Philip C. Andrews

Isolation and characterization of a drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.

A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis

A procedure is described which provides high yields of pyridylethylated cysteine during gas-phase sequencing of peptides. The method decreases transfer losses by reducing the number of transfer steps required for reduction, alkylation, prior to sequencing a peptide. Proteins bound to polybrene-coated glass-fiber filters used in a gas-phase sequenator may be reduced, pyridylethylated, and sequenced directly on the filter. Reduction of cystine-containing peptides is performed using the nonnucleophilic reductant, tributylphosphine [U. T. Rüegg and J. Rudinger (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 111-116, Academic Press, New York] with concomitant alkylation by 4-vinylpyridine in a buffer soluble in the organic phase. Excess reagents and the buffer are removed by drying the filter and briefly washing with chlorobutane. No N-alkylation is apparent under the conditions described, nor are any amino acid side chains modified. The procedure affords high yields and is particularly useful when subnanomole levels of material must be reduced and alkylated.

A new approach for detection and assignment of disulfide bonds in peptides

A new procedure is described for identification of disulfide bonds in peptides by fast atom bombardment mass spectrometry (FABMS). Prolonged bombardment of a disulfide-containing peptide in solution by a high-energy xenon beam results in gradual reduction of the disulfide bond. The reduction is the result of reaction intermediates initially produced by the xenon beam. The method for characterization of interchain disulfide bonds is based on the increase in the relative intensity of the pseudomolecular ions of the reduced peptides with a simultaneous decrease in the relative intensity of the protonated molecular ion of the oxidized peptide. This information allows one to identify peptide fragments covalently linked via intermolecular disulfide bonds. The intrachain disulfide bonds are identified by the increase in the relative intensity of the protonated molecular ion of the reduced peptide, relative to the intensity of the protonated molecular ion of the oxidized peptide. These results indicate that this method can be used to detect disulfide bonds of peptides and provides unambiguous information regarding disulfide bond assignment in peptides. Approximately 1 nmol of sample is required.

Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY

It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined.

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus).

SummaryResults from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.

Specific glucagon-related peptides isolated from anglerfish islets are metabolic cleavage products of (pre)proglucagon-II

Sequence analyses of cDNAs prepared from anglerfish islet mRNA have demonstrated the presence of mRNAs coding for two different preproglucagons, aPPG-I and aPPG-II. Each of these precursors was predicted to contain 29 residue and 34 residue glucagon-related peptides as potential cleavage products. Recently, several glucagon-related peptides found in extracts of anglerfish islets have been isolated and characterized. In order to determine whether any of these peptides could be identified as metabolic cleavage products in anglerfish islets, differentially radiolabeled Mr 2,500-8,000 peptides from islet extracts were subjected to reverse phase HPLC under varying conditions. The potential cleavage products aPPG-II[52-80] and aPPG-II[89-122] could be readily identified among the extract peptides. Both peptides became labeled appropriately (as predicted from their sequences) with 13 different amino acids and demonstrated glucagon-like immunoreactivity in a radioimmunoassay. Conversely, a third peptide (aPPG-II[89-119]) could be found among the labeled products in small amounts only. These results demonstrate that glucagon-II[52-80] and aGLP-II[89-112] are primary cleavage products of aPPG-II and suggest that aGLP-IIc[89-119] may be a peptide generated more slowly by post-translational modification of aGLP-II.

Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide ☆ ☆☆

The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide.

New fluorogenic substrates for a rat brain proline endopeptidase

Abstract Several compounds having the general formula, Z· X - l -prolyl-4-methoxy-β-naphthylamide have been examined as substrates for a homogeneous rat brain proline endopeptidase. The compounds examined included X equal to l -Ala-, l -His, l -Lys-, and l -Asp-, the latter compound being the poorest substrate of those examined. A simple assay was developed based upon the appearance of the fluorescent product, 4-methoxy-β-naphthylamine. The assay is highly sensitive, continuous, and affords the possibility of utilizing the product of the reaction for tissue or subcellular localization of the enzyme. The homogeneous enzyme hydrolyzed each substrate examined. The most favorable kinetic constants were noted with Z- l -Ala-, Z- l -His-, and Z- l -Lys, Pro-4-methoxy-β-naphthylamide. These substrates show a pH optimum at 8.3 with the homogeneous rat brain proline endopeptidase. The tissue distribution of the enzyme was examined using each substrate and the highest specific activity was noted in the brain. A monospecific antibody prepared against the homogeneous rat brain enzyme inactivates essentially all enzyme activity in every tissue examined except kidney, where as musch as 7% residual activity was noted.

Characterization of aPY-like peptides in anglerfish brain using a novel radioimmunoassay for aPY-Gly

Anglerfish peptide YG (aPY) was isolated from pancreatic islets of the anglerfish. Subsequent immunohistochemical and biochemical analyses demonstrated that anglerfish islet cells synthesize aPY. We have now developed and characterized a radioimmunoassay (RIA) for aPY and have examined extracts of anglerfish brain for aPY-like peptides. Brain extracts were subjected to gel filtration and high performance liquid chromatography (HPLC). Fractions from HPLC eluates were analyzed in the aPY RIA and also in a neuropeptide Y (NPY) RIA. A single peak of aPY-like immunoreactivity eluted from HPLC columns. The elution position of this aPY-like peptide coincided exactly with the aPY-Gly marker under several gradient conditions. Results from the NPY RIA confirmed the presence of several molecular forms of NPY-like immunoreactive peptides in the anglerfish brain. These results demonstrate the utility of the newly developed aPY RIA for studies of anglerfish brain peptides and extend our previous immunohistochemical demonstration of aPY-like staining in the anglerfish brain.

The use of plasma desorption time-of-flight mass spectrometry to screen for products of prohormone processing in crude tissue extracts

Californium-252 plasma desorption mass spectrometry (252Cf PDMS) of a crude, desalted, extract of piscine endocrine pancreas provided mass information for the major biologically active peptide hormones present in this tissue. An extraction procedure compatible with 252Cf PDMS analysis was developed. In extracts of catfish pancreas, strong molecular ions were identified in the positive mode for somatostatin-14 (1638 amu), O-glycosylated somatostatin-22 (2944 amu), glucagon (3512 amu), glucagon-like peptide (3785 amu), insulin (ca. 5550 amu), and other prohormone-derived peptides. Both protonated species and sodium adducts were apparent in the mass spectrum. A number of other molecular ions were observed including somatostatin-26, 1-10 (1014 amu) and the entire portion of prosomatostatin-22 remaining after removal of somatostatin-22 (6465 amu). The data obtained by this method also resulted in the identification of the third major product of proglucagon processing in catfish pancreas, glicentin-related polypeptide. Subtractive Edman degradation analyzed by 252Cf PDMS was also used to confirm a mass assignment.