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...

The storage granules of cholinergic nerve terminals, the synaptic vesicles, have a central role in the mechanism of cholinergic transmission, and direct evidence as to whether acetylcholine is stored in them in a free or partially immobilized form is therefore of interest. In some secretory systems it has become evident that low molecular weight secretion products are stored within the granules as complexes with proteins or proteoglycans. The application of high resolution NMR spectroscopy to isolated granules has recently elucidated the internal structure of secretory granules. We report here that proton NMR analysis of isolated cholinergic synaptic vesicles from Torpedo marmorata allows the identification of acetylcholine within the organelle. The spectrum shows that all the acetylcholine is dissolved in an essentially fluid phase within the core. Vesicular and uncompartmentalized acetylcholine can be distinguished from each other and in addition, any hydrolysis to choline and acetate can be monitored. The results open the possibility of studying dynamics of acetylcholine pools and their breakdown in cholinergic tissue by a direct and non-perturbing method.

Proton magnetic resonance spectra of Be(N0 3h solutions in mixtures of water and dimethylsulphoxide (DMSO) have been measured at different solvent compositions and temperatures at 270 MHz. The relative amounts of the solvated species Be(OH 2),.(DMSO)4_i have been determined. The total solvation number was 4. The kinetics of solvent exchange has been studied in DMSO. The proton exchange in pure aqueous solutions may be explained in terms of a protolysis reaction. The solvent molecule preferentially solvating the beryllium ion is DMSO in water rich solutions and water in DMSO rich solutions. Protonenresonanzmessungen von Be(N0 3h-Losungen in Wasserdimethylsulfoxid-(DMSO)-Mischungen wurden bei verschiedenen Losungsmittelzusammensetzungen und Temperaturen mit 270 MHz durchgefuhrt. Die relativen Mengen der solvatisierten lonen Be(OH 2),.(DMSO)4_i wurden bestimmt. Die Gesamtsolvatationszahl war 4. Die Solvensaustauschkinetik wurde in DMSO untersucht. Der Protonenaustausch in waBrigen Losungen laBt sich als eine Protolysereaktion interpretieren. Das bevorzugt das Berylliumion solvatisierende Solvensrnolekul ist DMSO in wasserreichen 'und Wasser in DMSO-reichen Losungen.usually proceed according to the Eigen-Tamm [1] mechanism:

Kinetic measurements of proton and oxygen exchange of the aquo beryllium(II) ion in concentrated solutions of beryllium(II) nitrate are reported. These measurements have been performed by means of 1H spin‐echo NMR and by 17O FT‐NMR using natural abundance 17G and europium(III) nitrate as a shift reagent for the 17O bulk water line. The combination of both methods allows one to separate the contributions from protolysis and water exchange. The data obtained for water exchange are k298ex = 1.8 · 103 sec−1 (rate constant for one water molecule), ΔH+ex = 41.5 kJ mol−1, and ΔS+ex = ‐44 JK−1 mol−1. Protolytic dissociation of the aquo beryllium(II) ion is found to be approximately one order of magnitude faster than water exchange: k298d = 8 · 104 sec−1 (rate constant for the tetra aquo beryllium(II) ion). ΔH+d = 31.2 kJ mol−1, and ΔS+d = −45 JK−1 mol−1 for beryllium concentrations up to 0.2 molal.

The relative amounts of the differently solvated species Be(HMPT)i(H2O)2+4‐i have been determined as a function of solvent composition using 31P‐NMR measurements. The formation of a Bc(NO3)+ inner‐sphere complex was established at higher mole fractions of HMPT. The consecutive stability constants of the Be‐HMPT complexes were obtained and compared with those calculated more indirectly using Bjerrum's method [1].