Extraction and Partial Characterization of Enkephalin-Like Peptides from Splanchnic Nerve

A method is described for the extraction of enkephalin-like peptides from peripheral nerve using chloroform and acidic methanol to facilitate a differential extraction of peptides and lipid. Porcine splanchnic nerve contains enkephalin-like peptides in low amounts compared to porcine adrenal medulla and striatum. Gel filtration chromatography reveals the presence of enkephalin-like peptides in both processed and cryptic forms. This is the first reported isolation and partial characterization of these peptides in splanchnic nerve. The presence of these peptides in this nerve provides support for the contention that the splanchnic nerve can modulate catecholamine release from the adrenal medulla through an effect on opiate receptors located on chromaffin cells.     

Mass Spectrometric Analyses of Brain Synaptic Peptides Containing Taurine

The structure of a number of low-molecular-weight acidic peptides containing taurine prepared from calf brain synaptosomes and their subcellular vesicles was studied using electron impact mass spectrometry. At least seven sequences could be identified: N-acetylaspartyl-glutamyl-taurine, N-acetylaspartyl-taurine, N-acetylglutamyl-taurine, glutamyl-taurine, aspartyl-taurine, seryl-glutamyl-seryl-taurine, and seryl-taurine.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.     

Factors to consider in performing survival studies with insect cells

Summary Insect cell lines are not well-suited to colony formation in liquid medium following low-density cell plating. The present studies demonstrate that the time of addition of fetal bovine serum to the culture medium and the number of γ-irradiated feeder cells added to each plate are important factors in developing a useful colony formation assay. TN-368 lepidopteran and WR69-DM-1 dipteran cell lines were used for these experiments. Both cell types display increased plating efficiencies if serum is added to the medium one or more days prior to plating as compared to adding serum immediately before plating. Growth curves obtained by seeding cells at higher densities also indicate that cell growth is slightly better if serum is added one or more days before seeding. These findings are especially important for survival and toxicity studies because the results demonstrate that even seemingly minor factors involved in cell survival assays may benefit treated cells to a greater degree than untreated control cells, thus providing an erroneous assessment of cell survival. This work was supported by USPHS grant R01-CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Correlation of neuronal size and peptide immunoreactivity in the guinea-pig trigeminal ganglion

Summary Frequency and size of guinea-pig trigeminal neurones immunoreactive with antisera to -neo-endorphin(-neo-END), dynorphin A-(DYN), vasoactive intestinal polypeptide-(VIP), somatostatin-(SOM), and substance P-(SP) are reported. Co-localisation of the various peptides to the same ganglion cells was investigated immunocytochemically in consecutive 7-m thick paraffin sections. According to their size, all peptide-immunoreactive neurones belong to the class of small ganglion cells. Within this cell group, SP-immunoreactive neurones appear to be the largest, followed by SOM-, VIP-, -neo-END- and DYN-immunoreactive ganglion cells. The observed differences in size are statistically significant with the exception of that between -neo-END and DYN. This finding correlates well with the observed co-occurrence of the two immunoreactive peptides. All -neo-END-immunoreactive perikarya are also reactive to VIP antisera. These neurones are significantly smaller than those containing VIP-immunoreactivity exclusively. Ganglion cells displaying co-existence of -neoEND- and SP-immunoreactivity or VIP- and SP-immunoreactivity are found too infrequently to allow morphometric analysis. Some non-immunoreactive ganglion cells are shown to be approached by dense baskets of VIP-, -neo-END- or SP-immunoreactive varicose fibres, indicating the presence of intraganglionic modulation sites. The combination of immunohistochemistry and morphometry presented in this study allows the differentiation of diverse populations of primary afferent neurones exhibiting peptide immunoreactivity, most likely reflecting their involvement in different central and peripheral reflex arcs and sensory modalities.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.