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B A Eipper

Publications and source records attributed to B A Eipper.

At least 127 records · Page 7Linked to original sources
PubMed1987

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

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

Animals↗
PubMed1987

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.

Aged↗
PubMed1986

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate.

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.

Animals↗
PubMed1986

Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide.

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.

Amides↗
PubMed1986

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

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

Animals↗
PubMed1986

Transport and stability of ascorbic acid in pituitary cultures.

Ascorbic acid uptake in AtT-20 tumor cells and primary cultures of rat anterior and intermediate pituitary was sodium-dependent and showed half-maximal saturation between 9 and 18 microM ascorbate. When incubated in [14C]ascorbic acid at concentrations similar to those in serum (50 microM), all of the cells concentrated ascorbate 20- to 40-fold, producing intracellular ascorbate concentrations of 1-2 mM. HPLC analyses showed that over 90% of the intracellular label comigrated with authentic ascorbic acid. Although ascorbate was rapidly oxidized in culture medium in the absence of cells, incubation of ascorbate in culture medium in the presence of cells stabilized the ascorbate substantially. Unlike systems that transport dehydroascorbic acid, the ascorbate transport systems in all three preparations were not inhibited by glucose. Thus all three systems possess similar saturable, high-affinity, sodium-dependent active transport systems for ascorbic acid.

Animals↗
PubMed1986

Long term culture of primary rat pituitary adrenocorticotropin/endorphin-producing cells in serum-free medium.

Insulin, transferrin, and serum albumin were found to be essential additives for maintenance of primary rat pituitary ACTH/endorphin cells in complete serum-free defined medium (CSFM). Primary anterior pituitary cultures maintained in CSFM exhibited a 3- to 5-fold increase in cell content of ACTH/endorphin-related peptide during the first 2 weeks in culture, and this level remained stable for at least the next week. Immunocytochemical and morphometric studies indicated that the number of corticotropes increased only slightly, so that hormone content per corticotrope increased. Anterior pituitary cultures maintained in CSFM exhibited a basal secretory rate of 0.3-0.4% of cell hormone content/h throughout the 3-week period in culture, and total hormone production increased 6-fold. Primary anterior pituitary cultures maintained chronically in CSFM containing 10 nM CRF demonstrated a 15- to 20-fold increase in total ACTH/endorphin production over 3 weeks in culture. Chronic treatment with CRF brought about a sustained 6-fold increase in secretory rate (2.5% of cell hormone content/h), and corticotrope content of hormone was diminished 3-fold. Corticotropes maintained chronically in CSFM containing CRF did not increase in number and exhibited a rim of immunocytochemically identifiable hormone around the cell periphery. Anterior pituitary cultures maintained chronically in CSFM containing 100 nM dexamethasone (DEX) exhibited decreased cell hormone content and an unaltered secretory rate. In the DEX-treated cultures the number of immunocytochemically identifiable corticotropes declined, as did the staining intensity per corticotrope. Primary cultures of rat intermediate pituitary cells maintained in CSFM exhibited a 1.5- to 2-fold increase in hormone content after 1-2 weeks in culture, maintained a constant basal secretory rate of 0.4-0.5% cell content/h, and were not responsive to chronic treatment with CRF or DEX.

Adrenocorticotropic Hormone↗
PubMed1986

Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus.

The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta-endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta-endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1-26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.

Acetylation↗
PubMed1985

Regulation of peptide amidation in cultured pituitary cells.

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.

Animals↗
PubMed1985

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.

Amides↗
PubMed1985

Peptidyl-glycine alpha-amidation activity in tissues and serum of the adult rat.

Bioactive peptides frequently terminate in a carboxyl-terminal alpha-amide. The tissue distribution of enzymatic activity capable of converting [125I]D-Tyr-Val-Gly into [125I]D-Tyr-Val-NH2 has been determined. Assay conditions have been established so that enzyme activity can be measured in crude homogenates. In adult male rats, the highest concentrations of activity are found in the anterior and neurointermediate lobes of the pituitary. Lower concentrations of activity are found in the hypothalamus, submandibular glands, and the rest of the brain. Enzyme activity is also easily assayed in serum. Taking into account the mass of each tissue, the submandibular glands and the brain are the major tissue sources of enzymatic activity; serum contains more enzymatic activity than is found in the pituitary gland. In all tissues and in serum, enzyme activity is stimulated by the addition of copper sulfate and ascorbate and is dependent on molecular oxygen. This activity is, therefore, referred to as peptidyl glycine alpha-amidating monooxygenase (PAM) activity. The pH optima for PAM activity in the pituitary and submandibular glands are near neutrality, whereas the pH optima for PAM activity in serum and hypothalamus are broad, with substantial activity at alkaline pH.

Animals↗
PubMed1985

Hormonal, drug, and dietary factors affecting peptidyl glycine alpha-amidating monooxygenase activity in various tissues of the adult male rat.

The factors controlling levels of peptidyl glycine alpha-amidating monooxygenase (PAM) activity in its major tissue sources in the adult male rat were investigated by carrying out a variety of endocrine, pharmacological, and dietary manipulations. Levels of PAM activity and alpha MSH immunoactivity in the neurointermediate lobe of the pituitary gland rose and fell in parallel in rats treated with the dopamine antagonist haloperidol or the dopamine agonist bromocriptine, respectively. PAM activity in the anterior pituitary lobe was increased after adrenalectomy or castration and decreased after thyroidectomy or treatment with haloperidol. PAM activity in the submandibular gland was increased after treatment with the alpha-adrenergic antagonist phenoxybenzamine and decreased after treatment with the alpha-adrenergic agonist phenylephrine. Serum levels of PAM activity were unaltered after hypophysectomy, adrenalectomy, sialectomy, or castration, but rose after thyroidectomy and declined after treatment with the ganglionic blocker chlorisondamine or phenoxybenzamine. Chronic dietary copper deficiency in rats resulted in increased PAM activity in homogenates of anterior pituitary lobe and submandibular gland assayed under optimized conditions; chronic dietary ascorbate deficiency in guinea pigs did not produce consistent changes in PAM activity in the tissues examined.

Animals↗
PubMed1984

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.

Amides↗
PubMed1984

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.

Amides↗
PubMed1984

Secretion and regulation of two biosynthetic enzyme activities, peptidyl-glycine alpha-amidating monooxygenase and a carboxypeptidase, by mouse pituitary corticotropic tumor cells.

Secretion of peptidyl-glycine alpha-amidating monooxygenase (PAM) activity and enkephalin convertase (a carboxypeptidase B-like enzyme) activity from AtT-20 mouse corticotrope tumor cells was compared with secretion of pro-ACTH/endorphin-derived peptides. Upon stimulation of secretion by corticotropin-releasing factor, a beta-adrenergic agonist, or barium ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone rose in parallel. During inhibition of stimulated secretion with glucocorticoids or cobalt ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone fell in parallel. No measurable secretion of cytosolic, mitochondrial, or lysosomal marker enzymes was detected, indicating that the secretion of PAM activity and enkephalin convertase activity was specific and not a result of cell damage or lysis. The kinetic characteristics, apparent mol wt, and enzymatic properties determined for the secreted enzymes were similar to those for the enzymes from secretory granules. In AtT-20 cells treated with glucocorticoids for up to 8 days, cellular levels of immunoactive ACTH/endorphin-related peptides decreased to one third of control levels. Levels of PAM activity in cell extracts declined in parallel with levels of hormone. In contrast, levels of enkephalin convertase activity, cell protein, lactate dehydrogenase, fumarase, and lysosomal carboxypeptidase did not decline in response to chronic glucocorticoid exposure.

Animals↗
PubMed1983

Selective loss of alpha-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells.

The production of alpha-melanotropin (alpha-N-acetyl-ACTH(1-13)NH2 (alpha MSH) in rat intermediate pituitary was investigated using reverse phase high performance liquid chromatography, immunoassays, and biosynthetic labeling. Extracts of rat intermediate pituitary contain primarily alpha-N,O-diacetyl-ACTH(1-13)NH2, with smaller amounts of des- and monoacetyl-ACTH(1-13)NH2. No significant amount of ACTH(1-14)OH- or ACTH(1-13)OH-related material was observed. Analysis of the newly synthesized alpha MSH-sized peptides produced by freshly prepared rat intermediate pituitary cell suspensions revealed labeled peptides co-migrating primarily with des-, mono-, and diacetyl-ACTH(1-13)NH2. In contrast, when extracts of 2-week-old intermediate pituitary cultures were analyzed, the major peak of material detected with the general NH2-terminal ACTH antiserum eluted at the position expected for alpha-N,O-diacetyl-ACTH(1-14)OH; smaller amounts of material co-migrating with des- and monoacetyl-ACTH(1-14)OH were also detected. Analysis of the newly synthesized alpha MSH-sized peptides produced by 16-day-old cultures of intermediate pituitary cells revealed labeled peptides co-migrating with des-, mono-, and diacetyl-ACTH(1-14)OH. Thus, rat intermediate pituitary cells in culture maintain the ability to perform several major proteolytic cleavages of pro-ACTH/endorphin as well as the ability to alpha-N-acetylate alpha MSH and beta-endorphin, but selectively lose the ability to alpha-amidate the carboxyl terminus of alpha MSH.

Adrenocorticotropic Hormone↗
PubMed1983

Adrenocorticotropin(1-14)OH-related molecules in primary cultures of rat intermediate pituitary cells. Identification and role in the biosynthesis of alpha-melanotropin.

The structures of the altered alpha-melanotropin (alpha MSH or alpha-N-acetyl-ACTH(1-13)NH2)-related molecules produced by cultured rat intermediate pituitary cells were investigated. Peptide analyses demonstrated that the alpha MSH-related molecules produced by acutely prepared intermediate pituitary cells were primarily des-, mono-, and diacetylated ACTH(1-13)NH2; in contrast, longer term cultures produced primarily des-, mono-, and diacetylated ACTH(1-14)OH. No significant amount of labeled ACTH(1-13)OH-related material was observed under any incubation conditions. Intermediate pituitary cells in culture stopped producing alpha-amidated alpha MSH-related molecules with a half-time of approximately 15 to 18 h; instead, ACTH(1-14)OH-related molecules were synthesized. In several pulse-chase experiments, performed under conditions where cultured intermediate pituitary cells were capable of synthesizing alpha MSH-related molecules terminating in -Val13-NH2, labeled molecules ending in -Val13-Gly14-OH were not found to give rise to major amounts of labeled molecules ending in -Val13-NH2. This failure to observe conversion of glycine-extended molecules into alpha-amidated products was not due to selective secretion from the cells, since the acetylation and amidation states of labeled molecules that were secreted under basal conditions reflected those of the molecules stored in the cells, and the basal rate of secretion was very low.

Adrenocorticotropic Hormone↗