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PubMed2000

Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis.

The fatty-acid primary amide, oleamide, is a novel signaling molecule whose mechanism of biosynthesis is unknown. Recently, the N(18)TG(2) cell line was shown to synthesize oleamide from oleic acid, thereby demonstrating that these cells contain the necessary catalytic activities for generating the fatty-acid primary amide. The ability of peptide alpha-amidating enzyme, peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3), to catalyze the formation of oleamide from oleoylglycine in vitro suggests this as a function for the enzyme in vivo. This investigation shows that N(18)TG(2) cells, in fact, express PAM and that cellular differentiation dramatically increases this expression. PAM expression was confirmed by the detection of PAM mRNA, PAM protein, and enzymatic activity that exhibits the functional characteristics of PAM isolated from mammalian neuroendocrine tissues. The regulated expression of PAM in N(18)TG(2) cells is consistent with the proposed role of PAM in the biosynthesis of fatty-acid primary amides and further establishes this cell line as a model for studying the pathway.

Animals↗
PubMed1996

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: a monitor of intramolecular hydrogen bonds in helical peptides.

2D 1H NMR spectroscopy of two alpha-helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from -10 to -6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the alpha-helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in alpha-helices in H20/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in alpha-helices.

Hydrogen↗
PubMed1984

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide.

The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.

Animals↗
PubMed1995

Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain, pituitary and islet organ of the anglerfish (Lophius americanus).

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediate and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and anglerfish peptide Y-containing cells, but no overlap with glucagon-producing cells. The results also indicate that PAM immunoreactivity is widely distributed in the brain, pituitary and islet organ of anglerfish in cells, that contain peptides that require presence of a C-terminal glycine for amidation.

Animals↗
PubMed1995

Insulinotropic actions of intravenous glucagon-like peptide-1 (GLP-1) [7-36 amide] in the fasting state in healthy subjects.

GLP-1 (7-36 amide) stimulates insulin and suppresses glucagon secretion in normal subjects and may, in pharmacological doses, normalize hyperglycaemia in type 2 diabetic patients. It is not known whether such pharmacological doses can actually lower blood glucose to hypoglycaemic levels. Therefore, in seven normal fasting subjects, GLP-1 (7-36 amide) was infused intravenously at 0.3, 0.9 and 2.7 pmol/kg per min for 30 min each. The plasma concentration of GLP-1 (7-36 amide) increased dose-dependently, but insulin secretion (insulin, C-peptide) was stimulated only marginally. Glucagon was slightly suppressed, and plasma glucose was reduced, but not into the hypoglycaemia range. In conclusion, when plasma glucose concentrations are in the normal fasting range, GLP-1 (7-36 amide) is not able to stimulate insulin secretion to a degree that causes hypoglycaemia. This should limit the risk of hypoglycaemic responses when GLP-1 (7-36 amide) is administered in pharmacological doses to reduce hyperglycaemia in type 2 diabetic patients.

Adult↗
PubMed1993

New Gaba-containing analogues of human growth hormone releasing hormone (1-30)-amide: II. Detailed in vivo biological examinations.

Analogues of human growth hormone-releasing hormone-(1-30)-amide [GH-RH(1-30)-amide] were tested for their ability to stimulate GH release in vivo by injecting the peptides intravenously (iv), subcutaneously (sc), and intramuscularly (im). The analogues involved derivatization with Nle27 and Gaba substituents at the C-terminus with or without D-amino acid(s) in the peptide chain. The potency of the analogues was compared to that of GH-RH(1-29)-amid testing their ability to release GH at 5, 15 and 30 min after the administration. In iv test the potency of the analogues was 1.2-2 times higher than that of the GH-RH(1-29)-amide, and no significant differences were detected between the potencies of the analogues with or without D-amino acid. In the sc test the analogue with D-Ala2, Nle27, and Gaba30 substitutions expressed 8.0-51.7 times higher potency than the GH-RH(1-29)-amide, however, the analogue with similar modifications but with L-Ala2 showed the same low potency (1.2-2.1) as in the iv test. Results from the im experiments were similar to those of SC test. The most potent analogues were those which had D-Ala2, Nle27, and Gaba30 substitutions with Gly15 or Leu15. Circular dichroism (CD) spectra of the analogues showed that Leu in position 15 increased the stability of the predominant alpha-helix conformation, which improved the absorption of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Inositolphosphoglycans are possible mediators of the glucagon-like peptide 1 (7-36)amide action in the liver.

A potent glycogenic effect for GLP-1(7-36)amide has been found in rat hepatocytes and skeletal muscle, and the specific receptors detected for GLP-1(7-36)amide in these tissue membranes do not seem to be associated to adenylate cyclase. On the other hand, inositolphosphoglycan molecules (IPGs) have been implicated as second messengers in the action of insulin. In a human hepatoma cell line (HEP G-2), we have observed the presence of [125I]GLP-1(7-36)amide specific binding, and a stimulatory effect of the peptide upon glycogen synthesis, confirming the findings in isolated rat hepatocytes. Also, GLP-1(7-36)amide modulates the cell content of radiolabelled glycosylphosphatidylinositols (GPIs), in the same manner as insulin, indicating hydrolysis of GPIs and an immediate and short-lived generation of IPGs. Thus, IPGs could be mediators in the GLP-1(7-36)amide glycogenic action in the liver.

Animals↗
PubMed1995

Failure of GLP-1(7-36)amide to affect glycogenesis in rat skeletal muscle.

Glucagon-like peptide-1(7-36)amide has been described as exerting potent glycogenic action and as stimulating glycolysis in skeletal muscle. We exposed isolated rat soleus muscle strips to various concentrations of glucagon-like peptide-1(7-36) amide (10(-11) - 10(-6) mol/l) or insulin (10(-10) - 10(-7) mol/l) and determined the respective effects on glucose metabolism. Insulin markedly increased the rate of glucose incorporation into glycogen with a maximal effect at 10(-8) mol/l insulin (348 +/- 46% of intraindividual control experiment, p < 0.005), while glucagon-like peptide-1(7-36)amide was without an effect (e.g. 10(-11) mol/l, 96 +/- 10%; 10(-9) mol/l, 104 +/- 9%; 10(-7) mol/l, 121 +/- 13%; not significant). Likewise, glucagon-like peptide-1(7-36)amide did not affect the rate of 3H-2-deoxy-glucose transport or glycogen content of soleus muscle strips. The rates of aerobic or anaerobic glycolysis were also not increased. The findings were independent of peptide source and of employed muscle size. Our results do not suggest any effect of glucagon-like peptide-1(7-36)amide on skeletal muscle glucose metabolism and, hence, are in contrast to data derived from similar experiments by others.

Animals↗
PubMed1998

Effects of glucagon-like peptide-1 (7-36)amide on insulin stimulated rat skeletal muscle glucose transport.

Glucagon-like peptide-1 binding sites have been reported in peripheral tissues including muscle. However, the potential extra-pancreatic effects of glucagon-like peptide-1(7-36)amide are controversial. To evaluate whether glucagon-like peptide-1(7-36)amide has any effects on skeletal muscle glucose transport, isolated rat soleus muscles were incubated in increasing concentrations of insulin (0-150 nmol/l) in the presence or absence of 1 nmol/l glucagon-like peptide-1(7-36)amide for 3 h. Subsequently glucose transport was measured as uptake of [3H]-O-methylglucose. It was found that glucagon-like peptide-(7-36)amide has a small but significant stimulating effect on skeletal muscle glucose transport independent of the insulin concentration (P<0.01). However, because of the magnitude of the observed effect, the physiological importance of glucagon-like peptide-1(7-36)amide on skeletal muscle glucose metabolism is questionable.

Animals↗
PubMed1985

Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules.

In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.

Adrenocorticotropic Hormone↗
PubMed1989

Secreted alpha amidating enzymes are generated by specific posttranslational processing of precursors containing transmembrane domains.

The biosynthesis and secretion of alpha amidating enzymes from CA-77 cells has been investigated to determine the relationship among the various forms of alpha amidating enzyme seen after purification of alpha amidating enzyme activity from conditioned cell culture media. Initially 2 proteins of 104 kD and 94 kD are synthesized. With time the 104 kD precursor is processed to 41 kD and 43 kD, and the 94 kD precursor is processed to 75 kD. The 41 kD, 43 kD, and 75 kD proteins are secreted into the medium as functional enzymes. In comparing these data with known cDNA sequence for alpha amidating enzyme we conclude that the 104 kD and 94 kD precursors are membrane bound proteins which are posttranslationally processed to generate secreted alpha amidating enzyme.

Animals↗
PubMed1990

Peptidylglycine alpha-amidating reaction: evidence for a two-step mechanism involving a stable intermediate at neutral pH.

In our previous study of the rat brain alpha-amidating activity, we suggested that a protein of 41 kdal (41K protein) that shows no alpha-amidating activity is required for the reaction at neutral pH in addition to an alpha-amidating enzyme of 36 kdal (36K enzyme). Here we report on the purification of both proteins to near homogeneity and provide evidence that alpha-amidation proceeds via a two-step mechanism involving a stable intermediate at neutral pH, which is initially formed by the 36K enzyme and then readily converted into an amide product by the 41K protein.

Amino Acid Sequence↗
PubMed1985

N-Ethyl-17(R,S)-methyl-(6aR,10aR)-delta 8-tetrahydrocannabinol-18-oic amide. Brain pharmacokinetics in mice, triglyceride/phospholipid partitioning and generalization to the discriminative stimulus properties of delta 9-THC in rats.

The title compound, designed as a model for the affinity moiety of a cannabinoid affinity gel was synthesized in tritiated form (sp. act. 7.27 mCi/mmole). To validate the affinity approach to isolate the putative THC receptor, the properties of the amide were studied. Upon i.p. injection in mice the amide reaches peak brain levels of 0.13% of the total dose after 15 min. Following i.v. injection, maximal brain concentrations of 1.9% are observed at 5 min. Compared to delta 9-THC, which distributes almost equally between triglyceride and phospholipid phases (51:49) the amide exhibits a strong preference for phospholipids (5:95) that can be interpreted as high relative membrane affinity. In rats trained in a water maze to discriminate between i.p. injections of 3 mg/kg delta 9-THC (ED50 = 1.8 mg/kg) and its vehicle, the amide was generalized to the training drug, being five times less potent (ED50 = 8.7 mg/kg) than delta 9-THC. This demonstration of cannabis-like activity indicates that the amide retains affinity to the postulated receptor and justifies the choice for the affinity ligand.

Animals↗
PubMed1983

Effects of procaine amide, quinidine and ethmozin on delayed afterdepolarizations.

We studied the effects of three chemically different antiarrhythmic drugs on ouabain-induced delayed afterdepolarizations (DAD) in canine Purkinje fibers. The three drugs, ethmozin, 4.6 X 10(-6) M; procaine amide, 1.1 X 10(-4) M; and quinidine, 1.13 X 10(-6) M reduced DAD amplitude equivalently at drive cycle lengths less than 500 ms. Quinidine and procaine amide in these concentrations had no effect on the action potential characteristics except for a prolongation of action potential duration (APD) induced by procaine amide. Ethmozin reduced action potential amplitude, maximum upstroke velocity of phase 0 (Vmax), and APD measured to 50% and full repolarization (APD50 and APD100). Rate dependent changes in Vmax and maximum diastolic potential (MDP) were not exaggerated by quinidine in the ouabain intoxicated Purkinje fibers. The DAD coupling interval was increased as DAD amplitude decreased with all three drugs. Although ethmozin, procaine amide and quinidine similarly reduced DAD amplitude; procaine amide and quinidine exerted these effects in the absence of other transmembrane potential effects, whereas ethmozin did so only in concentrations that depressed the action potential as well.

Action Potentials↗
PubMed1985

The effects of opioid and FMRF-amide peptides on thermal behavior in the snail.

Administration of methionine-enkephalin, beta-endorphin or, as previously shown, the opiate agonist, morphine sulfate (0.10-10.0 micrograms per snail), resulted in significant dose-dependent increases in the latency of thermal (40 degrees C hot plate) avoidance behavior of the terrestrial snail, Cepaea nemoralis. The analgesic effects could be blocked by the opiate antagonist, naloxone, as well as by the non-opioid peptides, FMRF-amide and YGG-FMRF-amide. When administered by themselves the FMRF-amide peptides had significant bimodal effects either decreasing (0.10 and 10.0 micrograms) or increasing (1.0 micrograms) the latency of the response to the thermal stimulus. These results indicate that opioid and FMRE-amide peptides may be involved in the determination of thermal behavior in the snail. They also suggest that FMRF-amide peptides may function as endogenous modulators of opioid activity.

Animals↗
PubMed1990

In vivo metabolic activity of des-(B26-B30)-insulin-B25-amide and related analogues in the rat.

Metabolic potency of des-(B26-B30)-insulin-B25-amide, [TyrB25]des- (B26-B30)-insulin-B25-amide and [HisB25]des-(B26-B30)-insulin-B25-amide was studied in anaesthetized rats. Compared to insulin, full potency for des-(B26-B30)-insulin-B25-amide and an enhanced potency for both substituted analogues has been described previously on rat adipocytes in vitro. Hypoglycaemic effects following i.v. injection of all of these analogues were almost identical to those of native insulin with a half-maximal effective dose of approximately 3 nmol.kg-1. Stimulation of glucose metabolism during euglycaemic hyperinsulin-/analogueaemic clamp studies was indistinguishable from that of the native hormone with a maximal stimulation of approximately 19 mg.kg-1.min-1 and half-maximal effective hormone concentrations of approximately 1 pmol.ml-1. Analogue action on individual peripheral tissues estimated by the uptake of 2-deoxyglucose as well as stimulation of lipogenesis in epididymal fat was not different to that of insulin. These data demonstrate that C-terminal amidation of des-(B26-B30)-insulin results in a shortened molecule with full in vivo metabolic potency. When substituting phenylalanine in position B25 by tyrosine or histidine, the insulin-identical potency is preserved.

Adipose Tissue↗
PubMed1986

Superactive amidated COOH-terminal glucagon analogues with no methionine or tryptophan.

The functions of the Trp-25 and Met-27 residues and the free carboxy terminus of glucagon have been debated for many years. Despite some semi-synthetic data to the contrary, comparison of the glucagon sequence with the other 5 members of this family of peptides, all of them amides and particularly growth hormone-releasing factor(1-29) amide and its recently described analogues, suggests that alterations to these positions should be quite well tolerated in terms of biological activity. To test this prediction, [Phe-25,Leu-27]-glucagon amide was synthesized in high yield and was found to actually have superior glycogenolytic activity (196%) to glucagon in the rat. Replacement of Gly-4 by D-Phe, which has been shown to give much enhanced glycogenolytic activity than glucagon itself, also increased the activity of [D-Phe-4,Phe-25,Leu-27]-glucagon amide (518%). The L-Phe-4-analogue, [Phe-4,25,Leu-27]-glucagon amide, in contrast, was 20 times less active (30%), strongly suggesting the presence of a beta-bend in this N-terminal region of glucagon. This was supported by Chou-Fasman structural predictions which indicate extensive folding in the 1-15 region. Indeed, additional conformational restriction by substitution of D-Ser in position 2 of glucagon also increased activity to 226%. [D-Gln-3]-glucagon was slightly less active (74%) than glucagon. Chou-Fasman calculations on glucagon were compared to similar treatments of the VIP, secretin, PHI, and GRF(1-29) sequences.

Amino Acid Sequence↗
PubMed1993

FMRF-amide modulates the electrical activity of the leech Retzius cell.

The effect of the peptide FMRF-amide on the electrical activity of the leech Retzius (R) cell was investigated using electrophysiological techniques. FMRF-amide and six structurally related analogs increased the excitability of the R cell in several distinct ways that could act in concert to modulate transmitter release. 'Puffs' of FMRF-amide transiently depolarized the cell leading to a barrage of action potentials. This depolarization was followed by a phase of rhythmical bursting that appeared intrinsic to the neuron. FMRF-amide also broadened the plateau of the Ca(2+)-dependent action potential. The results suggest that the terminal Phe and Arg as well as the C-terminal amide are critical for the activity of these peptides.

Action Potentials↗