Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.
1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)
Deupree
J D
JD
Department of Pharmacology, University of Nebraska College of Medicine, Omaha 68105.
Hitchcock
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D005033:Ethylmaleimide / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012110:Reserpine / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002352:Carrier Proteins; D013439:Sulfhydryl Reagents; D012110:Reserpine; D000255:Adenosine Triphosphate; D008274:Magnesium; D005033:Ethylmaleimide; D009638:Norepinephrine