Enkephalins in human phaeochromocytomas: localization in immunoreactive, high molecular weight form to the soluble core of chromaffin granules

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Enkephalins, endogenous opioid pentapeptides, are found in normal chromaffin tissue and may influence blood pressure regulation. We studied the subcellular localization and precursor-product status of enkephalin immunoreactivity in 11 human phaeochromocytomas (seven adrenal, four extra-adrenal). Enkephalin immunoreactivity was found in all phaeochromocytomas, it paralleled radio-immunoassay standard curves and was not destroyed by boiling or protease inhibitors i.e. ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF). Sucrose gradients localized enkephalin immunoreactivity to chromaffin granules (55 ± 17% of total immunoreactivity; n=6). In vitro granule lysis released 81% of the enkephalins and 91% of the catecholamines. Thus, phaeochromocytoma enkephalins are present in the soluble core of chromaffin granules, along with catecholamines. Enkephalin immunoreactivity was not contained in purified chromogranin A, either before or after trypsin cleavage. High pressure liquid chromatography (HPLC) elution of enkephalin immunoreactivity matched that of synthetic methionine-enkephalin, leucine-enkephalin, and methionine-sulfoxide-enkephalin standards. Enkephalin immunoreactivity was augmented by trypsin alone and by trypsin plus carboxypeptidase B (by 352 ±56%), suggesting that the majority of the enkephalins were present in higher molecular weight precursor form. Sephacryl S-200 gel filtration of chromaffin granule lysate revealed a trypsinaugmented putative human enkephalin precursor with a molecular weight of 2000-4000 daltons as well as product enkephalins. Enkephalin concentration in phaeochromocytoma closely paralleled the epinephrine, but not the norepinephrine content of the tumours. However, it was not statistically different in adrenal versus extra-adrenal tumours. Thus, these peptides are contained in high molecular weight form in the soluble core of catecholamine storage vesicles, predominantly epinephrine vesicles

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