications

References 17 publications

31P-NMR Analysis of Synaptic Vesicles. Status of ATP and Internal pH

31P-NMR Analysis of Synaptic Vesicles. Status of ATP and Internal pH

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

The role of thiols in nucleotide uptake into synaptic vesicles from Torpedo marmorata

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