Bovine intermediate pituitary alpha-amidation enzyme: preliminary characterization

A secretory granule-associated enzymatic activity that converts mono-[125I]-D-Tyr-Val-Gly into mono-[125I]-D-Tyr-Val-NH2 has been studied. The activity is primarily soluble and shows optimal activity at pH 7 to pH 8. Amidation activity was stimulated 9-fold by addition of optimal amounts of copper (3 microM). In the presence of optimal copper, ascorbate stimulated the reaction 7-fold; none of the other reduced or oxidized cofactors tested was as effective. Taking into account the dependence of the reaction on ascorbate and molecular oxygen and the production of glyoxylate [2], it is suggested that the alpha-amidation enzyme is a monooxygenase. Lineweaver Burk plots with D-Tyr-Val-Gly as the varied substrate demonstrated Michelis-Menten type kinetics with the values of Km and Vmax increasing with the addition of ascorbate to the assay. A variety of peptides ending with a COOH-terminal Gly residue act as inhibitors of the reaction. Two synthetic peptides, gamma 2MSH and ACTH(1-14), with carboxyl termini similar to the presumed physiological substrates for the enzyme, act as competitive inhibitors with similar K1 values. It is likely that this secretory granule alpha-amidation activity is involved in the physiological biosynthetic alpha-amidation of a wide range of bioactive peptides.     

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.     

Purification and characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.     

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.     

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.     

Evidence for presence of peptide alpha-amidating activity in pancreatic islets from newborn rats.

The peptide alpha-amidating activity of a homogenate of pancreatic islets from 5-7-day-old rats was investigated, using as substrate a glycine-extended tripeptide (D-Tyr-Val-Gly). The islet homogenates had a marked amidating activity, with a Km of 57 microM, a Vmax. of 185 pmol/h per mg and a pH optimum of 7.0. This activity was dependent on the presence of ascorbic acid (in the reduced form) and Cu2+, the optimum concentrations being 4 mM and 40 microM respectively. On fractionation of the homogenate, the highest specific activity was found in the soluble fraction. Exocrine pancreatic tissue showed very low levels of amidating activity.     

Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation

The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.     

The alpha-amidation of alpha-melanocyte stimulating hormone in intermediate pituitary requires ascorbic acid

Rat intermediate pituitary cells in primary culture display a time-dependent loss of the ability to produce COOH-terminally alpha-amidated alpha MSH (Glembotski, C.C., Eipper, B.A., and Mains, R.E. (1983) J. Biol. Chem. 258, 7299-7304). Instead of des-, mono-, and diacetyl-adrenocorticotropic hormone(ACTH) (1-13)NH2, the cells produce des-, mono-, and diacetyl-ACTH(1-14)OH. Since the pituitary secretory granule-associated alpha-amidation enzyme requires copper and ascorbic acid for optimal activity (Eipper, B.A., Mains, R.E., and Glembotski, C. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148), these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide alpha-amidation ability. When the cultures were supplemented with up to 100 microM cooper (II) there was very little change in the ability to alpha-amidate alpha-melanotropin. Ascorbic acid at concentrations of up to 500 microM resulted in a dramatic increase in the ability of the cells to form the alpha-amidated peptide. Various combinations of ascorbic acid and copper additions indicated that a relatively short exposure (hours) to ascorbic acid produced the maximal response. Ascorbic acid displayed a dose-dependent effect on the alpha-amidation ability with a half-optimal concentration of about 25 microM. Pulse-chase labeling experiments demonstrated the ascorbic acid-dependent conversion of labeled ACTH(1-14)OH-related peptides to ACTH(1-13)NH2-related peptides. These results correlate with the ascorbic acid requirement of the pituitary alpha-amidation enzyme and demonstrate that the direct precursors to ACTH(1-13)NH2-related peptides are ACTH(1-14)OH-related peptides. Combined with our previous data, the present studies support the notion that a wide range of neuro- and endocrine peptides become alpha-amidated in a similar ascorbic acid-dependent manner.     

Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.     

The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.     

Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.     

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.     

Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.

Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.     

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.     

Ability of cofactors to support peptide amidation is cell-type specific

The ability of various cofactors to substitute for ascorbate in the biosynthesis of alpha-aidated peptides from pro-ACTH/endorphin (PAE) was compared in corticotrope tumor cells (AtT-20) and in primary anterior and intermediate pituitary cultures. In all three systems, ascorbate was the most potent cofactor tested. In AtT-20 cells, dopamine, norepinephrine, epinephrine, dehydroascorbate and dihydro- and tetrahydrobiopterin supported significant alpha-amidation of joining peptide [PAE(77-94)NH2]. In contrast, amidation of joining peptide by primary corticotropes was stimulated only slightly by catecholamines and not by tetrahydrobiopterin. Neither catecholamines nor tetrahydrobiopterin stimulated peptide amidation by melanotropes. The ability of cofactors to support the synthesis of alpha-amidated peptides is cell-type specific.     

Expression of a peptide processing enzyme in cultured cells: truncation mutants reveal a routing domain.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme responsible for the alpha-amidation of peptides in secretory granules of neuroendocrine cells. The single gene encoding PAM undergoes tissue-specific alternative splicing and endoproteolytic processing to generate bifunctional membrane proteins with a single transmembrane domain as well as soluble proteins that are mono- or bifunctional. In order to examine the endoproteolytic processing and subcellular localization of the various forms of PAM in cells lacking regulated secretory granules, we established stably transfected hEK-293 cell lines expressing naturally occurring and mutant forms of PAM. As expected, newly synthesized soluble PAM proteins were rapidly secreted into the medium. Integral membrane protein forms of PAM were largely localized in the perinuclear region with punctate staining visible throughout the cell and 2-5% of the enzyme activity detectable on the cell surface. Bifunctional PAM proteins were slowly released into the medium after expression of integral membrane protein forms of PAM. Deletion of 77 amino acids from the COOH-terminus of the integral membrane forms of PAM resulted in a membrane-bound protein which retained both enzymatic activities but accumulated on the cell surface. Rapid internalization of full-length PAM proteins was observed by incubating live cells with antiserum to PAM; deletion of the COOH-terminal domain eliminated the ability of cells to internalize PAM. Thus the cytoplasmic domain of integral membrane PAM contains a routing determinant recognized by cells lacking the regulated secretory pathway.     

Gastrin-amidating enzyme in the porcine pituitary and antrum. Characterization of molecular forms and substrate specificity

As is the case with many other peptide hormones of the brain and intestine, the formation of biologically active gastrin from a glycine-extended processing intermediate occurs via the action of a peptidylglycyl alpha-amidating monooxygenase (PAM). The observation that gastrin exists primarily as unamidated precursors in the pituitary but as amidated gastrin in the antrum prompted this study to examine whether the amidating enzymes in the two organs were different in their characteristics. Amidating activity was quantified by measuring the conversion of glycine-extended tridecagastrin (G13-Gly) to amidated tridecagastrin and glycine-extended hexapancreatic polypeptide (PP6-Gly) to amidated hexapancreatic polypeptide by radio-immunoassay. Two molecular forms of amidating activity were identified in both the porcine antrum and pituitary. The first, PAM-A, had an apparent Mr of 51,000 and a net negative charge at pH 7.0, whereas PAM-B was smaller (Mr approximately 30,000) and had a net positive charge at pH 7.0. Both molecular forms were similar in their cofactor requirements (copper, ascorbic acid, and catalase) and pH optima in the antrum and pituitary. The Km was significantly lower and the Vmax higher for PP6-Gly than for G13-Gly in the pituitary and antrum. These data suggest that although there is no difference between antral and pituitary PAM, the selective affinity of PAM for certain substrates may provide a mechanism for the differential amidation of different hormones within a given tissue or cell.     

Characterization of gastrin amidation in the rat and porcine antrum: comparison with the pituitary

The formation of biologically active gastrin from glycine-extended processing intermediates occurs via the action of a peptide alpha-amidating enzyme. The observation that gastrin exists primarily as unamidated precursors in the pituitary but as amidated gastrin in the antrum prompted these studies to examine whether the amidating enzymes in the two organs were different in their characteristics. Furthermore, the amidating enzyme in the stomach has not previously been characterized in extensive detail. Amidating activity was quantified by measuring the conversion of Tyr-Gly-Trp-Met-Asp-Phe-Gly (glycine-extended hexagastrin) to Tyr-Gly-Trp-Met-Asp-Phe-NH2 (amidated hexagastrin) by radioimmunoassay. The activity of the antral enzyme in both the rat and hog had a similar apparent molecular weight (45,000-60,000), cofactor requirements (copper, ascorbic acid, and catalase), pH optima (5.5-8.5), and Km (12 microM) as the pituitary enzyme. These data suggest that antral and pituitary peptide alpha-amidating enzymes are the same enzyme, thus it is unlikely that differences in amidating enzymes can account for the observed differences in the tissue specific processing of gastrin.     

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.     

Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression

The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.