Abstract
Purified neurosecretory granules (NG) from bovine neurohypophysis stain metachromatically with acridine orange. NG were sonicated in 0.2% Triton X-100. The soluble protein, 83% of the total protein, was separated by ultracentrifugal flotation in KBr into two lipoprotein (LP) fractions, a high density lipoprotein fraction (HDLP, D < 1.35 g/ml), 37%, and a very high density LP fraction (VHDLP, D > 1.35 g/ml), 63% of the total. The HDLP contained 0.46 mg phospholipid and 0.13 mg cholesterol/mg protein. The VHDLP contained 0.08 mg phospholipid and 0.025 mg cholesterol/mg protein. The amino acid composition of the delipidated LPs resembled that of bovine neurophysins. Disc gel electrophoresis resolved two major and four minor components of identical mobilities in HDLP and VHDLP. The major anodic components, 90% of the protein, and one minor cathodic component were sudanophilic, reduced iodine and stained metachromatically with acridine orange. The NG lysates prepared without detergent or sonication contained 52% of the total protein and phospholipid. The insoluble residue consisted of membrane profiles and had a different lipid and amino acid composition. Gel electrophoretograms of water extracts of NG revealed the same protein components present in Triton X-100 extracts. The major anodic bands and the cathodic band were colored by Sudan black B and iodine and stained metachromatically with acridine orange. Prior extraction of gels with chloroform-methanol (2/1, v/v) abolished these staining reactions. Neurophysins prepared by extraction of acetone-dried tissue with 0.1 N HCl contained about 7% of the phospholipid present in Triton X-100 extracts. Gel electrophoretograms of neurophysins disclosed the same protein constituents seen in Triton X-100 and water extracts of NG. The major components did not stain with Sudan black B or iodine but stained metachromatically with acridine orange due to the presence of residual phospholipid. Neurophysins are essentially apoproteins of these soluble acidic LPs.