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PubMed1987

Alzheimer's disease: low levels of peptide alpha-amidation activity in brain and CSF.

Carboxyl terminal alpha-amidation confers biologic activity to many neuropeptides. Levels of alpha-amidating activity, peptidyl-glycine alpha-amidating monooxygenase (PAM), were reduced in the CSF of patients with dementia of the Alzheimer type (DAT) compared with healthy, age-matched controls. Repeat lumbar puncture data revealed a decline in CSF PAM activity of approximately 16% per year in DAT patients. Of the cerebral cortical regions examined, only the temporal pole showed reduced PAM activity in patients with Alzheimer's disease (AD) compared with controls. These studies may indicate selective dysfunction of neurons which normally synthesize biologically active, alpha-amidated peptides in the CNS of AD patients.

Aged↗

PubMed2000

Effect of APGW-amide on [Ca2+]i in rat pheochromocytoma PC12 cells.

In order to determine whether Ala-Pro-Gly-Try-NH2 (APGW-amide) could affect mammalian excitable cells, we investigated the effect of APGW-amide in PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely prevented by elimination of extracellular Ca2+ with EGTA and inhibited by two L-type Ca2+ channel blockers. [Ca2+]i elevation was also blocked by a specific PKC inhibitor and prolonged pretreatment of cells with PMA. These results indicate that APGW-amide elevates [Ca2+]i in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.

Animals↗

PubMed1989

Characterization of the receptor for glucagon-like peptide-1(7-36)amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking.

125I-Labelled glucagon-like peptide-1(7-36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at Mr63,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex. The band was not observed when 10nM glucagon-like peptide-1(7-36)amide was included in the binding assay, but 1 microM concentrations of glucagon-like peptide-1(1-36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7-36)amide, no cross-linked ligand-binding protein complex was observed.

Adenoma, Islet Cell↗

PubMed1989

Glucagonostatic and insulinotropic action of glucagonlike peptide I-(7-36)-amide.

We examined the effect of glucagonlike peptides (GLPs), which are cleaved from preproglucagon in the enteroglucagon cells, on rat endocrine pancreas with the isolated perfused system. GLP-I-(7-36)-amide, a truncated form of full-sequence GLP-I-(1-37), showed a potent inhibitory effect on glucagon secretion. This inhibitory effect of GLP-I-(7-36)-amide was demonstrated at concentrations of 0.25, 2.5, and 25 nM in 11.2 and 2.8 mM glucose. In contrast, insulin release was significantly stimulated by GLP-I-(7-36)-amide at its concentration from 0.025 to 25 nM in a high glucose concentration, whereas in a low glucose concentration, the stimulation was seen only at the highest concentration (25 nM). Neither GLP-I-(1-37) nor GLP-II showed any effect on glucagon and insulin release. Although several gastrointestinal hormones have been nominated as incretins, none of them may suppress the glucagon secretion. A truncated form of GLP-I, GLP-I-(7-36)-amide thus seems to be a unique incretin that exerts glucagonostatic action.

Animals↗

PubMed1999

Kinetic and inhibition studies on substrate channelling in the bifunctional enzyme catalysing C-terminal amidation.

A series of experiments has been conducted to investigate the possibility that substrate channelling might occur in the bifunctional forms of enzymes carrying out C-terminal amidation, a post-translational modification essential to the biological activity of many neuropeptides. C-terminal amidation entails sequential action by peptidylglycine mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), with the mono-oxygenase catalysing conversion of a glycine-extended pro-peptide into the corresponding alpha-hydroxyglycine derivative, which is then converted by the lyase into amidated peptide plus glyoxylate. Since the mono-oxygenase and lyase reactions exhibit tandem reaction stereospecificities, channelling of the alpha-hydroxy intermediate might occur, as is the case for some other multifunctional enzymes. Selective inhibition of the mono-oxygenase domain by competitive ester inhibitors, as well as mechanism-based mono-oxygenase inactivation by the novel olefinic inhibitor 5-acetamido-4-oxo-6-phenylhex-2-enoate (N-acetylphenylalanyl acrylate), has little to no effect on the kinetic parameters of the lyase domain of the AE from Xenopus laevis. Similarly, inhibition of the lyase domain by the potent dioxo inhibitor 2,4-dioxo-5-acetamido-6-phenylhexanoate has little effect on the activity of the monooxygenase domain in the bifunctional enzyme. A series of experiments on intermediate accumulation and conversion were also carried out, along with kinetic investigations of the reactivities of the monofunctional and bifunctional forms of PAM and PGL towards substrates and inhibitors. Taken together, the results demonstrate the kinetic independence of the mono-oxygenase and lyase domains, and provide no evidence for substrate channelling between these domains in the bifunctional amidating enzyme.

Acrylates↗

PubMed2001

Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines.

After therapeutic hormone deprivation, prostate cancer (CaP) cells often develop androgen-independent growth through not-well-defined mechanisms. The presence of neuroendocrine (NE) cells is often greater in prostate carcinoma than in normal prostate, and the frequency of NE cells correlates with tumor malignancy, loss of androgen sensitivity, increase of autocrine-paracrine activity, and poor prognosis. In some CaPs, neuropeptides have been previously implicated as growth factors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme producing alpha-amidated bioactive peptides from their inactive glycine-extended precursors. In the present work, we demonstrate that androgen-independent PC-3 and DU145 cell lines, derived from human CaP, express PAM in vitro and in xenografts implanted in athymic nude mice, indicating that they are able to produce alpha-amidated peptides. Contrarily, barely detectable levels of PAM were found in the androgen-sensitive LNCaP cell line. We also show that whereas PC-3 and DU145 cells produce and secrete adrenomedullin (AM), a multifunctional amidated peptide, no expression was found in LNCaP cells. We further demonstrate that AM acts as a growth factor for DU145 cells, which suggests the existence of an autocrine loop mechanism that could potentially drive neoplastic growth. PAM mRNA levels were found to be 3-fold higher in prostate adenocarcinomas compared with that of human benign prostate hyperplasia (BPH) as demonstrated by real-time quantitative reverse transcription-PCR. The analysis of AM message expression in BPH and CaP (Gleason's score, 6-9) shows a clear distinction between benign and CaP. The expression was detected only in adenocarcinomas tissues with a marked increase in samples with a high Gleason's score. Immunocytochemically, AM was localized in the carcinomatous epithelial compartment. NE phenotype, assessed after the immunocytochemical localization of neuron-specific enolase (NSE), was found in both the epithelial and the stromal compartments of cancers; in BPH, only some spare basal cells were NSE-labeled. Cancer progression could be accelerated by peptides secreted by a population of cells capable of inducing androgen-independent tumoral growth via autocrine-paracrine mechanisms.

Adenocarcinoma↗

PubMedDate not supplied

Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.

Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity. Methods to monitor the amidation of polypeptide substrates and analyze the final products are described, including the use of capillary zone electrophoresis. A transient alpha-hydroxyglycine intermediate, [Ala15]-GRF(1-29)-Gly(alpha-OH)-OH, was isolated and characterized. Kinetic studies with this intermediate demonstrate that the rat alpha-amidating enzyme from recombinant mouse C127 cells possesses both the monooxygenase and lyase activities needed to catalyze both steps of the amidation process.

Amino Acid Sequence↗

PubMed1990

Both amidated and nonamidated forms of glucagon-like peptide I are synthesized in the rat intestine and the pancreas.

Biologically active peptides are initially synthesized in the form of protein precursors, and the peptides are liberated by post-translational processing from the precursors in a tissue-specific manner. Mammalian proglucagon, which is synthesized in the neuroendocrine L-cells of the intestine and the alpha-cells of the pancreas, contains within its structure the sequences of glucagon and two glucagon-like peptides (GLP-I and GLP-II) flanked at their amino and carboxyl termini by dibasic residues. Tissue-specific processing liberates different peptides in the intestine compared with the pancreas. One of these intestinal peptides, glucagon-like peptide I(7-37) (GLP-I(7-37], is one of the most potent insulin secretagogues studied to date. It contains within its carboxyl-terminal domain an arginine residue that, because of an adjacent glycine residue, may alternatively be used during post-translational processing as a site for amidation. Using a chromatographic system and radioimmunoassays that discriminate among the closely related GLP-I peptides, we find that the processing of proglucagon in the rat intestine and to a lesser extent in the rat pancreas results in the formation of at least three GLP-I peptides, of 37, 31, and 30 residues. The 30-residue peptide is in the form of an alpha-carboxyl-terminal arginine amide, a modification that is not usually found in proteins. Remarkably, the relative potencies for the stimulation of insulin secretion from the perfused rat pancreas of the nonamidated (GLP-I(7-37] and the amidated (GLP-I(7-36) amide) peptides are the same (Weir, G. C., Mojsov, S., Hendrik, G. K., and Habener, J. F. (1989) Diabetes 38, 338-342; Suzuki, S., Kawai, K., Okashir, S., Mukal, H., and Yamashita, K. (1989) Endocrinology 125, 3109-3114).

Animals↗

PubMed1991

GLP-1 and GLP-1(7-36) amide: influences on basal and stimulated insulin and glucagon secretion in the mouse.

We studied the cellular distribution of glucagon-like peptide-1 (GLP-1) in the pancreas and gut and the effects of GLP-1 and its truncated form, GLP-1(7-36) amide, on basal and stimulated insulin and glucagon secretion in the mouse. Immunofluorescence staining showed that GLP-1 immunoreactivity occurred within peripheral islet cells and in cells located mainly distally in the small intestine and in the entire large intestine. Double-immunostaining revealed that the GLP-1-immunoreactive cells were identical to the glucagon/glicentin cells. Experiments in vivo revealed that basal insulin secretion was stimulated by GLP-1(7-36) amide at the dose levels of 8 and 32 nmol/kg, and by GLP-1 at 32 nmol/kg. Furthermore, GLP-1(7-36) amide showed additive stimulatory influence with glucose (2.8 mmol/kg), the cholinergic agonist carbachol (0.16 mumol/kg), and the C-terminal octapeptide of cholecystokinin (CCK-8, 5.3 nmol/kg), when injected at 8 or 32 nmol/kg. In contrast, stimulated insulin secretion was unaffected by GLP-1. Moreover, the glucagon secretory responses to carbachol and CCK-8 were inhibited by GLP-1(7-36) amide but were unaffected by the entire GLP-1. We conclude that GLP-1(7-36) has the potential for being a modulator of islet hormone secretion.

Animals↗

PubMed1990

[Comparative study of the toxicity of cyanic derivatives and/or aromatic amides].

The toxical potentiality of the cyanic radicle and the detoxicating characteristic of the amide group are studied following the evolution of the benzonitrile and benzamide toxicity when a cyanic or amide radicle is added to the original molecule. This comparative study shows that the presence of amide group has not always given a higher toxicity to the organic molecules that own it. It is the respective localization of the cyanic or amide radicles which appears to play the most important role.

Animals↗

PubMed1990

Comparison of GLP-1 (7-36amide) and GIP on release of somatostatin-like immunoreactivity and insulin from the isolated rat pancreas.

The effect of equimolar doses of GIP and GLP-1 (7-36amide) on insulin and somatostatin secretion in the isolated perfused rat pancreas was compared. At a perfusate glucose concentration of 70 mg/dl GLP-1 (7-36amide) 10(-9) and 10(-8) M and GIP 10(-9) M elicited a significant stimulation of insulin while GIP 10(-8) M and lower doses of both peptides (10(-11) and 10(-10) M) were ineffective. At elevated perfusate glucose levels of 150 mg/dl both peptides stimulated insulin release at 10(-11), 10(-10), 10(-9) and 10(-8) M but not at 10(-12) M. The insulin response at the higher glucose level was significantly greater compared to the effect of the same doses at normoglycemic conditions. Somatostatin release was stimulated significantly by GLP-1 (7-36amide) at 10(-10) and 10(-9) M at perfusate glucose level 70 mg/dl. At a glucose concentration of 150 mg/dl this effect was abolished. GIP did not alter somatostatin release at a perfusate glucose concentration of 70 mg/dl while at 150 mg/dl only the highest dose of GIP (10(-8) M) stimulated somatostatin release significantly. In conclusion, the present data demonstrate that in vitro in the rat pancreas both peptides are equally effective secretagogues of insulin release at normal and moderately elevated perfusate glucose levels. In contrast, somatostatin secretion is stimulated by GLP-1 (7-36amide) at normoglycemic conditions while only a rather high and presumably pharmacological dose of GIP is a stimulus of somatostatin secretion at moderate hyperglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Carbachol priming increases glucose- and glucagon-like peptide-1 (7-36)amide-, but not arginine-induced insulin secretion from the isolated perfused rat pancreas.

The priming effect of carbachol on glucose-, arginine- and glucose plus GLP-1 (7-36)amide induced insulin secretion was investigated. The isolated rat pancreas was perfused in vitro during a basal period of 10 min with Krebs-Ringer-bicarbonate buffer containing 2.8 mmol/l glucose. This medium was then supplemented with carbachol (10,1.0.1 mumol/l, respectively). After an additional 10 min period at 2.8 mmol/l glucose insulin secretion was stimulated for 44 min with 10 mmol/l glucose, or glucose plus GLP-1 (7-36)amide (0.5 nmol/l), or arginine (10 mmol/l). Pretreatment with carbachol resulted in a concentration dependent sensitization of B-cells to a consecutive glucose load (10 mmol/l). Both phases of the biphasic insulin secretory response were significantly enhanced. Priming experiments followed by a subsequent combined glucose / GLP-1 (7-36)amide or arginine stimulation were performed with 10 mumol/l carbachol. Prior exposure of the pancrease to carbachol enhanced the glucose / GLP-1 (7-36)amide induced insulin release, but left the arginine stimulated secretion unaltered. Our data suggest that in the regulation of postprandial insulin release cholinergic sensitizing effects might be involved which are mediated by muscarinic receptors.

Animals↗

PubMed1986

Analysis of FMRF-amide effects on Aplysia bursting neurons.

The peptide L-phenylalanyl-L-methionyl-L-arginyl-L-phenylalaninamide (FMRF-amide) was pressure-applied onto the somata of bursting neurons L4 and L6 in the Aplysia abdominal ganglion. FMRF-amide causes a biphasic response, first depolarizing and then hyperpolarizing the neuron. In voltage-clamp experiments, FMRF-amide induces an inward current that begins 100-200 msec after applying the peptide and peaks in 2-10 sec. This is followed by an outward current that begins with a latency of 2-5 sec and peaks in 15-65 sec. The entire response lasts 1-5 min. Experiments were done to separate the two currents induced by FMRF-amide on the basis of ion selectivity and kinetics and to determine their I(V) relationships. The currents were studied using a method to quickly measure I(V) curves. The inward current is caused by a conductance increase and has a reversal potential of approximately +18 mV. This current depends on the concentration of extracellular Na ions but not Ca, Cl, or K ions and is insensitive to tetrodotoxin, hexamethonium, and curare. The outward current is caused by a conductance increase and has a reversal potential of approximately -61 mV, which is similar to the reversal potential of the fast, transient K current (IA) in the same cells. This current is sensitive to changes in the external K ion concentration but not to changes in Cl, Ca, or Na concentration. The outward current is partially blocked by 1 mM 4-aminopyridine but not TEA or curare. Neither current is significantly voltage dependent within the range from -70 to -40 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

[Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules].

About 40-60% of the peptidylglycine alpha-amidating amonooxygenase activity in the lysates of secretory granules from bovine atria and adrenal medulla isolated and lyzed in the presence of pepstatin, phenylmethylsulfonyl gluoride, N-ethylmaleimide and catalase, was found to be in the soluble form. The remaining part bound to the membrane fraction was extracted with Triton X-100. The procedure of purification of the soluble form of peptidylglycine alpha-amidating monooxygenase from both atrial and chromaffin granules in electrophoretically homogeneous enzyme preparations was developed. The enzyme is made up of a single subunit with a molecular mass of 68 kDa and contains one copper atom per molecule. The EPR spectra of peptidylglycine alpha-amidating amonooxygenase and dopamine beta-monooxygenase were found to be practically identical, thus indicating that the copper environment in the both enzymes is the same. Both peptidylglycine alpha-amidating monooxygenase and dopamine beta-monooxygenase are inhibited by the neurocuprein apoform, an extremely acidic protein isolated from brain and secretory granules of different endocrine tissues.

Adrenal Medulla↗

PubMed1985

Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides.

Ubiquitin carboxyl-terminal hydrolase (formerly known as ubiquitin carboxyl-terminal esterase), from rabbit reticulocytes, has been shown to hydrolyze thiol esters formed between the ubiquitin carboxyl terminus and small thiols (e.g. glutathione), as well as free ubiquitin adenylate (Rose, I. A., and Warms, J. V. B. (1983) Biochemistry 22, 4234-4237). We now show that this enzyme hydrolyzes amide derivatives of the ubiquitin carboxyl terminus, including those of lysine (epsilon-amino), glycine methyl ester, and spermidine. It also hydrolyzes ubiquitin COOH-terminal hydroxamic acid, but is inactivated under the conditions for assaying ubiquitin-hydroxylamine adduct hydrolysis. Amide adducts formed between ubiquitin and epsilon-amino groups of protein lysine residues are much poorer substrates than is the ubiquitin amide of the epsilon-amino group of free lysine. The enzyme is thus a general hydrolase that recognizes the ubiquitin moiety, but is highly selective for small ubiquitin derivatives. It probably functions to regenerate ubiquitin from adventitiously formed ubiquitin amides and thiol esters. It also has the correct specificity to function in regenerating ubiquitin from small ubiquitin peptides that are probable end products of ubiquitin-dependent proteolysis. A simple, large-scale preparation of the enzyme from human erythrocytes is described.

Animals↗

PubMed1988

Effect of tolmetin glycine amide (McN-4366), a prodrug of tolmetin sodium, on adjuvant arthritis in the rat.

The glycine amide of tolmetin sodium (TGA) functions as a prodrug and was demonstrated to be more potent than the parent compound as an inhibitor of developing and established adjuvant arthritis in the female Lewis rat. In contrast, the glycine amide of indomethacin was less potent than indomethacin. The superiority of TGA relative to tolmetin sodium in alleviating this condition was demonstrated by inhibition of paw swelling and reduction of the degenerative bone changes that are associated with the progression of this chronic animal model of rheumatoid arthritis in humans. These properties were not evident when equimolar mixtures of tolmetin sodium and glycine were administered concurrently. Pharmacokinetic analyses revealed that TGA was absorbed completely and hydrolyzed to tolmetin in the female adjuvant arthritic rat. The combined effects of absorption, distribution and hydrolysis of TGA produced lower peak plasma tolmetin levels than an equivalent dose of tolmetin sodium, but plasma concentrations were sustained for a longer period of time contributing to an apparent increase in potency. Furthermore, TGA displayed a decreased propensity to cause gastrointestinal irritation compared to tolmetin sodium. Several additional amino acid amides of tolmetin were similar to the glycine amide in exhibiting increased potency and reduced gastrointestinal toxicity in comparison to equivalent doses of tolmetin sodium.

Animals↗

PubMed1987

Studies on the derivatives of aziridine. II. Synthesis and immunopharmacological analysis of substituted amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid.

Several new amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid were synthetized. The beta-/p-chlorobenzoyl/-acrylic acid 2 was used as the substrate. This compound was converted by reaction with appropriate amine into amides and anilides of beta-/p-chlorobenzoyl/-acrylic acid (3-10). These compounds react with ethylenoimine giving appropriate amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid (11-18). When pharmacologically analyzed, they appeared to possess marked immunotropic activity. The derivatives in question modulated both humoral as well as cellular immune response, the effect being related to the type of substitutent in the amide group.

Animals↗

PubMedDate not supplied

[Model studies of DNA-protein interaction. I. Effect of aminocarboxylic and amide groups of amino acids on DNA thermostability and conformation].

To elucidate interactions of amino-carboxylate dipole and amide group of amino acids with DNA, glycine and glutamine, concentration dependences of the melting curves and CD spectra of calf thymus DNA at low ionic strength (10(-4) M) Na-citrate have been studied. A considerable increase of the melting temperature delta t1/2 and a decrease of the temperature interval of melting delta t with the rise of glycine concentration were observed without changes in the CD spectrum. A comparison was made between the influence of dipolar glycine ion and isolated amino and carboxylate ions of ammonium acetate. The data obtained indicated the predominance of electrostatic interaction of glycine with DNA phosphates until the ligand concentration was approximately 0.6 M and, apparently, specific interactions of carboxylate ion with guanine at higher glycine concentrations. Destabilizing effect of glutamine on DNA at a concentration of 5.10(-3) M was observed, whereas at higher concentrations two-phase increase of delta t1/2 was shown. Small changes in DNA CD spectrum under the action of glutamine were registered. The comparison data for glutamine and acrylamide showed that DNA destabilizing effect was due to the amide group. The destabilizing effect of amide group can be explained by its interaction with the bases in single-stranded regions of DNA with the formation of two H-bonds. It is possible that the increase of DNA delta t1/2 is the result of the interaction of phosphates both with aminocarboxylate and amide groups of glutamine.

Acetates↗