Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.     

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.     

Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal rat cardiocytes: a model for studying regulation of PAM expression in the rat heart.

Primary cultures of neonatal rat atrial and ventricular cardiomyocytes were used to investigate the expression of peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme required for the production of alpha-amidated neuroendocrine peptides. The use of assays for the individual enzymes, peptidylglycine alpha-amidating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), demonstrated that the levels of expression observed in vitro approximated those observed in vivo. Both in vivo and in vitro, atrial and ventricular PAL activity greatly exceeded PHM activity. Atrial and ventricular cardiomyocytes secreted PHM and PAL activity at a constant rate throughout the culture period. Immunofluorescence studies localized PAM proteins to the perinuclear region, with intense punctate staining. Both in vivo and in vitro, PAM mRNAs encoding integral membrane proteins predominated throughout the neonatal period, with PAM-1 mRNA becoming more prevalent after the first week in culture. Although PAM-2 mRNA decreased in prevalence in vivo at the time when PAM-1 expression increased, levels of PAM-2 mRNA remained elevated throughout 2 weeks in vitro. Western blot analysis demonstrated intact PAM-1 and PAM-2 proteins in atrial cultures, with the prevalence of PAM-1 increasing in older cultures. Atrial cardiomyocytes secreted only bifunctional PAM proteins. Many of the features of PAM expression, processing, and storage that are unique to cardiomyocytes as opposed to endocrine cells are faithfully replicated by primary atrial and ventricular cultures.     

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.     

Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

Cnidarians are primitive animals that use neuropeptides as their transmitters. All the numerous cnidarian neuropeptides isolated, so far, have a carboxy-terminal amide group that is essential for their actions. This strongly suggests that alpha-amidating enzymes are essential for the functioning of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in mammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and the 3D structure of PHM are conserved between CP1-A, -B and the PHM part of rat PAM. Furthermore, eight introns in CP1 occur in the same positions and have the same intron phasing as eight introns in the rat PAM gene, showing that the sea anemone PHM is not only structurally, but also evolutionarily related to the PHM part of rat PAM.     

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase

We report the purification and characterization of human bifunctional peptidylglycine alpha-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 alpha-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 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L 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 1mM 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.     

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.     

Intermittent hypoxia activates peptidylglycine alpha-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.     

Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

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.     

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.     

The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation

A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine alpha-amidating monooxygenase (PAM)-A and -B to produce alpha-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the 108-kDa PAM precursor protein is cleaved to form a peptidylglycine alpha-hydroxylating monooxygenase and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase, which function sequentially in the 2-step formation of alpha-amidated peptides.     

Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules

About 40-60% of the peptidylglycine alpha-amidating amonooxygenase activity in the lysates of secretory granules from bovine atria and adrenal medulla isolated and lyzed in the presence of pepstatin, phenylmethylsulfonyl gluoride, N-ethylmaleimide and catalase, was found to be in the soluble form. The remaining part bound to the membrane fraction was extracted with Triton X-100. The procedure of purification of the soluble form of peptidylglycine alpha-amidating monooxygenase from both atrial and chromaffin granules in electrophoretically homogeneous enzyme preparations was developed. The enzyme is made up of a single subunit with a molecular mass of 68 kDa and contains one copper atom per molecule. The EPR spectra of peptidylglycine alpha-amidating amonooxygenase and dopamine beta-monooxygenase were found to be practically identical, thus indicating that the copper environment in the both enzymes is the same. Both peptidylglycine alpha-amidating monooxygenase and dopamine beta-monooxygenase are inhibited by the neurocuprein apoform, an extremely acidic protein isolated from brain and secretory granules of different endocrine tissues.     

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.     

Trafficking of a secretory granule membrane protein is sensitive to copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.     

Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium

The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [H?gberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical finding of the amidating enzymes in mouse pancreatic A-, B-, and D-cells: a comparison with human and rat.

alpha-Amidation is catalyzed by two enzymatic activities, peptidyl-glycine alpha-hydroxylating mono-oxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), denoted collectively as peptidyl-glycine alpha-amidating mono-oxygenase (PAM), which also may include transmembrane and cytoplasmic domains. PAM is present in mammalian pancreas, where it appears to be abundant in the perinatal period. Nevertheless, there is no agreement on the cell type(s) that produces PAM or even on its presence in adults. In the present study we found PAM (PHM and cytoplasmic domain) immunoreactivity (IR) in A-, B-, and D-cells of adult mouse pancreas. In contrast to previous reports, PAM IR was found in B-cells of human and rat. Most of the B/D-cells were PAM immunoreactive, although with variable intensity, whereas less than half of A-cells displayed IR. Immunocytochemistry and Western blotting suggested the existence of different PAM molecules. Differences in the cellular distribution of IR for PAM domains were also observed. Whereas PHM-IR was extended throughout the cytoplasm in the three cell types, presumably in the secretory granules, IR for the cytoplasmic domain in A/D-cells was restricted to a juxtanuclear region, perhaps indicating its cleavage in Golgi areas. Although glucagon, insulin, and somatostatin are non-amidated, amidated peptides (glucagon-like peptide 1, adrenomedullin, proadrenomedullin N-terminal 20 peptide) were found in the three cell types.     

In situ hybridization: mRNA levels of secretogranin II,VGF and peptidylglycine alpha-amidating monooxygenase in brain of salt-loaded rats

The mRNA levels of secretogranin II (SgII), VGF and peptidylglycine alpha-amidating monooxygenase (PAM) were studied in brains of salt loaded rats by in situ hybridization. In these rats the levels of the message for secretogranin II and VGF were increased in the paraventricular, supraoptic and retrochiasmatic nuclei and in the subfornical organ. The increases ranged from 416 to 721% for SgII and from 778 to 890% for VGF. The PAM message was also elevated in these brain regions; however, the maximal increase was only 221%. We conclude that the message for all secretory peptides investigated so far, i.e. vasopressin, galanin, secretogranin II and VGF are upregulated to a similar degree in the hypothalamus of salt-located rats. The relative increase in mRNA for the enzyme peptidylglycine alpha-amidating monooxygenase occurred to a much lower extent, and was comparable to the limited changes previously seen for carboxypeptidase H.