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L Ouafik

Publications and source records attributed to L Ouafik.

14 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Expression of peptidylglycine alpha-amidating monooxygenase: an in situ hybridization and immunocytochemical study.

Peptide alpha-amidation, an essential posttranslational modification that confers bioactivity to many neuroendocrine peptides, is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3). To complement our previous studies on the distribution of PAM in neuroendocrine organs, we have examined expression of the PAM gene in several endocrine tissues by in situ hybridization and immunocytochemistry. In all instances, the autoradiographic densities for PAM mRNA correlated with staining patterns for PAM immunoreactivity. Very high levels of PAM mRNA were found in all heart atrial cardiomyocytes, while much lower levels were present in ventricular cells. In the sublingual gland, PAM was expressed diffusely in both acinar and tubule cells. In contrast, expression of PAM was confined to granular convoluted tubule cells in the submaxillary gland. PAM was expressed at high levels in a subset of adrenal medullary chromaffin cells, and low levels of PAM mRNA and immunoreactivity were also detected in the adrenal cortex. PAM was found predominantly in the calcitonin-producing parafollicular C-cells in the thyroid gland and in the glucagon-containing A-cells in the endocrine pancreas. Collecting and distal tubule cells of the kidney expressed both PAM mRNA and immunoreactivity. The basal cells in testicular seminiferous tubules containing PAM may represent developing germ and Sertoli cells. The cellular localization of PAM within the thyroid gland, adrenal gland, testis, and pancreas correlated with known peptidergic systems, and some of the observed cellular heterogeneity in PAM mRNA expression and immunoreactivity may reflect differences in the levels of amidated peptide production. The expression of PAM in cells not known to produce high levels of alpha-amidated peptides may indicate the production of yet unidentified alpha-amidated bioactive peptides or alternative functions of the PAM protein.

Adrenal Medulla↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1990

Immunocytochemical and in situ hybridization studies of peptidylglycine alpha-amidating monooxygenase in pituitary gland.

The pituitary gland contains high levels of peptidylglycine alpha-amidating monooxygenase (PAM) activity and mRNA. Using affinity-purified rabbit polyclonal antisera generated to synthetic PAM peptides and PAM RNA probes, immunocytochemical and in situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM in the adult male rat pituitary gland. PAM immunoreactivity was present at varying levels in nearly all of the anterior pituitary cells; one cell population stained intensely, while others stained moderately or weakly. These results correlated well with in situ hybridization studies that demonstrated high levels of PAM mRNA in a subpopulation comprising approximately 10-15% of the anterior pituitary cells. Based upon immunocytochemistry, intermediate pituitary lobe cells were divided into an intensely stained and a moderately stained group. PAM staining was also present in neural lobe fibers. Immunocytochemical staining of serial pituitary tissue sections for PAM and other pituitary hormones demonstrated that the anterior pituitary cells intensely stained for PAM represented a subpopulation of the gonadotropes. PAM was also identified at moderate levels in corticotropes and at lower levels in sommatotropes and lactotropes. These results suggest that many anterior pituitary cells are capable of producing amidated peptides along with their major peptide hormone.

Adrenocorticotropic Hormone↗
PubMed1990

Thyroid hormone regulation of peptidylglycine alpha-amidating monooxygenase expression in anterior pituitary gland.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a copper-, molecular oxygen-, and ascorbate-dependent enzyme which catalyzes the COOH-terminal amidation of bioactive peptides. Expression of PAM in the adult male rat anterior pituitary was evaluated after experimental manipulation of thyroid status. Levels of PAM mRNA increased 4- to 7-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil or thyroidectomy and were not diminished below control levels in animals made hyperthyroid by treatment with T4. Treatment of thyroidectomized animals with T4 prevented the increase in PAM mRNA levels; similar doses of T4 returned serum TSH and anterior pituitary PAM mRNA to euthyroid values. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by reverse transcription and the polymerase chain reaction, thyroid status did not affect the distribution of PAM mRNA among its various alternatively spliced forms. The specific activity of PAM in the anterior pituitary was increased slightly in both the soluble and particulate fractions from chemically hypothyroid rats; the majority of the PAM activity in the rat anterior pituitary was soluble, and increased secretion of enzyme may account for the lesser effect of chemical thyroidectomy on specific activity compared to mRNA levels. Western blot analysis demonstrated a 104-kDa PAM protein in particulate fractions prepared from control, PTU-treated, and T4-treated animals. The soluble fraction contained major PAM proteins of 95 and 75 kDa, and PTU treatment brought about an increase in the prevalence of the 75-kDa form of PAM protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms.

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.

Aging↗
PubMed1987

Ontogenesis of TRH mRNA in the rat pancreas.

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day 0, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.

Animals↗
PubMed1987

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.

Aging↗
PubMed1986

Evidence for high peptide alpha-amidation activity in the neonatal rat pancreas.

A high peptide alpha-amidating activity is present in a mitochondrial/secretory granules preparation from 3-day old rat pancreas. It is dependent on copper, ascorbate and molecular oxygen. This preparation is able to generate TRH when incubated with Pyroglu-His-Pro-Gly, a sequence present in the TRH precursor molecular. The peptide alpha-amidating activity may be involved in the high rate of TRH biosynthesis in the pancreas during the neonatal period. In the pancreas of adult rats which contain low levels of TRH, the peptide alpha-amidating activity is barely detectable.

Amidohydrolases↗
PubMed1986

[Demonstration of a TRH precursor in the pancreas of the newborn rat].

This study allows the indirect demonstration of a precursor for TRH in pancreatic extracts of 2-days old rats. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a large increase in Pyroglutamyl Histidine Proline (TRH-OH). The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30,000 and 40,000 daltons. During ontogenesis. TRH levels decrease earlier and more rapidly than that of TRH-precursor levels. These data suggest that changes in the processing of TRH-precursor play a role in the diminution of TRH concentrations that is observed during the first two weeks of life.

Animals↗
PubMed1985

TRH and TRH-OH in the pancreas of adult and newborn rats.

TRH and its metabolite TRH-OH have been measured by specific radioimmunoassays in acid extracts of pancreas in adults and developing rats. TRH and TRH-OH immunoreactivity had the same ontogenic pattern with a maximal concentration on day 4 followed by a progressive return towards adult levels on day 20. A significant linear correlation was found between TRH levels and the TRH/TRH-OH ratio. The range of TRH/TRH-OH ratio varied from 136 +/- 1.6, at the peak of concentrations of both peptides, to 18 +/- 3.9 on day 20. Pancreatic TRH and TRH-OH had the same elution pattern as corresponding synthetic peptides both on Biogel P2 and high-pressure liquid chromatography. The origin of TRH-OH as well as its potential function need further investigations.

Age Factors↗
PubMed1985

Evidence for a precursor for TRH in the neonatal rat pancreas.

Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.

Animals↗
PubMedDate not supplied

Peptidylglycine alpha-amidating monooxygenase activity and TRH and CRF biosynthesis. Role of copper.

Carboxy-terminal amidation of biologically active peptides, an important characteristic of more than half of these substances, occurs during the maturation process of peptide precursors. It is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme that is copper-dependent. We show here that alterations of copper stores in cultured cells from different origins (pancreas and hypothalamus) affect the immunoreactivity of thyrotropin-releasing hormone (TRH) and corticotropin-releasing factor (CRF) (two alpha-amidated peptides). This suggests that copper can affect neuropeptide biosynthesis and may play a role in the endocrine or central nervous system function.

Amino Acid Sequence↗