The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

Abstract

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.

Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential

“…NEM did not have significant effect on catecholamine efflux. These results are comparable to those of Hasselbach and Taugner (3). The reduction in the SCN-concentration ratio by NEM corresponds to a 10-mV decrease in electrical potential.…”

“…Because the in vitro rate of uptake of catecholamines into chromaffin vesicles is increased severalfold by ATP and magnesium (1), it had been thought that the Mg2+-dependent ATPase associated with the vesicle membrane is a catecholamine transport enzyme. Although partial correlations have been obtained between ATPase activity and catecholamine transport (2,3), lack of a simple quantitative correlation suggests that the ATPase may be indirectly rather than directly involved.…”

Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles.

“…Isolated granules catalyze ATP-dependent uptake of massive amounts of epinephrine (5,6), and the process is inhibited by proton conductors (7). ATPase activity (8)(9)(10)(11), ATP-dependent proton translocation (12), and ATP-dependent changes in the fluorescence of 1-anilino naphthalene-8-sulfonate (ANS) (11) have been detected in intact granules or membrane vesicles prepared from chromaffin granules by osmotic shock. Moreover, it has been demonstrated that an uncoupler-sensitive pH gradient (ApH, interior acid) is maintained across the chromaffin granule membrane (13)(14)(15).…”

“…After 10 min, the vesicles were collected by centrifugation at 40,000 X g for 10 min. The procedure was repeated once, and the chromaffin granule membrane vesicles were suspended in 0.3 M sucrose containing 10 size). The filters were then washed with another 2 ml of the latter solution, removed from the suction apparatus, dried, and assayed for radioactivity by liquid scintillation spectrometry.…”

Norepinephrine uptake dependent upon apparent Mg++‐ATPase activity and proton transport in storage vesicles in axoplasm

“…Another inhibitor of Mg + +-ATPase activity, N-ethyl maleimide (NEM) also blocks the uptake of NE by isolated vesicles (Hasselbach and Taugner, 1970) and pre- Fig. 1.…”