Peptidylglycine Α-amidating Monooxygenase Research Articles

Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine α-amidating monooxygenase (PAM)

Peptidyglycine α-amidating monooxygenase is a copper- and zinc-dependent, bifunctional enzyme that catalyzes the cleavage of glycine-extended peptides or N-acylglycines to the corresponding amides and glyoxylate. This reaction is a key step in the biosynthesis of bioactive α-amidated peptides and, perhaps, the primary fatty acids amides also. Two clinically useful N-acylglycines are thiorphan and tiopronin, each with a thiol moiety attached to the acyl group. We report here that thiorphan and tiopronin are substrates for PAM, exhibiting relatively low K M,app and V MAX,app values. The low V MAX,app values result, most likely, from a decrease in active PAM · 2Cu(II) as the enzyme competes ineffectively with thiorphan and tiopronin for free copper.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine α-amidating monooxygenase

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding α-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine α-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a ( V/ K) amidation that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.

Molecular profile of androgen-independent prostate cancer xenograft LuCaP 23.1

After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9 ± 76.9 mm 3) and 63 slow growing (SG) tumors (229.6 ± 18.4 mm 3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5α-reductase 2 (5α-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine α-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.

PACAP, a New Target for Bipolar Disorder

Lithium is an effective treatment for bipolar disorder, a psychiatric mood disorder. Glycogen synthase kinase 3 (GSK3) and at least two inositol phosphatases (inositol monophosphatase and inositol polyphosphate 1-phosphatase) are inhibited by lithium at therapeutic concentrations. Brandish et al. examined the gene regulatory changes that occurred in rat brain cortical slices in response to inositol depletion. Inositol depletion, resulting from lithium's inhibition of the inositol phosphatases, which decreases recycling of these sugars into inositol lipids that are involved in signaling pathways, is believed to be one mechanism of action for lithium's beneficial effects. Microarray analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of rat brain cortical slices were performed on samples treated with lithium alone, lithium plus the muscarinic acetylcholine receptor agonist carbachol (to stimulate activity and thus deplete inositol), and lithium plus carbachol plus inositol (to provide an inositol replete sample). Genes that were regulated in the presence of carbachol and lithium and for which the regulation was reversed by the addition of inositol were counted as positive. Genes encoding proteins that had not previously been implicated in bipolar disorder and that may point to new therapeutic directions were highlighted. These included the gene encoding orphan G protein-coupled receptor (GPCR) GPR88 and the genes encoding pituitary adenylate cyclase activating polypeptide (PACAP) and one of the biosynthetic enzymes involved in PACAP processing, peptidylglycine α-amidating monooxygenase (PAM). PACAP is particularly interesting for several reasons: The gene is located near the bipolar disorder risk locus on chromosome 18. PACAP signaling regulates tyrosine hydroxylase abundance, thereby regulating dopaminergic activity. And finally, PACAP knockout mice exhibit altered behaviors consistent with a role for PACAP in circadian behavior and alterations in anxiety and activity levels. Consistent with the connection to dopaminergic signaling, Brandish et al. found that the gene encoding an enzyme involved in synthesis of a cofactor for tyrosine hydroxylase was also increased in response to inositol depletion. This analysis suggests that PACAP may be a target for development of new treatments for bipolar disorder. P. E. Brandish, M. Su, D. J. Holder, P. Hodor, J. Szumiloski, R. R. Kleinhanz, J. E. Forbes, M. E. McWhorter, S. J. Duenwald, M. L. Parrish, S. Na, Y. Liu, R. L. Phillips, J. J. Renger, S. Sankaranarayanan, A. J. Simon, E. M. Scolnick, Regulation of gene expression by lithium and depletion of inositol in slices of adult rat cortex. Neuron 45 , 861-872 (2005). [Online Journal]

Peptidylglycine-α-amidating monooxygenase activity and protein are lower in copper-deficient rats and suckling copper-deficient mice

Peptidylglycine-α-amidating monooxygenase (PAM) is a copper-dependent enzyme involved in peptide posttranslational activation. Dietary Cu deficiency (Cu−) was studied to determine if lower PAM activity was due to reduction in protein or cofactor limitation. PAM activity was lower in cardiac atria of Cu− rats than Cu-adequate (Cu+) rats and there was a 50% equivalent reduction in PAM protein. No reduction in Cu− rat midbrain PAM protein was detected although PAM activity was reduced 40%. In 12-day-old (P12) mouse pups derived from dams that began Cu deficiency on day 7 of gestation, there was a parallel reduction in brain PAM activity and protein of 40–50%. PAM mRNA levels assessed in atria and brains from Cu+ and Cu− rats and mice were not altered by dietary treatment, suggesting a posttranscriptional mechanism for lower PAM protein when Cu is limiting in the cell, perhaps due to enhanced apoprotein turnover.

Peptidyl-Glycine α-Amidating Monooxygenase Targeting and Shaping of Atrial Secretory Vesicles

ANP (atrial natriuretic peptide) is widely recognized as an important vasorelaxant, diuretic, and cardioprotective hormone. Little is known, however, about how ANP-secretory vesicles form within the atrial myocytes. Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing proANP-enhanced green fluorescent protein (EGFP), or N-terminal-mutated fusion proteins thought to suppress the calcium-dependent aggregation of proANP. Results showed the following: (1) aggregates of proANP and coexpressed proANP-EGFP recruited peptidylglycine alpha-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal-mutated (Glu23,24-->Gln23,24) and N-terminal-deleted proANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 mumol/L), a pharmacological inhibitor of the lumenal peptidylglycine alpha-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP-PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking. These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal proANP and on the lumenal domain of PAM-1. Conversely, PAM-1 participates in shaping the proANP-secretory vesicles. The full text of this article is available online at http://circres.ahajournals.org.

A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana

The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana. All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys 18-(Xaa) 4-Lys-Cys 24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa) 4-Ile-(Xaa) 2-Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys·NH 2] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally α-amidated residue. It is suggested, as one possibility, that the Cys 18 in the brevinin-1 consensus sequence has been deleted and the Cys 24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine α-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA 10KNLLAAGLDK 20LKCKISGTQC 30), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.

Investigation of the pathway for inter-copper electron transfer in peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase catalyzes the oxidative cleavage of glycine extended peptides at their terminus. In the course of the reaction, there is a requisite long-range electron transfer between the two copper centers (CuH and CuM) located in the hydroxylating domain. This communication presents data that argue against the participation of the extended peptide backbone of substrate in the long-range electron transfer. We propose that electron transfer occurs via the bulk solvent that separates CuH from CuM.

Cdk5 and Trio modulate endocrine cell exocytosis.

Hormone secretion by pituitary cells is decreased by roscovitine, an inhibitor of cyclin-dependent kinase 5 (Cdk5). Roscovitine treatment reorganizes cortical actin and ultrastructural analysis demonstrates that roscovitine limits the ability of secretory granules to approach the plasma membrane or one another. Trio, a multifunctional RhoGEF expressed in pituitary cells, interacts with peptidylglycine alpha-amidating monooxygenase, a secretory granule membrane protein known to affect the actin cytoskeleton. Roscovitine inhibits the ability of Trio to activate Rac, and peptides corresponding to the Cdk5 consensus sites in Trio are phosphorylated by Cdk5. Together, these data suggest that control of the cortical actin cytoskeleton, long known to modulate hormone exocytosis and subsequent endocytosis, involves Cdk5-mediated activation of Trio.

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine α-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.

An enzyme-coupled assay for glyoxylic acid

Herein, we present a new enzyme-linked spectrophotometric assay for glyoxylate that detects glyoxylate via the formation of an intensely colored formazan. This glyoxylate-specific assay is reliant upon the enzymatic conversion of glyoxylate to oxaloacetate coupled to the reduction of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide (NADH). The NADH-dependent reduction of a tetrazolium to the formazan enables the measurement of nanomole quantities of glyoxylate in an assay that is amenable to high-throughput screening methods. Assay validation was accomplished using two methods for glyoxylate generation, the base-catalyzed N-dealkylation of α-hydroxyhippurate to benzamide and glyoxylate and the oxidative cleavage of the glycyl C α-N bond in N-benzoylglycine (hippurate) by peptidylglycine α-amidating monooxygenase to again yield benzamide and glyoxylate. For both reactions, analysis of benzamide produced by reverse-phase high-performance liquid chromatography compared with glyoxylate measured using our glyoxylate assay showed a 1:1 molar ratio of benzamide to glyoxylate. These results indicate that the enzyme-linked spectrophotometric assay can quantitatively measure submicromole quantities of glyoxylate.

Androgen-independent expression of adrenomedullin and peptidylglycine alpha-amidating monooxygenase in human prostatic carcinoma.

Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues.

Peptidylglycine-α-amidating monooxygenase and pro-atrial natriuretic peptide constitute the major membrane-associated proteins of rat atrial secretory granules

Peptidylglycine-α-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of α-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1–126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99–126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.

Expression and characterization of human bifunctional peptidylglycine α-amidating monooxygenase

We report the purification and characterization of human bifunctional peptidylglycine α-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu , we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3 mg of the purified PAM, which showed a single band at 91 kDa on SDS–PAGE, from 1 L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn 2+ and 1.25 mol of Cu 2+ per mol of bifunctional PAM. Further, the addition of 1 mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn 2+. In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.

Glutathione, S-substituted glutathiones, and leukotriene C 4 as substrates for peptidylglycine α-amidating monooxygenase

The C-terminal α-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine α-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C 4 results in the consumption of O 2 and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal α-hydroxyglycine-extended peptide. Amidated glutathione (γGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/ K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest ( V/ K) app values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest ( V/ K) app of the substrates described in this report.

Adrenomedullin stimulates proliferation and inhibits apoptosis of immature rat thymocytes cultured in vitro

Adrenomedullin (AM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)AM, and acts through two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as either a calcitonin gene-related peptide (CGRP) receptor or a selective AM receptor depending on which member of a family of receptor-activity-modifying proteins (RAMPs) is expressed: RAMP1 generates CGRP receptors, while RAMP2 and RAMP3 produce AM receptors. Reverse transcription (RT)–polymerase chain reaction (PCR) consistently allowed the detection of pAM and peptidyl-glycine α-amidating monooxygenase (the enzyme converting immature AM to the mature peptide) mRNAs in the thymus cortex of immature (10-day-old) rats. Accordingly, radioimmune assay (RIA) measured low but sizeable AM concentrations in this tissue. RT–PCR also demonstrated the presence of the specific mRNAs of L1-R, CRLR and RAMPs. AM (from 10 −9 to 10 −7 M) increased proliferation index and lowered apoptotic index of cultured immature rat thymocytes, and the effects were annulled by the AM receptor antagonist AM(22–52). In conclusion, our study demonstrated that (1) immature rat thymus cortex expresses AM and the AM receptors L1-R and CRLR/RAMP; and (2) AM, acting via AM(22–52)-sensitive receptors, exerts a potent growth promoting effect on immature rat thymus, by enhancing proliferation and lowering apoptotic death of thymocytes. Taken together, these findings could suggest that AM may play a role in the development of immunity.

Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase.

Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.

Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli

The export of heterologous products into the conditioned medium of an Escherichia coli culture offers the advantages of a higher product yield, an increased probability of recovering an intact recombinant protein, proper folding for biological activity, and greater stability of a secreted product. In this report, we describe the development of an optimized direct expression system, designed to maximize the extracellular accumulation of recombinant glycine-extended salmon calcitonin peptide (sCTgly). We have used dual promoters, an ompA signal sequence, co-expression of homologous secretion factor genes, and multiple gene cartridges to express the sCTgly. High-density fermentation conditions have been developed that allow for the selective secretion and accumulation of the expressed sCTgly at very high levels. Purification and in vitro enzymatic conversion by peptidylglycine α-amidating monooxygenase yields authentic, biologically active salmon calcitonin. This recombinant production technology is applicable to a variety of amidated peptide hormones.