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PubMed1999

Characterization and regulation of peptidylglycine alpha-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts.

Peptidylglycine alpha-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160 +/- 12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive alpha-amidated hormones and/or neuropeptides.

Alternative Splicing↗
PubMed2005

Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide.

The 13C and 15N backbone-labeled proline was prepared using Oppolzer's method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the 13C, 15N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed 1H, 13C and 15N NMR study confirmed the assigned oxytocin conformation containing a beta-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.

Carbon Isotopes↗
PubMed1991

Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography.

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.

Amino Acid Sequence↗
PubMed1988

Purification of a peptidylglycine alpha-amidating enzyme from transplantable rat medullary thyroid carcinomas.

A peptidyl glycine alpha-amidating activity has been isolated from total tissue extracts of rat medullary thyroid carcinoma (MTC). Purification of the activity by ammonium sulfate fractionation, Sephacryl S-300 chromatography, and strong anion-exchange chromatography at pH 6.0 has resolved at least four peaks of activity. The activity associated with peak III has been further purified to apparent homogeneity by strong anion-exchange chromatography at pH 8.0. The purified peak III enzyme has an apparent molecular mass of 75,000 Da as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the 75,000-Da band as the alpha-amidating enzyme has been confirmed by recovery of activity from a nondenaturing polyacrylamide gel. The enzyme is catalytically active as a monomer, exhibits a pH optimum between 5.0 and 5.5, and has a turnover number of 300 min-1 for N-dansyl-Tyr-Val-Gly amidation at pH 5.5. The larger size, more acidic pH optimum, and higher specific activity distinguish the purified peak III rat MTC enzyme from the enzymes isolated from bovine and porcine pituitary or from frog skin.

Animals↗
PubMed1984

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets.

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.

Animals↗
PubMed1986

Evidence for high peptide alpha-amidation activity in the neonatal rat pancreas.

A high peptide alpha-amidating activity is present in a mitochondrial/secretory granules preparation from 3-day old rat pancreas. It is dependent on copper, ascorbate and molecular oxygen. This preparation is able to generate TRH when incubated with Pyroglu-His-Pro-Gly, a sequence present in the TRH precursor molecular. The peptide alpha-amidating activity may be involved in the high rate of TRH biosynthesis in the pancreas during the neonatal period. In the pancreas of adult rats which contain low levels of TRH, the peptide alpha-amidating activity is barely detectable.

Amidohydrolases↗
PubMed1987

Cloning and sequence of cDNA encoding a peptide C-terminal alpha-amidating enzyme from Xenopus laevis.

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. We have recently isolated an alpha-amidating enzyme, AE-I, from Xenopus laevis skin, which is the only enzyme ever purified to homogeneity. In this study, we report cloning and sequence of cDNA encoding AE-I. Our results indicate that enzyme AE-I is initially synthesized as a precursor with 400 amino acid residues, which is further processed to the mature enzyme consisting of 344 residues. Preliminary expression in E. coli of the cDNA corresponding to AE-I was found to produce an enzyme with appreciable alpha-amidating activity.

Amino Acid Sequence↗
PubMed1988

A new facile trinitrophenylated substrate for peptide alpha-amidation and its use to characterize PAM activity in chromaffin granules.

Carboxyl terminal alpha-amidation is a prevalent post translational modification in neuropeptide hormones, with amidation being essential for biological activity. We report a direct demonstration and characterization of peptidyl alpha-amidating monooxygenase (PAM) activity in chromaffin granules, secretory vesicles long known as loci for synthesis and storage of catecholamines but only recently recognized as processing and storage sites for neuropeptides. This finding, together with the recently recognized competence of dopamine-b-monooxygenase to carry out N-dealkylation, provides important information regarding the co-localization and co-secretion of multiple neuromodulators. In addition, we introduce a new substrate for both pituitary and chromaffin granule PAM--TNP-D-Tyr-Val-Gly. This substrate exhibits high turnover, and has the important advantage of allowing quantitative activity determinations using standard spectrophotometric techniques, thus facilitating mechanistic studies and inhibitor development.

Adrenal Medulla↗
PubMed1991

GLP-1(7-36)amide-stimulated insulin secretion in rat islets is sodium-dependent.

We examined the Na(+)-dependency of the effects of GLP-1(7-36)amide in normal, overnight cultured rat islets. It was found that GLP-1(7-36)amide stimulated insulin secretion, 45Ca(2+)-efflux, and 86Rb(+)-efflux from prelabelled islets. All these effects were abolished by omitting Na+ from the medium and replacing it with N-methyl-glucamine. This suggests that GLP-1(7-36)amide stimulates insulin secretion by depolarizing the beta-cells by increasing their permeability to Na+.

Animals↗
PubMed1983

Immunohistochemical studies of FMRF-amide-like immunoreactivity in rat brain.

The anatomical distribution of neuronal perikarya and nerve fibres containing FMRF-amide-like immunoreactivity in the brain, spinal cord and pituitary of the rat has been studied by immunohistochemistry. In animals pretreated with colchicine, the highest concentration of nerve cell bodies occurred in hypothalamic nuclei. Cells were also present in the cortex, striatum, septum, thalamus and in the brainstem. Beaded nerve fibres were abundant in the septum, nucleus of the striae terminalis, hypothalamus, medial regions of the thalamus, the parabrachial nucleus, the ventrolateral medulla, the substantia gelatinosa of the spinal trigeminal nucleus and the dorsal horn of the spinal cord. Fibres were also present in the cortex, striatum, amygdala, pons, ventral spinal cord and the neural lobe of the pituitary. The localization was specific in that preabsorbtion of the antisera with FMRF-amide, but not structurally related molecules such as Met-Enk-Arg6Phe7, APP or BPP, completely abolished the localization. The mammalian counterparts of FMRF-amide may have a neurotransmitter or neuromodulatory role.

Animals↗
PubMed1989

Peptidylglycine alpha-amidating monooxygenase activity in spinal cord, dorsal roots, and dorsal root ganglia of Macaca fascicularis.

Several peptides synthesized by primary sensory neurons are alpha-amidated at the C-terminal residue, including vasoactive intestinal polypeptide, neurokinin A and substance P, which is also abundant in spinal cord. In pituitary and other tissues, the C-terminal amidation is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM). In the present study, soluble PAM activity in spinal cord and in primary sensory neurons is quantified and characterized as to cofactor and cosubstrate requirements and substrate specificity.

Animals↗
PubMed1992

Cultured astrocytes express mRNA for peptidylglycine-alpha-amidating monooxygenase, a neuropeptide processing enzyme.

Cultured astrocytes have been previously found to express several neuropeptides, as well as the neuropeptide processing enzyme carboxypeptidase E (CPE). To investigate whether cultured astrocytes contain additional peptide-processing enzymes, Northern blots were screened for peptidylglycine-alpha-amidating monooxygenase (PAM) mRNA. PAM is involved with the formation of amide groups on the C-terminus of numerous peptide hormones and neurotransmitters. Primary cultures of astrocytes contain moderate levels of PAM mRNA, as determined by Northern blot analysis. The level of PAM mRNA in cultured hypothalamic astrocytes is similar to the level expressed in cultured hypothalamic neurons. The relative abundance of PAM mRNA differs up to 6-fold between astrocytes cultured from various brain regions. Astrocytes cultured from hypothalamus have high levels of PAM mRNA, those cultured from striatum, frontal cortex, and hippocampus have moderate levels, and those cultured from cerebellum have low levels. To investigate whether all cultured astrocytes express PAM mRNA, in situ hybridization analysis of cultured astrocytes was performed. Interestingly, virtually all of the astrocytes cultured from either hypothalamus or cerebellum express PAM mRNA, in contrast to a previous finding that only 20-40% of similarly cultured astrocytes express CPE. The presence of PAM mRNA in cultured astrocytes suggests that these cells have the capacity to produce amidated neuropeptides.

Animals↗
PubMed1983

Formation of the COOH-terminal amide group of thyrotropin-releasing-factor.

The precursors of peptide hormones that possess a COOH-terminal alpha-amide group contain a glycine residue following the amino acid which is amidated in the hormone. Less than Glu-His-Pro-Gly was synthesized as a putative precursor of thyroliberin. Bovine pituitary neurosecretory granules were shown to contain an amide group-forming activity associated with an Mr of about 62 000 protein(s) which converts the tetrapeptide to thyroliberin.

Animals↗
PubMed1988

Truncated glucagon-like peptide-1 (proglucagon 78-107 amide), an intestinal insulin-releasing peptide, has specific receptors on rat insulinoma cells (RIN 5AH).

We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding was obtained when the cells were incubated in the presence of 3.3 x 10(-9) mol/l unlabelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide). Neither glucagon, full-length glucagon-like peptide-1 (proglucagon 72-107 amide) nor gastric inhibitory peptide competed for binding in concentrations up to 10(-6) mol/l.

Adenoma, Islet Cell↗
PubMed1989

A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla.

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.

Adrenal Medulla↗
PubMed1992

Rapid fluorimetric assay for the detection of the peptidyl alpha-amidating enzyme intermediate using high-performance liquid chromatography.

Peptidylglycine alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to their corresponding amidated peptides via a stable alpha-hydroxyglycine intermediate. Using a new rapid fluorimetric reversed-phase high-performance liquid chromatographic assay, we have demonstrated that the substrate and product of the amidation reaction, as well as both stereoisomers of the alpha-hydroxyglycine intermediate, can be separated and detected in quantities as low as 1 pmol. The method is highly reproducible and requires less than 11 min for separation and quantification.

Amino Acid Sequence↗
PubMed1994

Rat gastric somatostatin and gastrin release: interactions of exendin-4 and truncated glucagon-like peptide-1 (GLP-1) amide.

The effect of exendin-4, a peptide of the secretin-glucagon family with high homology of amino acid sequence with glucagon-like peptide-1 (GLP-1), on gastric hormone release was investigated in the isolated perfused rat stomach. Exendin-4 dose dependently stimulated somatostatin release up to 9-fold at a concentration of 10(-7) M whereas gastrin release was inversely inhibited by up to 63%. These effects could partially be reduced by concomitant perfusion of truncated exendin-4, exendin(9-39)amide. Similarly, stimulation of somatostatin secretion and inhibition of gastrin release induced by GLP-1(7-36)amide was partially reversed by exendin-4 (9-39)amide. These data are consistent with the assumption that exendin-4 and truncated GLP-1amide exert their effects on gastric D and G cell by interaction with the same receptor.

Animals↗
PubMed1992

Arthropod toxins as leads for novel insecticides: an assessment of polyamine amides as glutamate antagonists.

In the search for new toxins, preferably with new sites of action, the polyamine amides represent a new class of compounds with potential as insecticides and as pharmaceutical agents due to their antagonism of ligand-gated cation channels. In particular, they are potent antagonists of the L-glutamate receptors of insect skeletal muscle. In this paper, we report on synthetic studies to produce hybrid analogues based upon the argiotoxin spider toxins and philanthotoxin-433 which is obtained from a solitary, parasitic wasp. We speculate upon possible modes and sites of action for these antagonists and we discuss their potential as insecticides and in the possible treatment of ischaemic damage. The synthesis and characterization of 4-hydroxyphenylpropanoylspermine is reported and the locust muscle biological assay is described. Using this pharmacological screen, structure-activity relationships have been determined in our laboratories. These are reviewed in the light of the current literature. Voltage clamp studies of the synthetic analogue philanthotoxin-343 and the effects of this polyamine amide on glutamate receptors expressed in Xenopus oocytes are outlined. In conclusion, a description of our current ideas and understanding of the many sites and modes of action of the polyamine amides, based both upon our own studies and also upon those recently reported, is presented.

Animals↗