Partial characterization of a phosphoryl group transferring enzyme in the membrane of catecholamine storage vesicles.
Phosphoryl group transfer from ATP to ADP occurred in the isolated membrane of catecholamine storage vesicles. The reaction was accelerated by extraction of the membranes with 50% (v/v) acetone and by treatment with 1% (v/v) Triton X-100. The phosphoryl group transfer reaction was activated by Mg2+ and by Mn2+. The activation profile differed from that obtained for the ATPase activity. The Michaelis-Menten kinetics of the phosphoryl transfer reaction were not entirely linear. From the linear parts of the double reciprocal plots KmATP approximately equal to 1 mM and KmADP approximately equal to 0.4 mM was obtained. All lines of the double reciprocal plots intersected indicating a sequential reaction mechanism. The reaction exhibited a narrow specificity for nucleoside diphospate and a broader one for nucleoside triphosphate indicating that ADP was the true substrate. The transfer reaction was slightly inhibited by AMP, orthophosphate and P1, P5-di(adenosine-5')pentaphosphate. The thiol reagents, N-ethylmaleimide and para-chloromercuribenzoate (PCMB), affected the ATPase activity and the phosphoryl transfer activity differently: with the blockade of 2.4 essential thiol equivalents by N-ethylmaleimide the ATPase was inhibited 50% and net uptake of catecholamine ceased, while the phosphoryl transfer remained unimpaired. PCMB affected both, the ATPase activity and phosphoryl transfer reaction. Treatment of the membranes with dithioerythritol prevented the PCMB-induced inhibition of the phosphoryl transfer, but was ineffective in protecting the ATPase activity, indicating that different thiol groups must be involved in the both enzymatic activities.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002395:Catecholamines / Q000378:metabolism*; D002412:Cations / Q000502:physiology; D002417:Cattle; D066298:In Vitro Techniques; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity
D002395:Catecholamines; D002412:Cations; D007293:Inosine Triphosphate; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases