Measurement of delta pH and membrane potential in secretory vesicles isolated from bovine pituitary intermediate lobe.

摘要: Pro-opiomelanocortin (ACTH/endorphin prohormone) is processed within the secretory vesicles of pituitary intermediate lobe cells to alpha-melanotropin and beta-endorphin. In order learn more about microenvironment in which processing occurs, a method was developed purify large quantities bovine (ILSV) using isoosmolar metrizamide-sucrose gradients. Analysis marker enzymes revealed that gradients provided 94-fold purification with respect lysosomes 15-fold mitochondria. Pro-opiomelanocortin-converting enzyme activity ILSVs assayed found be maximally active around pH 5. From measuring delta across intact ILSV membrane 9-aminoacridine fluorescence quenching, internal determined less than 5.6, consistent operating range converting activity. The gradient collapsed presence ammonium sulfate or by combination nigericin K+, when external medium 7. Using voltage-sensitive dye, oxanol VI, Mg2+ ATP shown cause marked change potential inside being positive reversed proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Thus, appear have ATP-dependent electrogenic proton-translocating system.