Functional diversity of bioactive peptides in the nervous system itself: "how the brain may understand".

The interactions involving cells of the nervous system are a complex form of intercellular communication. Biosynthesis of peptide hormones or active neuropeptides is generally through a precursor which provides increased product choices as a function of the processing pathway. Proteolytic processing as well as other molecular modifications lead to a wide range of mature products which may vary in different tissues even though they are derived from the same precursor. Also the same neuropeptide may exhibit different bioactivities for different target cells. Finally, by means of collective packaging in secretory organelles, a cell may be able by synergism to further broaden its biologic effects. In these ways, what is seen as added complication in the CNS, may be from the point of view of the cell, a successful attempt to increase its survival ability to adapt and influence its bioenvironment.

Immunoreactivity of vasopressin and a novel pituitary protein '7B2' in Long-Evans and Brattleboro rat hypothalamus and hypophysis.

The unlabeled antibody (peroxidase-anti-peroxidase) method was used to simultaneously localize vasopressin and a novel pituitary protein designated '7B2' in rat hypothalamus and pituitary. Results showed the common localization of both substances within magnocellular neurons of supraoptic, supraoptic retrochiasmic and paraventricular nuclei. The distribution was also similar in the inner zone of the median eminence and in the posterior lobe of the pituitary gland. Only 7B2 antiserum labeled the external zone of the median eminence and the intermediate and anterior lobes of the pituitary. In the Brattleboro rat the anterior and intermediate lobes were strongly labeled with 7B2-IR and there was some 7B2-staining in the hypothalamus and the posterior lobe, but the intensity of the reaction was diminished.

Subcellular fractionation of pituitary neurointermediate lobes: revelation of various basic proteases.

Homogenates of rat neurointermediate lobes were purified by centrifugation on a Percoll gradient. Lysates of the Percoll gradient fractions were incubated with a synthetic octapeptide and pentapeptide substrate (N-acetyl Lys-Arg-Tyr-Asn-Leu-Thr-Ser-Val-amide and N-acetyl Lys-Arg-Tyr-Asn-Leu-amide), and enzymatic characteristics were compiled. Early assays on nonamidated forms revealed carboxypeptidase activity, whereas with the amide derivatives no carboxypeptidase activity could be detected. These amide substrates were therefore used in all subsequent incubations. High levels of a Leu/Thr and Lys/Arg cleavage were present in fractions almost throughout the Percoll gradient. Cleavages at Tyr/Asn, Thr/Ser, and Arg/Tyr were localized at different regions of the Percoll gradient. Surprisingly, none of the five enzymatic activities appear to be localized in the secretory granule fractions as defined by the presence of immunoreactive beta-endorphin in the gradient. All of the five proteolytic activities have a basic pH optimum (pH 8-9), and four of them seem to be thiol proteases, as categorized by inhibitor studies. The fifth one, namely the Try/Asn cleavage, is more likely to be due to a metalloendopeptidase, since it is activated by Zn2+ and Co2+.

Galactose transfer to endogenous acceptors within Golgi fractions of rat liver.

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.

Membrane fusion and glycosylation in the rat hepatic Golgi apparatus.

When purified Golgi fractions were incubated with UDP-[3H]galactose in the absence of Triton-X-100, radioactivity was incorporated into an endogenous lipid and several peptide acceptors. Electron microscope analysis of Golgi fractions incubated in the endogenous galactosyl transferase assay medium revealed extensive fusion of Golgi saccules. Systematic removal of constituents in the galactosyl transferase assay medium showed enhanced (minus beta-mercaptoethanol) or reduced (minus ATP, minus sodium cacodylate buffer or minus MnCl2) fusion of Golgi membranes compared to the complete medium, Stereologic analysis revealed a correlation between membrane fusion and galactosyl transferase activity (r = 0.99, P less than 0.001). Electron microscope radioautography was carried out after incubation of Golgi fractions with UDP-[3H]galactose. Silver grains were not observed over trans elements of Golgi but were revealed mainly over large fused saccules with the number of silver grains being proportionate to membrane fusion (r = 0.92, P less than 0.001). Bilayer destabilization at points of Golgi membrane fusion may act to translocate galactose across the Golgi membrane and thereby provide a fusion regulated substrate for terminal glycosylation.

A morphologic analysis of insulin binding sites in isolated rat liver nuclei.

Under conditions which demonstrated a high degree of specific 125I-insulin binding to purified plasmalemma, no specific binding could be found in freshly prepared nuclei. Analysis of light and electron microscope radioautographs of nuclei incubated with 125I-insulin with and without additional excess unlabeled insulin also failed to provide any evidence for 125I-insulin binding to rat liver nuclei in vitro. It is concluded that current methods are too insensitive to detect insulin receptors in nuclei (if indeed such receptors exist in this organelle to any appreciable extent.