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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate factors governing proteolytic processing and routing of biologically active peptides in the secretory pathway, cDNAs for preproneuropeptide Y (preproNPY) and preproneuropeptide Y fused to a membrane anchor were transfected into pituitary cells. The anchor was the transmembrane and COOH-terminal cytoplasmic domain of peptidylglycine alpha-amidating monooxygenase (PAM); these domains are essential for correct routing of integral membrane forms of PAM. Like proneuropeptide Y (proNPY), the integral membrane form of proNPY was a good substrate for the endogenous prohormone convertases, yielding soluble NPY stored in regulated secretory granules. Tethering of proNPY to the membrane resulted in only a small delay in the rate of cleavage to produce mature NPY and in the arrival of NPY in regulated secretory granules. In contrast, the COOH-terminal region of proNPY remained attached to the transmembrane/COOH-terminal domain of PAM and was rerouted to the vicinity of the trans-Golgi network, where integral membrane forms of PAM are concentrated. Thus, the COOH-terminal of proNPY cannot override the signals in the PAM membrane anchor.
Mol Endocrinol 1996 Jul
PMID:Processing and routing of a membrane-anchored form of proneuropeptide Y. 881 24

The expression of catecholamine-synthesizing enzymes in the adrenal medulla is upregulated in parallel by stress and pharmacological treatments. In this study we examined whether a neuropeptide and its processing enzyme are regulated in parallel with catecholamine enzyme genes after drug treatment. Because the main effect of stress on the adrenal medulla is via splanchnic nerve stimulation of nicotinic receptors, we used nicotine to stimulate the medulla and visualized expression of catecholamine enzyme genes, the medullary peptide neuropeptide Y (NPY), and the neuropeptide-processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) by in situ hybridization quantified by image analysis of autoradiographic images. Rats received a single injection of nicotine (0, 1, or 5 mg/kg sc). Six hours later, rats were transcardially perfused. Free-floating adrenal gland sections were hybridized with 35S-labeled cDNA probes for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), PAM, and NPY. Nicotine treatment upregulated the expression of TH, PNMT, and NPY genes in a dose-dependent fashion. Small but nonsignificant increases were observed in DBH and PAM mRNA levels. These results suggest that common transcriptional activation mechanisms may upregulate both catecholamine and neuropeptide synthesis in the adrenal medulla after nicotinic stimulation.
J Mol Neurosci 1997 Feb
PMID:Expression of catecholamine-synthesizing enzymes, peptidylglycine alpha-amidating monooxygenase, and neuropeptide Y mRNA in the rat adrenal medulla after acute systemic nicotine. 906 14

We have tried to approach the nature of the last common ancestor to Haemophilus influenzae and Escherichia coli and to determine how each bacterium could have diverged from this putative organism. The approach used was exhaustive analysis of the homologous proteins coded by genes present in these bacteria, using as criteria for sequence relatedness an alignment of at least 80 amino acid residues and a PAM distance (number of accepted point mutations per 100 residues separating two sequences) below 250. Evolutionarily significant similarities were found between 1,345 H. influenzae proteins (85% of the total genome) and 3,058 E. coli. proteins (75% of the total genome), many of them belonging to families of various sizes (from 666 doublets to 35 large groups of more than 10 members). Nearly all the genes found by this approach to be duplicated in both bacteria were already duplicated in their last common ancestor. This was deduced from (1) the comparison of the respective distributions of evolutionary distances between orthologs (genes separated only by speciation events) and paralogs (genes duplicated in the same genome) and (2) the analysis of the phylogenetic trees reconstructed for each family of paralogs containing at least two members belonging to each bacterium. The distributions of the different categories of homologs show a significant loss of paralogous genes in H. influenzae (reduction proportional to the genome size), of many sequences which are still present in one copy in E. coli, and of some entire gene families. Phylogenetic trees also confirmed this recent loss of paralogous genes in H. influenzae. Thus, the genome size of the last common ancestor of these two bacteria would have been close to that of present-day E. coli, and the evolution of H. influenzae toward a parasitic life led to an important decrease in its genome size by some mechanism of streamlining. During this recent evolution, the memory of the gene order present in the last common ancestor has been blurred, but a few short conserved chromosomal fragments can still be detected in present-day E. coli and H. influenzae.
Mol Biol Evol 1998 Jan
PMID:The evolutionary relationships between the two bacteria Escherichia coli and Haemophilus influenzae and their putative last common ancestor. 949 1

Distances between amino acids were derived from the polar requirement measure of amino acid polarity and Benner and co-workers' (1994) 74-100 PAM matrix. These distances were used to examine the average effects of amino acid substitutions due to single-base errors in the standard genetic code and equally degenerate randomized variants of the standard code. Second-position transitions conserved all distances on average, an order of magnitude more than did second-position transversions. In contrast, first-position transitions and transversions were about equally conservative. In comparison with randomized codes, second-position transitions in the standard code significantly conserved mean square differences in polar requirement and mean Benner matrix-based distances, but mean absolute value differences in polar requirement were not significantly conserved. The discrepancy suggests that these commonly used distance measures may be insufficient for strict hypothesis testing without more information. The translational consequences of single-base errors were then examined in different codon contexts, and similarities between these contexts explored with a hierarchical cluster analysis. In one cluster of codon contexts corresponding to the RNY and GNR codons, second-position transversions between C and G and transitions between C and U were most conservative of both polar requirement and the matrix-based distance. In another cluster of codon contexts, second-position transitions between A and G were most conservative. Despite the claims of previous authors to the contrary, it is shown theoretically that the standard code may have been shaped by position-invariant forces such as mutation and base content. These forces may have left heterogeneous signatures in the code because of differences in translational fidelity by codon position. A scenario for the origin of the code is presented wherein selection for error minimization could have occurred multiple times in disjoint parts of the code through a phyletic process of competition between lineages. This process permits error minimization without the disruption of previously useful messages, and does not predict that the code is optimally error-minimizing with respect to modern error. Instead, the code may be a record of genetic process and patterns of mutation before the radiation of modern organisms and organelles.
J Mol Evol 1998 Jul
PMID:On error minimization in a sequential origin of the standard genetic code. 966 91

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated alpha chains into alphaA and alphaD types, which are present in all reptiles except crocodiles, where only alphaA chains are expressed. The occurrence of the alphaD chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of beta chains (betaI and betaII), while only one type of beta chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both alpha and beta sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon + squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate alphaA and alphaD chains and crocodilian beta chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (alphaA) and 3.3 (beta) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate left and right arrow sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved approximately 3.5 (alphaA) or approximately 1.7 times (beta) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times.
J Mol Evol 1998 Oct
PMID:Phylogenetic analysis of reptilian hemoglobins: trees, rates, and divergences. 976 92

alpha-Amidation is essential for the function of many peptides in intercellular communication. This C-terminal modification is mediated in a two-step process by the hydroxylase and lyase activities of the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The first step, catalyzed by peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3), is rate limiting in the process, and therefore subject to regulation. Dexamethasone and disulfiram (tetraethylthiuram disulfide; Antabuse) were used as in vivo treatments to study the regulation of PHM expression and activity in cardiac atrium. Our findings show that both dexamethasone and disulfiram treatment increase the activity of PHM in atrial tissue but that they do so by distinctly different mechanisms. Dexamethasone elevated tissue levels of PAM mRNA and protein concurrently, suggesting that glucocorticoids regulate PAM expression at the level of gene transcription. In contrast, disulfiram treatment, which depletes stores of alpha-amidated peptides, increased the specific activity of PHM without affecting the level of PAM expression. The catalytic efficiency of PHM was enhanced by raising the Vmax of the enzyme. Importantly, this increase in Vmax was retained through purification to homogeneity, indicating that either a covalent modification or a stable conformational change had occurred in the protein. These novel findings demonstrate that the rate-limiting enzyme in the bioactivation of peptide messengers is differentially regulated by transcriptional and post-transcriptional mechanisms in vivo. It is proposed that regulation of PHM's expression and catalytic efficiency serve as coordinated physiologic mechanisms for maintaining appropriate levels of alpha-amidating activity under changing conditions in vivo.
Mol Pharmacol 1999 Jun
PMID:Differential regulation of peptide alpha-amidation by dexamethasone and disulfiram. 1034 50

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyse the alpha-amidation of neuroendocrine peptides. Previous studies have demonstrated that alpha-adrenergic stimulation results in an increase in intracellular volume and protein content of cultured neonatal rat myocardial cells. The present study examined the regulated expression of PAM during alpha-adrenergic stimulation. Alpha1-adrenergic stimulation activates the expression and release of PAM from myocytes. Following phenylephrine treatment, myocardial cells displayed a several fold increase in PAM activity, and a 2-4-fold increase in the steady state levels of PAM mRNA. This effect of alpha-adrenergic stimulation was dependent on the concentration and duration of exposure to the agonist, and displayed alpha1-adrenergic receptor specificity. The transcription rate experiments indicated that these alpha-adrenergic effects were not due to increased PAM gene activity, suggesting that a post-transcriptional mechanism was involved. The most common mechanism of post-transcriptional regulation affects cytoplasmic mRNA stability. Cardiomyocytes cultures from atria and ventricles in the presence of 5,6 dichloro-1-beta ribofuranosyl benzamidazole (DRB) showed that phenylephrine treatment increased the half-life of PAM mRNA from 13 +/- 1 to 21 +/- 1 h in atrial cells and from 8 +/- 1 to 12 +/- 1 h in ventricle cells. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that increased PAM expression after phenylephrine treatment was not due to intranuclear stabilisation of the primary transcript. Protein kinase C inhibitors H7 and GF109203x, completely blocked the phenylephrine stimulated PAM expression. These results suggest that alpha-adrenergic agonist induces PAM mRNA levels by increasing its stability in the cytoplasm. They indicate that PAM gene expression augments through a H7 and GF109203x sensitive pathway, involving the activation of protein kinase C.
Mol Cell Endocrinol 1999 Aug 20
PMID:Alpha1-adrenergic regulation of peptidylglycine alpha-amidating monooxygenase gene expression in cultured rat cardiac myocytes: transcriptional studies and messenger ribonucleic acid stability. 1050 4

The process by which analogs in peptide chemistry are currently designed does not include any quantitative basis for amino acid substitutions from pharmacological leads. Here, we show that substitution matrices such as PAM 250 can provide quantitative constraints compatible with biological activity. This article describes its use in a strategy of rational amino acid substitution in peptides and proteins: we have computed a chemically derived matrix equivalent to the well-known PAM 250 matrix, reflecting the natural mutability rates of amino acids in protein evolutions but that can be extended to all the noncoded amino acids. Some of these noncoded amino acids are widely used to mimic secondary structure, to constrain backbone conformation, or to evade protease degradation. An automated sequence mutation (ASM) strategy has been defined to generate mutations within constraints. Application of such a substitution matrix to quantitative structure-function relationship studies will be of use in the design of proteins and peptides destined to become pharmaceutical drugs. In particular, issues such as which functionally conserved substitutions are able to satisfy conformational restrictions, oral bioavailability, or formulation demands can be quantitatively addressed.
J Mol Graph Model
PMID:Peptides quantitative structure-function relationships: an automated mutation strategy to design peptides and pseudopeptides from substitution matrices. 1084 Jun 89

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.
Cell Mol Life Sci 2000 Aug
PMID:New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. 1102 16

One of the most common mechanisms of posttranslational modifications to generate biologically active (neuro)peptides is the process of peptide alpha-amidation. The only enzyme known to catalyze this important modification is peptidylglycine alpha-amidating monooxygenase (PAM): a (bifunctional) zymogen, giving rise to a monooxygenase (PHM) and a lyase (PAL). The highly peptidergic central nervous system and endocrine system of the marine mollusk Aplysia has homologs of various mammalian peptide processing enzymes, including furin, Afurin2, prohormone convertase 1 (PC1), PC2, carboxypeptidase E (CPE) and CPD. Previously, it has been shown that the abdominal ganglion of Aplysia, which contains approximately 800 peptidergic bag cell neurons, contains the highest specific alpha-amidating activity. We have identified and cloned multiple overlapping central nervous system and bag cell cDNAs that encode a predicted 748-residue protein that is a member of the PAM family. The protein sequence contains the contiguous sequence of the catalytic domains of PHM and PAL, clearly demonstrating the existence of bifunctional Aplysia PAM, the first invertebrate PAM zymogen with an organization similar to that in vertebrates. None of the characterized clones encoded the so-called exon A domain between the PHM and PAL domains. Furthermore, in a specific search by reverse transcription-polymerase chain reaction of RNA from multiple tissues we could only detect exon A-less transcripts. PAM expression was detected in the central nervous system, and in several endocrine and exocrine organs. Aplysia PAM is a candidate prohormone processing enzyme that plays an important role in the processing of Aplysia prohormones in the secretory pathway.
Brain Res Mol Brain Res 2000 Oct 20
PMID:Neuropeptide amidation: cloning of a bifunctional alpha-amidating enzyme from Aplysia. 1104 55


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