A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla.

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.

Neuropeptide Y is localized together with enkephalins in adrenergic granules of bovine adrenal medulla.

The subcellular localization of neuropeptide Y in the bovine adrenal medulla was studied. After differential centrifugation, a large part of total neuropeptide Y immunoreactivity (66%) was recovered in the large granule fraction. This fraction also contained 42% of the total catecholamines and 52% of the total free [Met]enkephalin. Thus neuropeptide Y co-sedimented with these chromaffin granule markers. The large granule fraction was analysed by the technique of rate zonal centrifugation in hypertonic sucrose media (molarities ranging from 1.6 to 2.2 M). Noradrenaline vesicles were preferentially enriched at high sucrose concentration. Adrenaline vesicles as well as enkephalin and neuropeptide Y immunoreactivity pelleted mainly at lower sucrose concentrations. The large granule fraction was also separated by successive centrifugation in self-generating gradients of Percoll-sucrose into two subpopulations, one containing lighter adrenergic vesicles and the other the dense noradrenergic vesicles. Like [Met]enkephalin immunoreactivity, neuropeptide Y immunoreactivity was concentrated in fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to free [Met]enkephalin to neuropeptide Y was 5000:12:1. This biochemical study supports immunohistochemical studies which described co-localization of neuropeptide Y in adrenaline cells in the rat, mouse, cat, guinea-pig and man and co-localization of neuropeptide Y with enkephalins in bovine adrenal chromaffin cells. Our results are however in contrast with the report of other immunohistochemical work which claimed co-localization of neuropeptide Y in noradrenaline cells of rat, cat, dog, horse and cow.

Subcellular fractionation of bovine ganglion stellatum: co-storage of noradrenaline, Met-enkephalin and neuropeptide Y in large 'dense-cored' vesicles.

The subcellular localization of noradrenaline, Met-enkephalin and neuropeptide Y was studied in homogenates of bovine ganglion stellatum. After differential centrifugation most of the noradrenaline (70%) was found soluble, while both neuropeptide Y and Met-enkephalin were sedimented for more than 65%. However, the 3 substances co-sedimented mainly in the microsomal fraction. The microsomal fraction was further analyzed by differential and equilibrium density gradient centrifugation. In both types of gradient, Met-enkephalin and neuropeptide Y were found in the more dense region of the gradient, coinciding with the main peak of noradrenaline. In this fraction, the molar ratio of Met-enkephalin to noradrenaline was 1:95. The corresponding molar ratio for neuropeptide Y to noradrenaline was 1:253. These results indicate that neuropeptide Y and Met-enkephalin are stored with noradrenaline in 'heavy' or large 'dense cored' vesicles in the cell bodies of sympathetic neurons of bovine ganglion stellatum. We present here for the first time biochemical evidence for the co-localization of neuropeptides and a classical transmitter in a neuronal cell body.

Enkephalin containing peptides in the peripheral sympathetic nervous system.

The processing pathway of enkephalins along the sympathetic neuron was studied. While in vasa deferentia terminal parts of peripheral sympathetic neurons are found, sympathetic ganglia contain the cell bodies of these neurons. Biochemical evidence was obtained for the colocalization of met-enkephalin and noradrenaline in large dense-cored vesicles of sympathetic neurons of bovine vasa deferentia and bovine ganglia stellata. Acetic acid extracts of these tissues were analysed by a combination of chromatography, proteolytic digestion with trypsin and carbonxypeptidase B and specific radioimmunoassays. High molecular weight species of enkephalin containing peptides were detected in ganglia stellata. In contrast with the ganglia, only low molecular weight enkephalin containing peptides could be found in the vasa deferentia. When these peptides extracted from vasa deferentia were further analysed on reversed phase fast protein liquid chromatography, the met- to leu-enkephalin ratio was found to be 4.8 to 1, which is close to the 4 to 1 ratio found in the proenkephalin precursor. After digestion with trypsin and carboxypeptidase B, met-enkephalin immunoreactivity appeared in fractions probably containing met-enkephalin-arg-6-phe-7, met-enkephalin-arg-6-gly-7-leu-8 and peptide E.