Ascorbic acid regeneration in chromaffin granules. In situ kinetics.

Abstract

We have investigated in intact chromaffin secretory vesicles the kinetics, specificity, and mechanism of intragranular ascorbic acid regeneration by extragranular ascorbic acid.The apparent K,,, of internal ascorbic acid regeneration for external ascorbic acid was 280 pM by Lineweaver-Burk analysis and 287 p~ by Eadie-Hofstee analysis.Intragranular ascorbic acid regeneration was specifically mediated by extragranular ascorbic acid or its isomer isoascorbic acid; the reducing agents glutathione, thiourea, homocysteine, NADH, and NADPH did not support regeneration.The structural analog D-glucose did not inhibit regeneration by external ascorbic acid, suggesting specificity at the membrane site of electron transfer.The driving force for regeneration of intragranular ascorbic acid was independent of membrane potential, absolute intragranular and extragranular pH, and ATPase activity, but might be coupled to the pH difference across the chromaffin granule membrane.Since the apparent K,,, of regeneration was approximately 10-fold below the cytosolic concentration of ascorbic acid, the reaction may proceed at V,,, in situ.