Use of reverse-phase high-performance liquid chromatography in structural studies of neurophysins,photolabeled derivatives,and biosynthetic precursors

The neurophysins are a class of hypothalamo-neurohypophyseal proteins that function as carriers of the neuropeptide hormones oxytocin and vasopressin. Currently, we are using reverse-phase high-performance liquid chromatography for structural characterization of the neurophysins, their chemically modified derivatives, and biosynthetic precursors. A cyanopropylsilyl (Zorbax CN) matrix has been found to be efficient and convenient for separation of major tryptic peptides of performic acid, oxidized or reduced, and alkylated neurophysins. Using this peptide mapping system we have studied the site of modification of a photoaffinitylabeled derivative of bovine neurophysin II by separation and identification of covalently modified peptides. In addition, this system has been used for mapping subfemtomole amounts of radioactively labeled biosynthetic precursors of the neurophysins. This procedure has allowed identification of neurophysin sequences within both pre-pro-neurophysins produced by in vitro translation and rat pro-neurophysins produced by in vivo pulse labeling.     

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.