Double-antibody immunoprecipitation procedures with antisera to endorphins and corticotropin (ACTH) were used study the biosynthesis of these peptides in a mouse pituitary tumor cell line. Cultures incubated 3 H-labeled amino acid, aliquots culture medium immunoprecipitated. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis [ H]phenylalanine-labeled immunoprecipitates prepared endorphin resolved three forms apparent molecular weights 31,000, 11,700, 3500; ACTH antiserum contained...

Rat sympathetic ganglia were disrupted by mechanical agitation to yield dissociated primary neurons, and the conditions for long-term growth in culture of isolated neurons examined. The grown with or without non-neural cells, simply addition deletion bicarbonate during culture. Fluorescence histochemistry indicated that contained catecholamines; incubations radioactive precursors used verify synthesis accumulation both dopamine norepinephrine. also produced octopamine using tyramine as...

Copper active sites play a major role in enzymatic activation of dioxygen. We trapped the copper-dioxygen complex enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM) by freezing protein crystals that had been soaked with slow substrate and ascorbate presence oxygen. The x-ray crystal structure this precatalytic complex, determined to 1.85-angstrom resolution, shows oxygen binds one coppers an end-on geometry. Given structure, it is likely dioxygen directly involved electron...

An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules rat anterior, intermediate, and neural pituitary bovine intermediate pituitary. High levels endogenous inhibitors this alpha-amidation have also found tissue homogenates. The is totally inhibited by addition divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, EDTA; restored to above control upon copper. reaction...

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed cell suspensions. Control studies indicated that the isolated cells were obtained in good yield after more than 24 h culture continued to synthesize a collection similar those found freshly extracted tissue. Rat synthesized molecule (Mr = 30,000; called 30K) contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), 16K fragment (the NH2-terminal region...

A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor cell line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis extracts resolved four forms with apparent molecular weights 4,500, 13,000, 23,000, and 31,000. These can be detected by radioimmunoassay or following incubation cultures [3H] tryptophan, lysine, tyrosine. The scheme developed is...

Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes of these diverse physiological regulators. The amino-terminal domain bifunctional PAM protein is a peptidylglycine α-hydroxylating (PHM) with two coppers that cycle through cupric cuprous oxidation states. anomalous signal endogenous was used to determine structure catalytic core oxidized rat PHM without bound substrate. These structures...

Graded, reversible suppression of neuronal excitability represents a logical goal therapy for epilepsy and intractable pain. To achieve such suppression, we have developed the means to transfer “electrical silencing” genes into neurons with sensitive control transgene expression. An ecdysone-inducible promoter drives expression inwardly rectifying potassium channels in polycistronic adenoviral vectors. Infection superior cervical ganglion did not affect normal electrical activity but...

Two subtilisin-like endoproteases called PC1 and PC2 are distributed in a tissue-specific manner the pituitary brain. AtT-20 cells corticotropes of anterior express primarily perform limited number cleavages proopiomelanocortin (POMC) precursor during biosynthesis. Melanotropes intermediate both more extensive set biosynthetic processing POMC. To investigate role POMC, mouse were stably transfected with full length cDNA. The expressing acquired ability to all additional seen pituitary, but...

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, question is still open as which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes processing with biochemical molecular biological studies, we cloned rat pituitary cDNAs for PC1 PC2. The amino acid sequence homologies among rat, human, mouse PC1, furin are consistent each...

Abstract Extracts of rat anterior and intermediate‐posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis assayed for immunoactive ACTH endorphin. In both lobes the major forms have apparent molecular weights 31,000 (31K), 20–21K, 14K, 4.5K, endorphin 31K (coincident with peak ACTH), 13K (a βLPH‐like peptide), 3.5K β‐endorphin‐like peptide). However, quantitative distribution immunoactivity among various differs greatly between lobes. Assays using...

Growth of axons and dendrites is a dynamic process that involves guidance molecules, adhesion proteins, neurotrophic factors. Although neurite extension stimulated by the neurotrophin nerve growth factor (NGF), we found precursor NGF, proNGF, induced acute collapse cones cultured hippocampal neurons. This retraction was initiated an interaction between p75 receptor (p75NTR) sortilin family member SorCS2 (sortilin-related VPS10 domain-containing 2). Binding proNGF to p75NTR-SorCS2 complex...

proceed at all using D-Tyr-Val-Gly as peptide substrate under the assay conditions used in these studies.

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We used affinity-purified antisera that recognize only the products and immunoelectron microscopy to identify compartments rat pituitary corticotropes mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling cryostat sections immunogold ultrathin frozen were for localization sites. By both procedures we detected processed peptides Golgi cisternae...

Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), predominant splice form of multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain localizes to postsynaptic side excitatory synapses. In vitro studies demonstrated that overexpression Kal7 increased dendritic spine density, whereas reduced expression endogenous decreased...

Many bioactive peptides terminate with an amino acid α-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine α-amidating monooxygenase or PAM. We identified cDNAs encoding the by using antibodies to screen a bovine intermediate pituitary λgt11 expression library. Antibodies β-galactosidase/PAM fusion protein removed PAM activity from homogenates. 108,207 dalton predicted complete cDNA approximately twice size of...

Mouse pituitary tumor cells were incubated in medium containing [3H]glucosamine or ['Hlmannose.By analyzing immunoprecipitates of cell extracts and culture it was shown that ['Hlglucosamine [SH]mannose incorporated into all three high molecular weight forms ACTH; label not M, = 4,500 ACTH (which is thought to be similar the 39 amino acid polypeptide form ACTH, Lu(l-39)).Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent weights these glycoprotein 31,000, 23,000,...