α-Amidated Peptides: Approaches for Analysis.

α-Amidation is a terminal modification in peptide biosynthesis that can itself be rate limiting in the overall production of bioactive α-amidated peptides. More than half of the known neural and endocrine peptides are α-amidated and in most cases this structural feature is essential for receptor recognition, signal transduction, and thus biologic function. This chapter describes methods for developing and using analytical tools to study the biology of α-amidated peptides. The principal analytical method used to quantify α-amidated peptides is the radioimmunoassay (RIA). Detailed protocols are provided for (1) primary antibody production and characterization; (2) radiolabeling of RIA peptides; (3) sample preparation; and (4) performance of the RIA itself. Techniques are also described for the identification and verification of α-amidated peptides. Lastly, in vivo models used for studying the biology of α-amidation are discussed.

In vitro synthesis of arachidonoyl amino acids by cytochrome c.

Arachidonoyl amino acids are a class of endogenous lipid messengers that are expressed in the mammalian central nervous system and peripherally. While several of their prominent pharmacologic effects have been documented, the mechanism by which arachidonoyl amino acids are biosynthesized has not been defined. We have previously observed that the mitochondrial protein, cytochrome c, is capable of catalyzing the formation of the prototypic arachidonoyl amino acid, arachidonoyl glycine, utilizing arachidonoyl CoA and glycine as substrates, in the presence of hydrogen peroxide. Here we report that cytochrome c is similarly able to catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid from arachidonoyl CoA and the respective amino acids. The identities of the arachidonoyl amino acid products were verified by mass spectral fragmentation pattern analysis. The synthetic reactions exhibited Michaelis-Menten kinetics and continued favorably at physiologic temperature and pH. Spectral data indicate that both cytochrome c protein structure and a +3 heme iron oxidation state are required for the reaction mechanism to proceed optimally. Reactions designed to catalyze the formation of N-arachidonoyl dopamine were not efficient due to the rapid oxidation of dopamine substrate by hydrogen peroxide, consuming both reactants. Finally, under standard assay conditions, arachidonoyl CoA and ethanolamine were found to react spontaneously to form anandamide, independent of cytochrome c and hydrogen peroxide. Accordingly, it was not possible to demonstrate a potential role for cytochrome c in the biosynthetic mechanism for either arachidonoyl dopamine or anandamide. However, the ability of cytochrome c to effectively catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid in vitro highlights its potential role for the generation of these lipid messengers in vivo.

Biosynthesis of oleamide.

Oleamide (cis-9-octadecenamide) is the prototype long chain primary fatty acid amide lipid messenger. The natural occurrence of oleamide was first reported in human serum in 1989. Subsequently oleamide was shown to accumulate in the cerebrospinal fluid of sleep-deprived cats and to induce sleep when administered to experimental animals. Accordingly, oleamide first became known for its potential role in the mechanisms that mediate the drive to sleep. Oleamide also has profound effects on thermoregulation and acts as an analgesic in several models of experimental pain. Although these important pharmacologic effects are well establish, the biochemical mechanism for the synthesis of oleamide has not yet been defined. This chapter reviews the biosynthetic pathways that have been proposed and highlights two mechanisms which are most supported by experimental evidence: the generation of oleamide from oleoylglycine by the neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), and alternatively, the direct amidation of oleic acid via oleoyl coenzyme A by cytochrome c using ammonia as the nitrogen source. The latter mechanism is discussed in the context of apoptosis where oleamide may play a role in regulating gap junction communication. Lastly, several considerations and caveats pertinent to the future study oleamide biosynthesis are discussed.

alpha-Amidated peptides: approaches for analysis.

alpha-Amidation is a terminal modification in peptide biosynthesis that can itself be rate-limiting in the overall production of bioactive alpha-amidated peptides. More than half of the known neural and endocrine peptides are alpha-amidated and in most cases, this structural feature is essential for receptor recognition, signal transduction, and thus, biologic function. This chapter describes methods for developing and using analytical tools to study the biology of alpha-amidated peptides. The principle analytical method used to quantify alpha-amidated peptides is the radioimmunoassay (RIA). Detailed protocols are provided for 1) primary antibody production and characterization; 2) radiolabeling of RIA peptides; 3) sample preparation; and 4) the performance of the RIA itself. Techniques are also described for the identification and verification of alpha-amidated peptides. Lastly, in vivo models used for studying the biology of alpha-amidation are discussed.

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine.

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.

In vitro synthesis of oleoylglycine by cytochrome c points to a novel pathway for the production of lipid signaling molecules.

Long chain fatty acyl glycines represent a new class of signaling molecules whose biosynthetic pathway is unknown. Here we report that cytochrome c catalyzes the formation of oleoylglycine from oleoyl-CoA and glycine, in the presence of hydrogen peroxide. The identity of oleoylglycine product was confirmed by isotope labeling and fragmentation mass spectrometry. Synthesis of oleoylglycine by cytochrome c was dependent upon substrate concentration and time. Other heme-containing proteins, myoglobin and hemoglobin, did not catalyze oleoylglycine synthesis. The functional properties of the reaction closely resemble those observed for the ability of cytochrome c to mediate the synthesis of oleamide from oleoyl-CoA and ammonia, in the presence of hydrogen peroxide (Driscoll, W. J., Chaturvedi., S., and Mueller, G. P. (2007) J. Biol. Chem. 282). The ability of cytochrome c to catalyze the formation of oleoylglycine experimentally indicates the potential importance of cytochrome c as a novel mechanism for the generation of long chain fatty acyl glycine messengers in vivo.

Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide.

Oleamide (cis-9-octadecenamide) is a member of an emerging class of lipid-signaling molecules, the primary fatty acid amides. A growing body of evidence indicates that oleamide mediates fundamental neurochemical processes including sleep, thermoregulation, and nociception. Nevertheless, the mechanism for oleamide biosynthesis remains unknown. The leading hypothesis holds that oleamide is synthesized from oleoylglycine via the actions of the peptide amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The present study investigated this hypothesis using pharmacologic treatments, physiologic assessments, and measurements of serum oleamide levels using a newly developed enzyme-linked immunosorbant assay (ELISA). Oleamide and oleoylglycine both induced profound hypothermia and decreased locomotion, over equivalent dose ranges and time courses, whereas, closely related compounds, stearamide and oleic acid, were essentially without effect. While the biologic actions of oleamide and oleoylglycine were equivalent, the two compounds differed dramatically with respect to their effects on serum levels of oleamide. Oleamide administration (80mg/kg) elevated blood-borne oleamide by eight-fold, whereas, the same dose of oleoylglycine had no effect on circulating oleamide levels. In addition, pretreatment with the established PAM inhibitor, disulfiram, produced modest reductions in the hypothermic responses to both oleoylglycine and oleamide, suggesting that the effects of disulfiram were not mediated through inhibition of PAM and a resulting decrease in the formation of oleamide from oleoylglycine. Collectively, these findings raise the possibilities that: (1) oleoylglycine possesses biologic activity that is independent of its conversion to oleamide and (2) the increased availability of oleoylglycine as a potential substrate does not drive the biosynthesis of oleamide.

De novo biosynthetic profiling of high abundance proteins in cystic fibrosis lung epithelial cells.

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Murine atrial HL-1 cells express highly active peptidylglycine alpha-amidating enzyme.

Peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the rate limiting step in peptide alpha-amidation, a posttranslational modification that is essential for receptor recognition and signal transduction. Secretory granules of the cardiac atrium contain the highest natural concentration of PHM and clearly demonstrate regulation of PHM expression and activity. The HL-1 atrial myocyte cell line faithfully maintains the differentiated phenotype of native atrial cells and thus provides an in vitro model system for investigating the mechanisms that regulate PHM. We observed that the specific activity of PHM expressed in HL-1 cells is five times higher than that found in rat atrium. The increased activity of HL-1 cell PHM was not reflected by a difference in Km for peptide substrate, change in copper optimum, altered sensitivity to inactivation by suicide inhibitor or variance in response to limited proteolysis by trypsin. Additionally, mixing experiments indicated that the increased activity in HL-1 cells versus rat atrium was not due to a diffusible factor. Based upon these findings we propose that the increased Vmax of HL-1 cell PHM results from a structural or conformational difference that involves either differential posttranslational modification and/or a high affinity chaperone that serves to regulate enzymatic activity by protein-protein interaction. The mechanism involved may participate in physiologic regulation of PHM.

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 alpha-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.

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

Peptidylglycine-alpha-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 alpha-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.