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1. A method of preparing isolated membranes of the catecholamine storing vesicles of the adrenal medulla is described. The membrane protein amounts to 23% of the total vesicular protein and still contains 0.1% of the catecholamine. 2. At 31‡C in the presence of ATP the membrane preparation accumulates catecholamine; in the absence of ATP, however, catecholamine is released into the medium. At 0‡C the catecholamine content of the membrane preparation remains unchanged, whether ATP is present or not, although a low catecholamine turnover is observed. 3. From the kinetics of the catecholamine turnover in the absence or presence of ATP a low ATP-independent catecholamine influx can be distinguished from a considerably higher ATP-dependent influx of catecholamine. 4. The catecholamine influx as well as the ATPase activity of the membrane preparation decreases with time; in contrast, the catecholamine efflux remains constant and is higher in the presence of ATP than in its absence. 5. Catecholamine is to a great extent accumulated within a membrane-enclosed space into newly formed particles; only a minor amount of the catecholamines is actually bound within the membrane

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.

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