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PubMed1988

Peptidyl-glycine alpha-amidating mono-oxygenase activity towards a gonadotropin-releasing-hormone C-terminal peptide substrate, in subcellular fractions of sheep brain and pituitary.

The amidation of a synthetic peptide D-Tyr-Pro-Gly-Gly by sheep hypothalamic and pituitary preparations was measured. This substrate was designed as a glycine-extended C-terminal peptide analogue of gonadotropin-releasing hormone (GnRH) to test the ability of these tissues to convert the product produced by cleavage of the GnRH prohormone into the active amidated decapeptide. An alpha-amidating activity capable of converting D-125I-Tyr-Pro-Gly-Gly into D-125I-Try-Pro-Gly-NH2 was identified in crude synaptosomal and neurosecretory-granule fractions from hypothalamus and anterior-pituitary secretory-granule preparations. This activity was stimulated by the addition of Cu2+ and reduced ascorbate, and was maximal at neutral pH in sulphonic acid buffers. Highest activity was measured in synaptosomes from the median eminence and medial basal hypothalamus and in pituitary granules. Lower activity was found in synaptosomes prepared from anterior hypothalamic tissue. Negligible activity was measurable in cerebral cortex and none in pineal synaptosomes. Direct comparison of alpha-amidation with D-125I-Try-Pro-Gly-Gly and a previously reported substrate D-125I-Tyr-Val-Gly showed that, although the latter was 15-20-fold more reactive, the optimal concentration of Cu2+ for amidation was similar with both substrates in medial-basal-hypothalamic synaptosomes and pituitary granules. Activity measured with 1 microM-D-125I-Tyr-Val-Gly was inhibited by increasing concentrations of D-Tyr-Pro-Gly-Gly, with 50% inhibition at 25 microM-D-Tyr-Pro-Gly-Gly, whereas activity with 3.3 microM-D-125I-Tyr-Pro-Gly-Gly was abolished by addition of 1 microM-D-Tyr-Val-Gly, evidence that the two substrates were competing for the same enzyme activity. Synaptosomal preparations demonstrated Michaelis-Menten kinetics for D-Tyr-Pro-Gly-Gly as substrate, with values of Km and V decreasing upon removal of ascorbate. We conclude that D-Tyr-Pro-Gly-Gly-directed alpha-amidation in sheep hypothalamic synaptosomes resembles the activity with D-Tyr-Val-Gly as substrate, as well as that demonstrated by others with D-Tyr-Val-Gly as substrate in rat hypothalamic and pituitary tissue. Although reactivity towards D-Tyr-Pro-Gly-Gly cannot be assumed to assess amidation solely of GnRH, the negligible D-Tyr-Pro-Gly-Gly-directed activity in the pineal gland and cerebral cortex, areas that are known to synthesize other alpha-amidated peptides, suggests some substrate specificity in alpha-amidating enzymes from different tissues.

Animals↗
PubMed1984

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves.

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [(15)N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no (15)N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [(15)N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH(3) accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of (15)N into glutamine-amide, NH(3), and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.

Journal Article↗
PubMed1989

Changes in Amide-Linked and Ester Indole-3-Acetic Acid in Cotton Fruiting Forms during Their Development.

The concentration of free indoleacetic acid (IAA) is high in cotton (Gossypium hirsutum L.) fruiting forms before anthesis, but is low at and for a few days after anthesis. Amide-linked and ester IAA were measured in fruiting forms at 9, 6, and 3 days before anthesis; at anthesis; and at 2, 4, 7, and 9 days after anthesis to determine if free IAA decreased because it was converted to a conjugated form. That did not appear to be the case. While the major decrease in free IAA occurred during the 6 days before anthesis, ester IAA increased only a small amount and amide-linked IAA decreased even more than free IAA. During the 6 days before anthesis free IAA decreased from 0.62 to 0.12 micrograms per gram and amide-linked IAA decreased from 19.14 to 1.16 micrograms per gram dry weight. No evidence was found that a large amount of amide-linked IAA was converted to an insoluble form; flowers contained less than 1 microgram per gram of insoluble IAA. The free and amide-linked IAA must have been converted to other forms, perhaps by oxidation. Soluble amide-linked IAA remained low after anthesis. No ester IAA was detected 6 days before anthesis and only 0.08 microgram per gram dry weight was measured at anthesis. The concentration of ester IAA increased thereafter to 4.43 micrograms per gram at 9 days after anthesis. Therefore, amide-linked IAA was the major form of IAA in flower buds and ester IAA was the major form in young fruits (bolls). Minimum concentrations of free and total IAA occurred during the 4 days after anthesis, a stage when cotton fruiting forms are most likely to abscise. The large decreases in free and amide-linked IAA during the 6 days before anthesis may indicate a rapid turnover of IAA in flower buds. But, the decrease in free IAA was not accompanied by a comparable increase in ester or amide-linked IAA.

Journal Article↗
PubMed1985

Characterization of FMRF amide-like immunoreactivity in rat spinal cord by region-specific antibodies in radioimmunoassay and HPLC.

Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe-Met-Arg-Phe-NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N-terminally extended analogue, Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C-terminus; both required the C-terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C-terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed-phase HPLC using Techsil C18 and Spherisorb-phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C-terminal tripeptide of FMRF amide and are probably extended at the N-terminus by sequences immunochemically distinct from other known peptides.

Animals↗
PubMed1989

Signal transmission after GLP-1(7-36)amide binding in RINm5F cells.

Glucagon-like peptide-1(7-36)amide [GLP-1(7-36)amide], probably representing an important incretin, binds to receptors on RINm5F cells resulting in an adenosine 3',5'-cyclic monophosphate increase. Guanine nucleotides (GTP, GTP-gamma-S, GDP-beta-S) decreased the binding of GLP-1(7-36)amide to receptors on RINm5F cell membranes. Further analysis revealed that GTP (10(-4) M) decreased the receptor affinity with an increase of the Kd from 2.5 +/- 0.99 x 10(-10) M to 9.43 +/- 2.16 x 10(-10) M. In cross-linking experiments the amount of labeled peptide linked to receptors was reduced in the presence of GTP (10(-4) M). Further studies investigated the involvement of membrane depolarization or changes in the cytosolic free calcium level in the intracellular signaling of GLP-1(7-36)amide-induced insulin secretion. In contrast to fuel and nonfuel secretagogues, GLP-1(7-36)amide did not cause a depolarization of the membrane potential. This was unaffected by various glucose concentrations (0-20 mM) or by previous cell depolarization by D-glyceraldehyde. Similarly, the cytosolic calcium concentration remained unchanged after addition of GLP-1(7-36)amide (10(-12)-10(-8) M). The effect of guanine nucleotides on binding of GLP-1(7-36)amide indicates that the action of the peptide is mediated by the adenylate cyclase system. GLP-1(7-36)amide binding neither changed the membrane potential nor altered the intracellular calcium concentration, making an involvement of the inositol 1,4,5-trisphosphate pathway or an activation of protein kinase C in the postreceptor signaling after GLP-1(7-36)amide binding unlikely.

Adenoma, Islet Cell↗
PubMed1994

Rat parietal cell receptors for GLP-1-(7-36) amide: northern blot, cross-linking, and radioligand binding.

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal cells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isolated rat gastric mucosal cells (F0) were fractionated by counterflow elutriation, resulting in five fractions (F1-F5) according to increasing cell diameter and parietal cell content (3, 5, 4, 27, 81%). Additional density gradient centrifugation of F4 yielded enriched chief cells (74%; parietal cells: 1%; F6), whereas density gradient centrifugation of F5 almost purified parietal cells (97%; chief cells: 1%; F7). Northern blot of total cellular RNA from F0-F7 with a probe specific for the GLP-1-(7-36) amide receptor revealed two RNA species of 2.7 and 3.6 kb. These messages were present to some extent in small cells (F1, F2), much more pronounced in F5, abundant in F7, barely detectable in F3 and F4, and absent from F6. Cross-linking of 125I-labeled GLP-1-(7-36) amide to parietal cell membranes revealed a single 59-kDa band that was abolished by unlabeled GLP-1-(7-36) amide. Throughout fractions F1-F7 specific binding of 125I-GLP-1-(7-36) amide was correlated with parietal cell content (r = 0.99; P < 0.01) and H+ production ([14C]aminopyrine accumulation) in response to GLP-1-(7-36) amide or histamine (r = 0.96; P < 0.01). Binding was maximal in purified parietal cells (F7), whereas almost no binding was detectable in enriched chief cells (F6). In F7, Scatchard analysis revealed a single class of high-affinity binding sites (KD = 2.8 +/- 0.6 x 10(-10) M, Bmax = 6.8 +/- 1.4 fmol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The following half-maximal inhibition values were found for GLP-1-(7-36) amide and (1-37) and (1-36) amide: 6.6 +/- 0.9 x 10(-10), 1.4 +/- 0.7 x 10(-7), and 2.6 +/- 0.4 x 10(-7) M, respectively. Pancreatic glucagon, GLP-2, and oxyntomodulin, products of the proglucagon gene, were 3-4 log units less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that rat parietal cells are equipped with specific high-affinity receptors for GLP-1-(7-36) amide, which, in addition, are present in as yet unidentified small cells (F1, F2) but not in chief cells.

Aminopyrine↗
PubMed1986

The characterization of the ascorbic acid-mediated alpha-amidation of alpha-melanotropin in cultured intermediate pituitary lobe cells.

Previous studies have shown that cultured rat intermediate pituitary lobe cells lose the ability to form ACTH-(1-13)NH2-related molecules (alpha MSH) and instead produce ACTH-(1-14)-related peptides. In vitro studies have shown that peptidylglycine alpha-amidating monooxygenase the enzyme responsible for the conversion of ACTH-(1-14) to ACTH-(1-13)NH2, requires ascorbate, CuSO4, and molecular oxygen as cofactors. In the present study we have demonstrated that cultured intermediate pituitary lobe cells require long term supplementation of the medium with ascorbate for continued production of immunoactive alpha-amidated alpha MSH. When the relative quantities of alpha-amidated and COOH-terminally glycine-extended forms of alpha MSH were assessed in biosynthetic labeling experiments, it was shown that either L-ascorbate or an epimer of the vitamin, D-isoascorbate, was capable of supporting cellular alpha-amidation. However, the potency of isoascorbate was approximately 4- to 5-fold lower than that of ascorbate. The ascorbate-mediated reestablishment of alpha-amidation ability was shown to be dependent on the presence of sodium in the medium; at physiological levels of ascorbate (35 microM), the EC50 for sodium was about 40 mM. Time-course experiments indicated that the time of exposure of the cultured cells to ascorbate could be decreased to as little as 30 min. However, a total incubation time of 6 h was required after such an exposure to convert biosynthetically labeled ACTH-(1-14)-related peptides to labeled ACTH-(1-13)NH2-related peptides. The time course of the effects of ascorbic acid on the reestablishment of alpha-amidation, as well as the relative stereospecificity for L-ascorbic acid and the sodium requirement, are all consistent with the hypothesis that ascorbic acid must be transported into the intermediate pituitary lobe cells to participate in the peptidyl alpha-amidation reaction. Moreover, it is apparent that the length of time required to reestablish alpha-amidation ability (6 h) is a function of transport of the cofactor into the granules, the rate of the peptidylglycine alpha-amidating monooxygenase-catalyzed reaction, or both.

Adrenocorticotropic Hormone↗
PubMed1985

Regulation of peptide amidation in cultured pituitary cells.

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.

Animals↗
PubMed1985

Amidation of C3 at the thiolester site: stimulation of chemiluminescence and phagocytosis by a new inflammatory mediator.

We studied the ability of particle-bound and fluid-phase C3b monomers and monomeric amidated C3 (prepared by treatment of purified human C3 with ammonium chloride or methylamine) to stimulate chemiluminescence of human polymorphonuclear leukocytes (PMN) and monocytes and to promote phagocytosis in the absence of antibody. Particle-bound C3b evoked chemiluminescence from both PMN and monocytes, and fluid-phase C3b (0.5 mg/ml) elicited significant chemiluminescence from PMN but not from monocytes. Amidated forms of C3, both particle bound and fluid phase, were potent stimulators of chemiluminescence from phagocytic cells and caused a significantly greater response than did C3b. The phagocytosis of 1-micron microspheres by PMN and monocytes was enhanced by coating them with purified C3b in an antibody-free system. Microspheres coated with amidated C3 were avidly phagocytized, and to a greater degree than were C3b-coated microspheres. In a direct binding assay with tritiated monomeric C3b and amidated C3, the affinity of the PMN complement receptor type 1 for C3b (Ka = 4.9 X 10(7) L/M) was similar to that for amidated C3 (Ka = 5.7 X 10(7) L/M). However, there was a fourfold increase in the number of apparent binding sites for amidated C3. This increase did not reflect binding of amidated C3 to the complement receptor type 3, because blocking of CR3 with the monoclonal antibody OKM 10 failed to decrease binding of amidated C3. In sites of increased ammoniagenesis, such as the kidney in chronic renal failure, amidated C3 may play a role as an inflammatory mediator by stimulating oxidative metabolism in phagocytic cells.

Ammonium Chloride↗
PubMed1995

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.

The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that 'anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.

Amides↗
PubMed1995

Gadolinium and dysprosium chelates of DTPA-amide-dextran: synthesis, 1H NMR relaxivity, and induced 23Na NMR shift.

In this study the conjugated macromolecular ligand, diethylenetriaminepentaacetic acid (DTPA)-amide-dextran, was synthesized by attaching DTPA to the dextran macromolecule (M(r) approximately 6000) by a covalent amide bond. Subsequently, DTPA-amide-dextran was complexed with either of the two lanthanide metal ions dysprosium (Dy) or gadolinium (Gd). The paramagnetic 23Na NMR shift induced by Dy(DTPA-amide-dextran) and the relaxivity (rho 1) induced by Gd(DTPA-amide-dextran) were characterized. Dy(DTPA-amide-dextran) induced a 25% larger 23Na NMR shift than that induced by Dy(DTPA). Neither the shift induced by Dy(DTPA-amide-dextran) nor the shift induced by Dy(DTPA) was affected by increasing levels of calcium ions in the solution. offDTPA-amide-dextran) exhibited an in vitro rho 1 of 8.4 (mM s)-1 at a 0.23 T magnetic field and 9.3 (mM s)-1 at a 0.47 T magnetic field, thus indicating a positive magnetic field dependence.

Amides↗
PubMed1986

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons.

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.

Amides↗
PubMed1984

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment.

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.

Amides↗
PubMed1986

Use of Pico-Tag methodology in the chemical analysis of peptides with carboxyl-terminal amides.

A chemical method has been established for the detection of carboxyl-terminally amidated peptides in tissue extracts. Tissue was homogenized in an acidic medium designed to solubilize peptides while precipitating high-molecular-weight protein. The homogenate supernatant was in turn subjected to reversed-phase extraction with C18 Sep-Pak cartridges. The eluates were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Individual fractions were exhaustively digested with thermolysin, derivatized with phenylisothiocyanate (PITC), and then subjected to ethyl acetate extraction under basic conditions. The phenylthiocarbamyl (PTC)-amino acid amide derivatives were selectively taken up into the organic phase, while the other digestion products remained in the aqueous phase. The organic phase was analyzed by RP-HPLC on a Pico-Tag amino acid analysis column, monitoring eluates at 254 nm. PTC-amino acid amides were identified and quantitated by comparing their elution positions and peak areas, respectively, with those of standards. Their identities were confirmed by amino acid analysis, following hydrolysis with hydriodic acid. The technique was applied to extracts of bovine posterior pituitaries and a human medullary thyroid carcinoma. Vasopressin (-Leu-Gly-amide), oxytocin (-Gly-amide), Lys1 gamma 1-melanotropin (-Phe-amide), and various acetylated and non-acetylated forms of alpha-melanotropin (-Val-amide) were identified in the posterior pituitary extract. Various forms of calcitonin (-Val-Gly-Ala-Pro-amide) were detected in the tumour extract. For vasopressin and calcitonin the thermolytic digest resulted in di- and tetra-peptides, respectively, reflecting thermolytic cleavage at more favoured sites.

Amides↗
PubMed1993

Efficient amidation of C-peptide deleted NPY precursors by non-endocrine cells is affected by the presence of Lys-Arg at the C-terminus.

Post-translational processing of peptide precursors producing amidated, biologically active peptides generally occurs in specially differentiated endocrine or neural cells. However, we have previously shown that a C-peptide-deleted precursor of neuropeptide Y (NPY1-39) in which the precursor terminates in the sequence Gly-Lys-Arg was partially amidated by the non-endocrine cell line, CHO. In the present study we show that two other non-endocrine cell lines, NIH 3T3 and BHK, also possess amidating activities and that the NPY1-39 precursor was completely converted to NPY1-36 amide by the NIH 3T3 cell line. The role of the two basic residues (Lys-Arg) in the C-terminus was studied by transfection of a construct encoding a NPY precursor terminating with glycine alone. Both the CHO and NIH 3T3 cell lines, transfected with this construct, secreted a significantly smaller fraction of NPY reactive material as amidated NPY compared to the fraction of amidated NPY secreted by the cells transfected with the NPY1-39 precursor. It is concluded that the capacity to perform C-terminal amidation appears to be a universal feature of eukaryotic cells and that the carboxypeptidase E-like enzyme influences the amidation process, beyond its known ability to remove the C-terminal basic residues.

3T3 Cells↗
PubMed2006

The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions.

Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1-37/38-108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from (1)H(15)N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293-3302]. The (1)H(15)N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH-midpoint (pH(m)) can be obtained by fitting. In segment A, the pH(m) of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the pK(a)). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pH(m) of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pH(m) of its backbone amide is larger than in the pK(a) of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pH(m) of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its pK(a) whenever there are none or few neighboring backbone amides with similar pH-dependence.

Amides↗
PubMed2006

Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity.

Oxidative stress caused by various stimuli lead to oxidation of glutathione (GSH), the major redox power of the cell. Amyloid beta [Abeta(1-42)] is one of the key components of senile plaques and is involved in the progress initiation and triggers of Alzheimer's disease (AD). Lower GSH levels correlated with the activation of mitogen-activated proteins kinases (MAPK) have been demonstrated in AD, Parkinson's disease (PD) and other neurodegenerative disorders and have been proposed to play a central role in the deterioration of the aging and neurodegenerative brain. In this study, we evaluated the ability of low molecular weight thiol amides, N-acetyl cysteine amide (AD4) that replenishes GSH levels, N-acetyl glycine cysteine amide (AD7) and N-acetyl-Cys-Gly-Pro-Cys-amide (CB4) to protect primary neuronal culture against the oxidative and neurotoxic effects of Abeta(1-42) and to inhibit cisplatin- and hydrogen-peroxide-induced phosphorylation of two MAP kinases (MAPK), p38 and ERK1/2, in NIH3T3 cells. Cell death induced by Abeta(1-42) in primary neuronal cells was reversed by the thiol amides. Likewise, protein oxidation, loss of mitochondrial function and DNA fragmentation all returned to control levels by pretreatment with the three thiol amides. Elevated phosphorylation of ERK1/2 and p38 induced by cisplatin or H2O2 in NIH3T3 cells was lowered by AD4, AD7 and CB4 in a dose-dependent manner. Taken together, these results suggest that the thiol amides AD4, AD7 and CB4 protect neuronal cells against Abeta(1-42) toxicity by attenuating oxidative stress in correlation with inhibiting the MAPK phosphorylation cascade. These results are consistent with the notion that these small molecular thiol amides may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.

Acetylcysteine↗
PubMed1999

Identification of beta-turn and random coil amide III infrared bands for secondary structure estimation of proteins.

Fourier transform infrared spectroscopy is increasingly becoming an important method to determine secondary structure of peptides and proteins. Among the spectral regions arising out of coupled and uncoupled stretching and bending modes of amide bonds, amide I and amide III spectral bands have been found to be the most sensitive to the variations in secondary structure folding. Amide I spectral region (1700-1600 cm-1), although most commonly used primarily because of its strong signal, suffers from several limitations, including a strong interference from water vibrational band, relatively unstructured spectral contour, and overlap of revolved bands correspondingly to various secondary structures. In contrast, amide III spectral region (1350-1200 cm-1), albeit relatively weak in signals, does not have the above limitations. Easily resolved and better defined amide III bands are quite suitable for quantitative analysis of protein secondary structure. While amide III region has been successfully used for determination of alpha-helix and beta-sheets (Fu, F.-N., et al. (1994) Appl. Spectrosc. 48, 1432-1441), bands corresponding to beta-turns and random coils have not been identified, so far. In this paper, we describe, for the first time, identification of amide III bands corresponding to beta-turns and random coils by selectively enhancing random coils by treatment with a denaturing reagent, and secondary structure estimation of several proteins by using the band assignments. The assignments of spectral bands were as follows: 1330-1295 cm-1, alpha-helix; 1295-1270 cm-1, beta-turns; 1270-1250 cm-1, random coils; and 1250-1220 cm-1, beta-sheets. The estimations of secondary structural elements by the above assignments correlated quite well with secondary structure estimations from X-ray crystallography data.

Amides↗