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B A Eipper

Publications and source records attributed to B A Eipper.

At least 73 records · Page 4Linked to original sources
PubMed1994

Purification and characterization of PAM-1, an integral membrane protein involved in peptide processing.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the two-step alpha-amidation of peptidylglycine intermediates. PAM-1, a Type I integral membrane protein, was solubilized from the membranes of stably transfected hEK-293 cells and purified to homogeneity by antibody affinity chromatography. Purified PAM-1 exhibits an acidic pH optimum and a lower maximal velocity than soluble bifunctional PAM. Limited tryptic digestion of this integral membrane protein releases monofunctional peptidylglycine alpha-hydroxylating monooxygenase, increasing its specific activity almost fourfold and shifting its pH optimum to coincide with the pH optimum of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.

Animals↗
PubMed1994

Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neuro-endocrine peptides through the sequential action of monooxygenase and lyase domains contained within this bifunctional protein. Alternative splicing leads to the expression of soluble and integral membrane bifunctional PAM proteins as well as a soluble monofunctional monooxygenase. In order to determine how alternative splicing affects post-translational modification of PAM proteins, we investigated the sulfation of PAM proteins expressed in stably transfected hEK-293 cells. Metabolic labeling with [35S]SO4(2-) or [35S]methionine and immunoprecipitation demonstrated that [35S]SO4(2-) was efficiently incorporated into PAM proteins that have the noncatalytic exon A region following the monooxygenase domain (PAM-1 and PAM-4) and into a soluble bifunctional PAM protein (PAM-3). Alkaline hydrolysis, radiosequencing, and deglycosylation experiments demonstrated the presence of a sulfated tyrosine (Tyr965) in the COOH-terminal domain of PAM-3 and multiple sulfated O-glycans in the exon A region of PAM-1 and PAM-4. A mutant PAM-3 protein in which Tyr965 was changed to Ala965 (PAM-3/Y965A) was not sulfated and exhibited monooxygenase and lyase activities similar to those of wild type PAM-3. Pulse-chase and temperature block experiments showed that the PAM-3/Y965A protein exits the trans-Golgi network faster than wild type PAM-3. Thus inclusion of exon A results in the sulfation of O-glycans, while elimination of the transmembrane domain results in the sulfation of Tyr965.

Alternative Splicing↗
PubMed1994

RESP18, a novel endocrine secretory protein transcript, and four other transcripts are regulated in parallel with pro-opiomelanocortin in melanotropes.

The homogeneous nature of the rat intermediate pituitary makes it a powerful model system in which to study peptide hormone secretion. Adult male rats were treated with bromocriptine, a dopamine agonist, or haloperidol, a dopamine antagonist, for 3 weeks. In cDNA libraries prepared from the neurointermediate pituitaries of these rats, pro-opiomelanocortin (POMC) expression exhibited the expected decrease in response to bromocriptine, and increase in response to haloperidol. We report the identification of six transcripts that are coregulated with POMC in the intermediate pituitary by these dopaminergic agents. In addition to demonstrating parallel dopamine-regulated expression of carboxy-peptidase E, chromogranin B, binding protein/glucose-regulated protein, and tenascin, two novel regulated transcripts are described. The expression of one of these novel transcripts, RESP18, is limited to neural and endocrine tissue. The RESP18 transcript is approximately 800 nucleotides in length; its cognate translation product is 20 +/- 1 kDa, contains a putative signal sequence, and has many characteristics of a secreted protein. Cell-free translation experiments in the presence of microsomal membranes demonstrate that the 20 +/- 1-kDa RESP18 protein is cleaved to an 18 +/- 1-kDa protein and sequestered within the lumen of the rough endoplasmic reticulum. Tissue in situ hybridization analysis shows that RESP18 mRNA is highly expressed in both the intermediate and anterior pituitary, as well as in the paraventricular and supraoptic nuclei of the hypothalamus.

Amino Acid Sequence↗
PubMed1994

Differential trafficking of soluble and integral membrane secretory granule-associated proteins.

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Amino Acid Sequence↗
PubMed1994

Regulated endocrine-specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein.

Regulated endocrine-specific protein-18 (RESP18) is an 18-kilodalton endocrine-specific transcript whose expression is regulated by a number of different physiological and pharmacological stimuli in different tissues. RESP18 messenger RNA was identified in all cell types in the anterior pituitary, at levels that varied 2-fold from the lowest (corticotropes and thyrotropes) to the highest (gonadotropes, somatotropes, and mammotropes); the melanotropes of the intermediate pituitary have levels of RESP18 messenger RNA comparable to the highest levels in cells in the anterior pituitary. Mouse RESP18 was cloned and used as the basis for biosynthetic studies on RESP18 in AtT-20 cells, which express RESP18 endogenously; mouse RESP18 was highly homologous to rat RESP18. Pulse-chase biosynthetic labeling studies showed that AtT-20 cells expressed much less RESP18 than the endogenous prohormone, POMC, but that glucocorticoid treatment lowered POMC and raised RESP18 biosynthetic rates so that they were nearly equimolar. Surprisingly, RESP18 was not processed to smaller peptides to any significant extent, nor was RESP18 or any smaller peptide secreted. Newly synthesized RESP18 normally disappeared from AtT-20 cell extracts with a half-life of less than 15 min; the intracellular half-life of RESP18 was increased strikingly after glucocorticoid treatment of the cells. Upon subcellular fractionation, RESP18 was found to be entirely particulate and to cofractionate with markers for the endoplasmic reticulum, rather than with markers for secretory granules, such as POMC and prohormone-processing enzymes. Therefore, RESP18 is a major glucocorticoid-responsive protein in the secretory pathway of corticotropes, but its function may be entirely within the neuroendocrine cell.

Amino Acid Sequence↗
PubMed1994

High levels of expression of the tumor suppressor gene APC during development of the rat central nervous system.

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is mutated in human familial adenomatous polyposis, an autosomal dominant condition with predisposition to colorectal carcinoma and brain tumors. Although tumor suppressor genes appear to play a general role in regulating cellular proliferation, the normal biological function of the APC gene product is unknown. In the present study, we cloned fragments of the rat homolog of the APC gene and examined its tissue distribution by Northern blot analysis. These studies demonstrated particularly high levels of APC mRNA in brain. To gain clues to the role of the APC gene in brain function, we examined the neuroanatomical distribution of APC mRNA using in situ hybridization. In the adult, prominent expression of APC mRNA was observed in the olfactory bulb, hippocampus, and cerebellum, with low levels of hybridization in other regions of adult rat brain. In contrast, during embryonic and early postnatal development (1-2 weeks), high levels of APC expression were found throughout the brain and then decreased to adult levels by 6 weeks after birth, except in the olfactory bulb where the high levels of APC mRNA found in development persist in the adult. During development of cortex, cerebellum, and retina, APC mRNA expression was particularly prominent in layers containing newly formed postmitotic neurons, with lower levels observed in the proliferative zones where neurogenesis occurs. The high levels of APC expression from early neurogenesis until late stages of neuronal maturation suggest that APC may contribute to suppressing neuronal proliferation during this period of intense growth.

Adenomatous Polyposis Coli↗
PubMed1993

Peptidylglycine alpha-amidating monooxygenase and other processing enzymes in the neurointermediate pituitary.

Studies on the mRNAs encoding PAM and on the various PAM proteins have begun to reveal some of the intricate mechanisms used to optimize the ability of this enzyme to carry out the alpha-amidation of peptides. Comparison of the regulatory elements governing expression of the various enzymes involved in peptide processing should reveal common elements. Knowledge of the processing enzymes themselves should help us to understand how these enzymes function in the secretory granule environment. In addition to their catalytic domains, other processing enzymes, like PAM, may well have processing domains and routing domains designed to optimize their ability to function in secretory granules.

Amino Acid Sequence↗
PubMed1993

Use of endoproteases to identify catalytic domains, linker regions, and functional interactions in soluble peptidylglycine alpha-amidating monooxygenase.

The production of alpha-amidated peptides is accomplished through the sequential action of two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), that are contained within the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein. Tissue-specific alternative splicing and endoproteolysis are known to generate both soluble and integral membrane mono- and bifunctional PAM proteins. In order to investigate the functional consequences of these differences we purified PAM-3, a soluble 95-kDa bifunctional form of the enzyme, from the spent medium of stably transfected hEK-293 cells. Using NH2-terminal sequence analysis of products of limited endoproteolysis and antibody cross-reactivity we identified protease-sensitive regions at the NH2 terminus, between the 35-kDa PHM and 42-kDa PAL domains and at the COOH terminus of the protein. Endoproteolytic removal of the COOH-terminal region from the bifunctional PAM-3 protein shifted the pH optimum of PHM to a more alkaline pH, increased the turnover number (kappa(cat)) of PHM and decreased its KM for alpha-N-acetyl-Tyr-Val-Gly; the catalytic properties of PAL were not altered. Since peptide amidation can be a rate-limiting step in the biosynthesis of neuropeptides, similar increases in PHM activity in vivo may play an important role in regulating the extent of peptide alpha-amidation.

Alternative Splicing↗
PubMed1993

Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing.

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.

Amino Acid Sequence↗
PubMed1993

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.

Alternative Splicing↗
PubMed1993

Localization of the gene encoding peptidylglycine alpha-amidating monooxygenase (PAM) to human chromosome 5q14-5q21.

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. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).

Blotting, Southern↗
PubMed1993

COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.

Animals↗
PubMed1993

Neurosecretory vesicles contain soluble and membrane-associated monofunctional and bifunctional peptidylglycine alpha-amidating monooxygenase proteins.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of neuropeptides in a reaction requiring the sequential action of two enzymes contained within this bifunctional protein. The CNS contains primarily transcripts encoding rPAM-1 and rPAM-2, integral membrane proteins differing by the presence or absence of a noncatalytic domain separating the two enzymes. Subcellular fractionation of adult rat hypothalamus and hippocampus demonstrated the localization of both enzymatic activities to fractions enriched in neurosecretory vesicles. Upon separation of the soluble contents from the membranes of neurosecretory vesicles, 30-40% of both enzymatic activities was recovered in the soluble fraction. Over 40% of both enzymatic activities remained membrane-associated following removal of peripheral membrane proteins. Antisera specific to different regions of PAM were used to identify intact rPAM-1 and rPAM-2, a monofunctional integral membrane peptidyl-alpha-hydroxy-glycine alpha-amidating lyase protein generated from rPAM-1, and a noncatalytic COOH-terminal fragment as the major PAM proteins in carbonate-washed membranes. Endoproteolytic processing generated large amounts of soluble, monofunctional forms of both enzymes from rPAM-1 and smaller amounts of a soluble, bifunctional PAM protein from rPAM-2. A significant amount of both monofunctional enzymes lacking the transmembrane domain was tightly associated with membranes. Whereas soluble mono- and bifunctional enzymes may be released upon exocytosis of neurosecretory vesicles, membrane-associated PAM proteins may remain on the cell surface or be internalized.

Amidine-Lyases↗
PubMed1993

Rapid increases in peptide processing enzyme expression in hippocampal neurons.

Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine alpha-amidating monooxygenase (PAM) converts peptidylglycine substrates into alpha-amidated products and prohormone convertases 1 and 2 perform the tissue-specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide-processing enzymes.

Animals↗
PubMed1993

Maturation, internalization, and turnover of soluble and membrane proteins associated with atrial myocyte secretory granules.

Primary cultures of neonatal atrial myocytes were used to study the biosynthesis of a prominent secretory granule enzyme that occurs naturally in soluble and integral membrane forms. The two most prominent forms of peptidylglycine alpha-amidating monooxygenase PAM) in atrial myocytes are type I integral membrane proteins (PAM-1 and -2); smaller amounts of a soluble form, PAM-3, are also found. All three PAM proteins are N-glycosylated, and PAM-1 also has sialylated O-linked oligosaccharide. Two hours after their biosynthesis, approximately half of the newly synthesized PAM-1 and PAM-2 proteins have acquired N-linked oligosaccharide chains resistant to digestion with endoglycosidase-H. Secretion of newly synthesized PAM-3 is detectable within 90 min after biosynthesis and is largely complete within 4 h. Release of the catalytic domains of PAM-1 and PAM-2, which requires endoproteolytic cleavage, occurs at a slow rate for many hours after biosynthesis. Release of PAM-3 and the soluble PAM proteins derived from PAM-1 and PAM-2 can be stimulated by secretagogue. Integral membrane PAM proteins that reach the surface of atrial myocytes are internalized and enter the endocytic pathway. The turnover of newly synthesized PAM-1 and PAM-2 is only partially accounted for by the release of soluble PAM protein into the medium and may involve a significant contribution from intracellular degradation.

Animals↗
PubMed1993

Peptide alpha-amidation and peptidylglycine alpha-hydroxylating monooxygenase: control by disulfiram.

The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). These enzymes are derived from the bifunctional precursor protein, peptidylglycine alpha-amidating monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL. Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low. Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of peptidylglycine alpha-amidating monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme.

Amides↗
PubMed1993

Biochemical characterization of peptide alpha-amidation enzyme activities of human neuroendocrine lung cancer cell lines.

Peptide alpha-amidation is a posttranslational modification of approximately half of all endocrine and neuroendocrine peptide hormones, including several hormones with mitogenic effects for tumor cells, and is typically essential for complete hormonal bioactivity. alpha-Amidated peptide hormones have been reported to be autocrine growth factors for small cell lung cancer cells. We report here that a variety of human lung tumor cell lines express both enzymes required for the two-step conversion of inactive glycine-extended peptides into their active COOH-terminal alpha-amide analogues. Human tumor cell peptidylglycine alpha-amidation enzymes are present in multiple molecular forms. Both proteins are metalloenzymes which are present at highest concentrations in secretory granules in neuroendocrine cell lines. The expression of these enzymes is positively correlated with expression of other markers of the neuroendocrine phenotype, such as DOPA decarboxylase. Peptidylglycine alpha-amidating enzyme-specific activities are approximately 50-fold higher in extracts of endocrine cell lines (lung small cell and carcinoid) than of nonendocrine lines. Biochemical characterization of the peptidylglycine alpha-amidating enzymes will enable development of tools for detection of endocrine processes in the early stages of neoplasia and for interruption of autocrine stimulation pathways in tumor cells.

Amidine-Lyases↗