Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.
Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.
D000313:Adrenal Medulla / Q000648:ultrastructure*; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002837:Chromaffin Granules / Q000648:ultrastructure; D004299:Dopamine beta-Hydroxylase / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008861:Microsomes / Q000648:ultrastructure; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000648:ultrastructure
D011506:Proteins; D004299:Dopamine beta-Hydroxylase