Alpha-amidating Activity Research Articles

Differential regulation of peptide alpha-amidation by dexamethasone and disulfiram.

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

The source of the oxygen atom in the alpha-hydroxyglycine intermediate of the peptidylglycine alpha-amidating reaction.

Peptidylglycine alpha-amidating activity catalyses the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O2 in the presence of Cu2+. The reaction involves oxidative N-dealkylation within the terminal glycine residue, with retention of the glycine N atom and release of the remainder as glyoxylate. Recent studies by us and others have revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyl alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein catalyse these steps, formation of the carbinolamide and its conversion into alpha-amide and glyoxylate. As for the mechanism of carbinolamide formation, two distinct pathways can be considered: direct mono-oxygenation at the glycine alpha-C atom and dehydrogenation leading to an imine followed by hydration. To draw a distinction between them, we carried out the reaction with D-Tyr-Val-Gly as the substrate either in the H2(18)O-enriched medium or under an atmosphere of 18O2, and isolated the alpha-hydroxylglycine intermediate. The fast-atom-bombardment mass-spectral analysis demonstrated that the hydroxy O atom comes from O2, but not from H2O, indicating that the alpha-hydroxylation should be a monooxygenase reaction.

Distribution and characterization of peptidylglycine alpha-amidating monooxygenase activity in the ovine brain and hypothalamo-pituitary axis.

The production of alpha-amidated peptide hormones from their glycine-extended precursors is catalyzed by the specific enzyme peptidylglycine alpha-amidating monooxygenase (PAM). In the present study, the distribution and subcellular localization of PAM activity in the sheep brain was examined and compared with known sites of amidated peptide synthesis and release. Of the brain regions assayed, the preoptic anterior and medial basal areas of the hypothalamus contained the greatest concentration of amidating activity. Lower concentrations (greater than 3-fold less) were found in the anterior and neurointermediate pituitary, median eminence, cerebral cortex, hippocampus, pons-medulla, and brainstem. Very low amounts of activity were present in the cerebellum and pineal gland. In most tissues tested, PAM activity was 40-75% higher in the membrane-associated fraction than in the soluble fraction. In the hypothalamus, affinity constants were identical for both membrane-associated and soluble fractions, and ranged from 12.3-13.3 microM. Maximal velocity was higher in the membrane fraction (4.7-4.8 pmol/microgram.h) than in the soluble fraction (2.6-2.9 pmol/microgram/h). Levels of amidating activity in hypophysial-portal and jugular plasma were similar and were 20- to 25-fold lower than in hypothalamic extracts. Insulin-induced hypoglycemia did not significantly alter PAM levels in portal or peripheral plasma, suggesting that amidating activity is not released during this stress. These results indicate that the hypothalamus is the richest source of amidating activity in the sheep brain, and suggest that amidation of neurohypophysial and hypothalamic releasing peptides may occur before axonal transport, given the much lower levels in median eminence, neurointermediate pituitary, and portal plasma.

Distribution and characterization of peptidylglycine alpha-amidating monooxygenase activity in the ovine brain and hypothalamo-pituitary axis

Distribution and characterization of peptidylglycine alpha-amidating monooxygenase activity in the ovine brain and hypothalamo-pituitary axis

Effect of cell density on growth hormone release from, cyclic AMP levels in and peptide alpha-amidation activity of primary cultured rat anterior pituitary cells.

To investigate the effect of cell density on rat somatotrophs, dispersed adult rat anterior pituitary cells were plated at several densities (0.6, 1.2, 2.4 and 6.0 x 10(5) cells/cm2) or at two densities (6.07 x 10(5) and 1.21 x 10(5) cells/cm2) as high and low densities respectively. A static incubation system was used to study the release of GH and peptidyl glycine alpha-amidating enzyme (PAM; a component of secretory granules) and the cellular concentration of cyclic AMP (cAMP) in basal and stimulated cells. In addition, a perifusion system was used to characterize the sensitivity to GH-releasing factor (GRF) at both high and low densities. The high density of cells in both the static incubation and perifusion systems caused a low basal secretion rate of GH and PAM. When the cultured cells were stimulated with human GRF (hGRF) in perifusion, GH secretion from cells at high density was markedly higher than that from cells at low density. The cAMP content in the static incubation system showed a similar tendency to that observed for basal and stimulated GH secretion; that is, the basal cAMP content was increased as the cell density decreased. The cellular concentration of cAMP was increased by about threefold by 1 nmol hGFR/l and by more than tenfold by 10 nmol hGRF/l. When the medium from cells cultured at low density was replaced by that from the cells at high density, there was no change in the basal cellular cAMP content of the cells at low density.(ABSTRACT TRUNCATED AT 250 WORDS)

CHARACTERIZATION OF AN α‐AMIDATING ACTIVITY IN A HUMAN PANCREATIC TUMOUR SECRETING VASOACTIVE INTESTINAL PEPTIDE (VIP)

A case of watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocytochemistry, and ultrastructural analysis of tumour sections, is reported. Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic alpha-amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D-Tyr-Val-Gly, the enzyme displayed an optimal activity at pH 7.0, under aerobic conditions and with 35 microM CuSO4 and 3 mM ascorbate as co-factors. The Kmax and Vmax values of the enzymatic activity were 133.7 microM and 26.9 pmol/h/micrograms protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the alpha-amidating activity in such tumours is discussed.

Evidence for presence of peptide α-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.

Purification and Characterization of a Peptide C-Terminal α-Amidating Enzyme from Porcine Atrium1

In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. Here we report the purification and characterization of an alpha-amidating enzyme from porcine atrium, in which a high concentration of alpha-amidating activity was detected. The enzyme was purified to homogeneity from the membrane fraction of porcine atria by five steps of chromatography, including an affinity chromatography using a Sepharose column coupled with a substrate, Tyr-Phe-Gly. The purified enzyme was found to be composed of a single polypeptide chain with an apparent molecular weight of 92,000. This enzyme converted several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides.

Thyrotropin releasing hormone in the pancreas of newborn rats from streptozotocin — Treated mothers

The effect of maternal diabetes (induced by i.p.injections of 40–50 mg/kg BW Streptozotocin on the day of mating) on TRH in the pancreas of newborn rats was studied. Determination of peptide alpha amidation activity and TRH precursor level on the day of birth revealed decreased biosynthesis of TRH resulting in profoundly (10 times) lower pancreatic TRH and TRH-OH concentrations in pups of diabetic rats. Pancreatic His-Pro-diketopiperazine (His-Pro-DKP) remained unaffected by maternal diabetes. The depression of pancreatic TRH was less profound 24 h later, and even elevated TRH was measured in the pancreas of pups of diabetic mothers on postnatal day 5. Short term postnatal starvation or nursing of intact pups by the diabetic foster mother did not affect pancreatic TRH. It could be postulated that postnatal TRH development in the rat pancreas is retarded by maternal diabetes, while His-Pro-DKP remains unaltered.

Thyroid hormone modulation of TRH precursor levels in rat hypothalamus, pituitary, thyroid and blood

In the present study we have examined the in vivo effects of thyroid hormones and TRH on tissue and blood levels of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Using specific radioimmunoassays (RIAs), we measured TRH immunoreactivity (TRH-IR) and TRH-Gly-IR concentrations in blood, hypothalamus, anterior and posterior pituitary, and thyroid in euthyroid, hypothyroid and thyroxine (T4)-treated 250 g male Sprague-Dawley rats. TRH-Gly-IR and TRH-IR were detected in all of these tissues. Highly significant positive correlations between whole blood TRH-Gly-IR levels and the corresponding serum TSH values (p less than 0.01), whole blood TRH-IR versus serum TSH (p less than 0.01) and whole blood TRH-Gly-IR versus whole blood TRH-IR (p less than 0.01) are consistent with cosecretion of TRH and TRH precursor peptides into the circulation. Euthyroid rats injected with TRH IP (1 microgram/100 g b.wt.) and hypothyroid rats had 4-fold higher whole blood TRH-Gly-IR levels compared to euthyroid controls (p less than 0.0005). Injection of TRH into euthyroid rats significantly increased the TRH-Gly-IR concentration in the hypothalamus, anterior and posterior pituitary and thyroid. The increase in blood TRH-Gly-IR following intravenous TRH may be due, in part, to partial saturation of TRH-degrading enzymes in blood and cell membranes. The ratio of TRH-Gly to TRH was significantly increased in the anterior pituitary by hypothyroidism and TRH injection, suggesting that thyroid hormones and TRH regulate the alpha-amidation of TRH-Gly to form TRH in this tissue. TRH-Gly levels of pooled pituitary and thyroid extracts quantitated by a combination of TRH-Gly RIA and high performance liquid chromatography (HPLC) revealed several-fold increases following incubation at 60 degrees C. Heating at this temperature may block the alpha-amidation activity in extra-hypothalamic tissues but not the "trypsin-like" enzymes which cleave prepro-TRH into TRH-Gly-immunoreactive peptides.

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-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.

Alzheimer's disease: low levels of peptide alpha-amidation activity in brain and CSF.

Carboxyl terminal alpha-amidation confers biologic activity to many neuropeptides. Levels of alpha-amidating activity, peptidyl-glycine alpha-amidating monooxygenase (PAM), were reduced in the CSF of patients with dementia of the Alzheimer type (DAT) compared with healthy, age-matched controls. Repeat lumbar puncture data revealed a decline in CSF PAM activity of approximately 16% per year in DAT patients. Of the cerebral cortical regions examined, only the temporal pole showed reduced PAM activity in patients with Alzheimer's disease (AD) compared with controls. These studies may indicate selective dysfunction of neurons which normally synthesize biologically active, alpha-amidated peptides in the CNS of AD patients.

Evidence for high peptide alpha-amidating activity in the pancrease from neonatal rats.

A high peptidylglycine alpha-amidating mono-oxygenase (PAMase) activity has been measured in the pancreas of neonatal rats. A significant fraction of this activity is contained in the beta cells of the islets of Langerhans and is colocalized with thyrotropin-releasing hormone (TRH) and its precursor in secretory granules. The ontogenetic variation of PAMase activity in the pancrease parallels that of TRH concentrations, suggesting that this enzymatic activity is directly related to TRH biosynthesis. In addition, PAMase activity is able to generate TRH when incubated with less than Glu-His-Pro-Gly, a tetrapeptide present as a repetitive sequence in the TRH precursor. The perinatal evolution of the TRH precursor levels in the pancreas is similar to that of PAMase activity (unpublished results). Thus, the neonatal rat pancreas offers an endocrine model in which the levels of a neuropeptide precursor and an enzyme activity, involved in the posttranslational modification of this precursor, are similarly regulated. Our results suggest also that a fraction of PAMase activity may be produced outside of the beta cells and related to the biosynthesis of COOH-terminally amidated peptide(s) other than TRH. The ontogenetic changes in PAMase activity imply that the synthesis of this peptide(s) is high during the neonatal period, decreasing thereafter.

Tissue distribution and characterization of peptide C-terminal alpha-amidating activity in rat.

The C-terminal alpha-amide formation of peptides is one of the most important events in prohormone processing. Recently, we developed a simple and sensitive assay for detecting alpha-amidating activity in tissues by using (125I)-Ac-Tyr-Phe-Gly as a substrate. Using this assay method, we have determined the tissue distribution of alpha-amidating enzyme activity in adult male rat. High concentrations of alpha-amidating activity were found in pituitary, brain, thyroid, gastrointestinal tract, pancreas, heart, submaxillary glands and parotid glands. Alpha-amidating enzyme activities in all tissues examined exhibit very similar copper and ascorbate requirements, pH dependence, and behavior on gel-filtration.

Characterization of a peptide alpha-amidation activity in human plasma and tissues

Peptidyl glycine α-amidation activity has been detected in human plasma and in several human tissues known to synthesize biologically active α-amidated peptides. Activity was monitored by measuring conversion of mono-[ 125I]-D-Tyr-Val-Gly into mono-[ 125I]-D-Tyr-Val-NH 2. The plasma α-amidation activity is dependent on molecular oxygen, copper, and ascorbic acid and appears to recognize a variety of peptide substrates which contain carboxyl terminal glycine residues. Kinetic analyses demonstrated Michaelis-Menten kinetics with a Km of 14 μmol/L for D-Tyr-Val-Gly. Based on gel filtration, the apparent molecular weight of the peptidyl glycine α-amidation activity in human serum is 60,000. The level of peptidyl glycine α-amidation activity in adult plasma (N = 17) was 106 ± 3 pmol/mL/h (Mean ± SEM) with no difference between male and female subjects (range 84 to 126 pmol/mL/h). In subjects under 15 years old (N = 10), mean plasma activity was 128 ± 10 pmol/mL/h, higher than values for adult control plasma ( P < .05). In serum from hypothyroid adults (N = 13), mean serum activity was 141 ± 11 pmol/mL/hr, higher than euthyroid controls ( P < .025). The most striking elevations in α-amidation activity occurred in plasma from patients with peptide-secreting tumors. Patients with medullary thyroid carcinoma (N = 19) had a mean plasma peptidyl glycine α-amidation activity of 142 ± 52 pmol/mL/h (range 84 to 435 pmol/mL/h). The level of plasma α-amidation activity in one patient with metastatic carcinoid tumor was 560 pmol/mL/h. Peptidyl glycine α-amidation activity was detected in extracts of pituitary and central nervous system tissue, but was not detected in significant amount in extracts of various peripheral tissues. Extracts of peptide-secreting tumors also contained peptidyl glycine α-amidation activity. Human peptidyl glycine α-amidation activity may be co-secreted from tissue along with amidated peptide hormones and serve as a useful marker for certain endocrine tumors.

Characterization of peptide alpha-amidation activity in human cerebrospinal fluid and central nervous system tissue.

Peptidyl-glycine alpha-amidation activity has been detected in human cerebrospinal fluid (CSF) and in several regions of the central nervous system. Activity was monitored by measuring conversion of mono-125I-D-Tyr-Val-Gly into mono-125I-D-Tyr-Val-NH2. The alpha-amidation activity in CSF is dependent on molecular oxygen, copper ions and ascorbic acid and appears to recognize a variety of peptide substrates which contain carboxyl terminal glycine residues. Kinetic analyses demonstrated Michaelis-Menten kinetics with a Km of 4.6 microM for D-Tyr-Val-Gly. The level of peptidyl-glycine alpha-amidation activity in 14 samples of CSF averaged 43 +/- 5 pmol/ml/h (mean +/- SEM; range 11-85 pmol/ml/h) or 1.9 +/- 0.2 pmol/Mg protein/h. No difference was noted between samples from male and female subjects. Extracts of central nervous system tissue contained alpha-amidation activity. The highest levels of enzyme activity were found in the hypothalamus with lower levels in the neurohypophysis and the cerebral cortex. Still lower but detectable activity was found in the cerebellum and pons. Human peptidyl-glycine alph-amidation activity is found in central nervous system tissues known to synthesize alpha-amidated neuropeptides and may be secreted from these tissues along with alpha-amidated peptides into CSF.

Hypothalamic peptidyl-glycine alpha-amidating monooxygenase: preliminary characterization

An enzymatic activity capable of converting mono-[125I]-D-Tyr-Val-Gly into mono-[125I]-D-Tyr-Val-NH2 was identified in a crude mitochondrial/synaptosomal preparation from rat hypothalamus. Further subcellular fractionation studies localized a majority of this enzymatic activity to fractions enriched in synaptic vesicles. The alpha-amidation activity demonstrated optimal activity at pH 7.5 to 8, was stimulated by the presence of copper ions and reduced ascorbate and required the presence of molecular oxygen. Endogenous alpha-amidation activity was inhibited by the addition of ascorbate oxidase. Kinetic analyses demonstrated Michaelis-Menten type kinetics for D-Tyr-Val-Gly as the varied substrate with the values of Km and Vmax increasing as the ascorbate concentration in the reaction increased. A variety of peptides possessing carboxyl-terminal glycine residues were potent inhibitors of the reaction, while peptides lacking a carboxyl-terminal glycine residue were not, suggesting that many glycine-extended peptides may serve as substrates in the alpha-amidation reaction. The characteristics of hypothalamic alpha-amidation activity are similar to those previously reported for the alpha-amidation activity in rat pituitary and mouse corticotropic tumor cells suggesting the presence of closely related enzymes in these tissues.

Characterization of a peptide alpha-amidation activity from rat anterior pituitary.

A secretory granule associated enzymatic activity from the rat anterior pituitary that can convert the synthetic peptide D-Tyr-Val-Gly into D-Tyr-Val-NH2 is described. Due to the presence of inhibitory activity in the cytosol and rough endoplasmic reticulum/Golgi apparatus fractions, the alpha-amidation activity is barely detectable in crude rat anterior pituitary homogenates. The alpha-amidation activity was primarily soluble, displayed a pH optimum of about 7.0, and showed a requirement for molecular oxygen. The activity was stimulated 7-fold by the addition of optimal concentrations of copper. Of the cofactors tested, only reduced ascorbate produced a severalfold stimulation of activity. Studies with varied D-Tyr-Val-Gly or varied monoiodo-D-Tyr-Val-Gly demonstrate Michaelis Menten kinetics with indistinguishable kinetic constants for both peptides. Upon addition of ascorbate, the apparent Km and Vmax for the synthetic substrate, as estimated from Lineweaver-Burk and Eadie-Hofstee plots, increased by 30- and 60-fold, respectively. Several alpha-melanotropin- and gamma-melanotropin-related peptides with COOH-terminal glycine residues were effective competitive inhibitors of the reaction while the corresponding alpha-amidated peptides were very poor inhibitors. The rat anterior pituitary alpha-amidation activity appears to be very similar to the alpha-amidation activity in rat intermediate and neural pituitary, and mouse anterior pituitary tumor cells.

Peptide alpha-amidation activity in mouse anterior pituitary AtT-20 cell granules: properties and secretion.

Mouse anterior pituitary corticotropic tumor cells (AtT-20/D-16v) were homogenized and subjected to subcellular fractionation. A secretory granule associated enzyme activity was detected which could convert D-Tyr-Val-Gly into D-Tyr-Val-NH2. The enzyme activity was dependent on the presence of molecular oxygen, copper ions, and reduced ascorbate. Potent endogenous inhibitors of the enzyme obscured the activity unless appropriate levels of copper ions were added. The production of radiolabeled D-Tyr-Val-NH2 from labeled D-Tyr-Val-Gly was inhibited by a wide variety of peptides possessing COOH-terminal glycine residues but not by a number of other peptides, suggesting that many peptides with COOH-terminal glycine residues can function as substrates in the alpha-amidation reaction. Kinetic studies with varied concentrations of D-Tyr-Val-Gly demonstrated Michaelis-Menten kinetics; both the Km for D-Tyr-Val-Gly and maximum velocity (Vmax) increased upon addition of ascorbate to the reaction. Under optimized assay conditions, the secretory granule pool contains enough alpha-amidation activity to alpha-amidate all the relevant peptides in granules in a small fraction of the total time required for complete biosynthetic processing. Secretion of alpha-amidation activity was stimulated along with secretion of pro-ACTH/endorphin-derived peptides upon addition of synthetic corticotropin releasing factor or 8-bromo-cAMP.