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C C Glembotski

Publications and source records attributed to C C Glembotski.

72 records · Page 4Linked to original sources
PubMed1986

The characterization of the ascorbic acid-mediated alpha-amidation of alpha-melanotropin in cultured intermediate pituitary lobe cells.

Previous studies have shown that cultured rat intermediate pituitary lobe cells lose the ability to form ACTH-(1-13)NH2-related molecules (alpha MSH) and instead produce ACTH-(1-14)-related peptides. In vitro studies have shown that peptidylglycine alpha-amidating monooxygenase the enzyme responsible for the conversion of ACTH-(1-14) to ACTH-(1-13)NH2, requires ascorbate, CuSO4, and molecular oxygen as cofactors. In the present study we have demonstrated that cultured intermediate pituitary lobe cells require long term supplementation of the medium with ascorbate for continued production of immunoactive alpha-amidated alpha MSH. When the relative quantities of alpha-amidated and COOH-terminally glycine-extended forms of alpha MSH were assessed in biosynthetic labeling experiments, it was shown that either L-ascorbate or an epimer of the vitamin, D-isoascorbate, was capable of supporting cellular alpha-amidation. However, the potency of isoascorbate was approximately 4- to 5-fold lower than that of ascorbate. The ascorbate-mediated reestablishment of alpha-amidation ability was shown to be dependent on the presence of sodium in the medium; at physiological levels of ascorbate (35 microM), the EC50 for sodium was about 40 mM. Time-course experiments indicated that the time of exposure of the cultured cells to ascorbate could be decreased to as little as 30 min. However, a total incubation time of 6 h was required after such an exposure to convert biosynthetically labeled ACTH-(1-14)-related peptides to labeled ACTH-(1-13)NH2-related peptides. The time course of the effects of ascorbic acid on the reestablishment of alpha-amidation, as well as the relative stereospecificity for L-ascorbic acid and the sodium requirement, are all consistent with the hypothesis that ascorbic acid must be transported into the intermediate pituitary lobe cells to participate in the peptidyl alpha-amidation reaction. Moreover, it is apparent that the length of time required to reestablish alpha-amidation ability (6 h) is a function of transport of the cofactor into the granules, the rate of the peptidylglycine alpha-amidating monooxygenase-catalyzed reaction, or both.

Adrenocorticotropic Hormone↗
PubMed1986

Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland.

Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-125I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.

Adrenal Glands↗
PubMed1986

Cross-linking of atrial natriuretic peptide to binding sites in rat olfactory bulb membranes.

Binding sites for 125I-atrial natriuretic peptide (ANP)2 in rat olfactory bulb membranes have been studied using pharmacological and biochemical methods. Various unlabeled ANP-related peptides were tested for the ability to inhibit the binding of the radioligand in membrane binding assays. ANP(92-126) and ANP(99-126) were the most potent inhibitors tested, both exhibiting an IC50 value of 0.40 nM. ANP(103-126) and ANP(103-123) were 3 and 70 times less potent, respectively. ANP(111-126) was unable to inhibit the binding of the radioligand at a concentration of 1 microM. Several peptides unrelated to ANP were unable to inhibit the binding of the radioligand to rat olfactory bulb membranes. Membranes labeled with 125I-ANP were incubated with cross-linking agents and subjected to SDS-PAGE followed by autoradiography. A band possessing an apparent molecular mass of 116 kDa was identified. The labeling of this band was progressively decreased by increasing concentrations of unlabeled ANP(99-126) (IC50 = 0.6 nM) and by several other ANP-related peptides at nanomolar concentrations. For comparison purposes, ANP binding sites in rat aorta membranes were labeled with 125I-ANP and cross-linked using identical techniques. Three bands possessing molecular masses of 120, 72, and 62 kDa were identified. These results indicate that the ANP binding site in rat olfactory bulb membranes displays pharmacological and biochemical properties similar to peripheral ANP receptors.

Animals↗
PubMed1985

Molecular forms of immunoactive atrial natriuretic peptide released from cultured rat atrial myocytes.

Primary cultures of atrial myocytes were prepared from newborn rats and maintained for 8 days in complete serum-free medium. The culture content of immunoactive atrial natriuretic peptide (ANP) increased from 10 to 25 ng/culture during this time. The cells released immunoactive ANP at a rate of 2 to 3% of culture content per hour in a linear fashion for at least 6 hours. When analyzed by gel filtration the major immunoactive material released by and contained within the cells displayed a molecular weight of approximately 15,000 daltons. The medium and cellular ANP-related peptides were further shown to be indistinguishable by reversed-phase HPLC. When the 15,000 dalton material was incubated with rat serum it was converted to ANP-related material possessing a molecular weight of approximately 3,000 daltons. These results suggest that under basal conditions, atrial myocytes release a large molecular weight form of ANP that is converted in the circulation to a low molecular weight form of ANP, which has been previously identified in plasma.

Animals↗
PubMed1985

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium.

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.

Animals↗
PubMed1985

Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules.

In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.

Adrenocorticotropic Hormone↗
PubMed1984

The alpha-amidation of alpha-melanocyte stimulating hormone in intermediate pituitary requires ascorbic acid.

Rat intermediate pituitary cells in primary culture display a time-dependent loss of the ability to produce COOH-terminally alpha-amidated alpha MSH (Glembotski, C.C., Eipper, B.A., and Mains, R.E. (1983) J. Biol. Chem. 258, 7299-7304). Instead of des-, mono-, and diacetyl-adrenocorticotropic hormone(ACTH) (1-13)NH2, the cells produce des-, mono-, and diacetyl-ACTH(1-14)OH. Since the pituitary secretory granule-associated alpha-amidation enzyme requires copper and ascorbic acid for optimal activity (Eipper, B.A., Mains, R.E., and Glembotski, C. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148), these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide alpha-amidation ability. When the cultures were supplemented with up to 100 microM cooper (II) there was very little change in the ability to alpha-amidate alpha-melanotropin. Ascorbic acid at concentrations of up to 500 microM resulted in a dramatic increase in the ability of the cells to form the alpha-amidated peptide. Various combinations of ascorbic acid and copper additions indicated that a relatively short exposure (hours) to ascorbic acid produced the maximal response. Ascorbic acid displayed a dose-dependent effect on the alpha-amidation ability with a half-optimal concentration of about 25 microM. Pulse-chase labeling experiments demonstrated the ascorbic acid-dependent conversion of labeled ACTH(1-14)OH-related peptides to ACTH(1-13)NH2-related peptides. These results correlate with the ascorbic acid requirement of the pituitary alpha-amidation enzyme and demonstrate that the direct precursors to ACTH(1-13)NH2-related peptides are ACTH(1-14)OH-related peptides. Combined with our previous data, the present studies support the notion that a wide range of neuro- and endocrine peptides become alpha-amidated in a similar ascorbic acid-dependent manner.

Adrenocorticotropic Hormone↗
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↗
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↗
PubMed1983

Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.

An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.

Aerobiosis↗
PubMed1982

Acetylation of alpha-melanotropin and beta-endorphin in the rat intermediate pituitary. Subcellular localization.

The subcellular site of the further processing (NH2-terminal acetylation and COOH-terminal proteolysis) of beta-endorphin-sized molecules in the rat intermediate pituitary has been studied. Rat intermediate pituitary primary cultures that had been incubated in radioactively labeled amino acids were homogenized and the secretory granule fraction was separated from the rough endoplasmic reticulum/Golgi apparatus fraction. The labeled beta-endorphin-sized molecules in each subcellular fraction were analyzed by immunoprecipitation, gel filtration chromatography, and ion exchange chromatography. A large percentage of the labeled beta-endorphin-sized molecules became NH2 terminally acetylated after becoming associated with the secretory granule fraction; most of the further COOH-terminal proteolytic processing of alpha-N-acetyl-beta-endorphin(1-31) to form alpha-N-acetyl-beta-endorphin(1-27) and alpha-N-acetyl-beta-endorphin(1-26) also occurred when the labeled beta-endorphin-sized molecules were associated with the secretory granule fraction. The acetylation of alpha-melanocyte-stimulating hormone (alpha MSH)-sized molecules was also investigated in rat intermediate pituitary primary cell cultures by immunoprecipitation, gel filtration chromatography, and reverse-phase high pressure liquid chromatography. Pulse-chase labeling experiments showed that newly synthesized molecules co-migrating with adrenocorticotropic hormone ((ACTH)(1-13)NH2) were converted first to molecules similar to alpha MSH (alpha-N-acetyl-ACTH(1-13)NH2) and then to molecules similar to alpha-N,O-diacetyl-alpha MSH. These results demonstrate that the enzyme activity(s) responsible for the NH2-terminal acetylation of beta-endorphin alpha MSH-sized molecules is located in the secretory granules of the rat intermediate pituitary.

Acetylation↗
PubMed1981

Subcellular fractionation studies on the post-translational processing of pro-adrenocorticotropic hormone/endorphin in rat intermediate pituitary.

The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.

Adrenocorticotropic Hormone↗
PubMed1981

Adenylate energy charge in Escherichia coli CR341T28 and properties of heat-sensitive adenylate kinase.

Escherichia coli strain CR341T28 will not grow at temperatures above 34 degrees C in liquid medium, and the adenylate kinase of this strain is heat sensitive. When a culture was shifted from a permissive (30 degrees C) to a nonpermissive (36 degrees C) temperature, the adenylate energy charge fell from 0.9 to 0.2, with a concurrent decrease in the number of viable cells and in the specific activity of adenylate kinase. When cultures of the temperature-sensitive strain were grown at temperatures above 30 degrees C, the adenylate energy charge, the specific activity of adenylate kinase, and the growth rate were lower than the corresponding parameters for the parental strain. By isotopic labeling of the adenine nucleotides in vivo, it was determined that increasing growth temperatures between 30 and 34 degrees C for the heat-sensitive strain resulted in a decrease in the adenosine triphosphate-to-adenosine monophosphate and adenosine triphosphate-to-adenosine diphosphate ratios. Between 26 and 30 degrees C the adenosine triphosphate-to-adenosine diphosphate ratio was essentially normal in the temperature-sensitive strain, but the adenosine triphosphate-to-adenosine diphosphate ratio was decreased. The adenylate ratios in the parental strain did not change between 30 and 34 degrees C. The adenylate kinase mass action ratio for each strain was essentially constant under all growth conditions. When assayed at 30 degrees C, the affinities of the enzyme from the mutant strain were somewhat lower than those of the parent adenylate kinase. The mutant enzyme also did not exhibit the substrate inhibition that was observed at high adenosine monophosphate concentrations with the parental enzyme. An increase in the assay temperature from 30 degrees to 40 degrees C had little or no effect on the Km values determined for the parental adenylate kinase, but caused the Km values determined for the mutant adenylate kinase to increase by a factor of two or more.

Adenosine Diphosphate↗
PubMedDate not supplied

Bovine intermediate pituitary alpha-amidation enzyme: preliminary characterization.

A secretory granule-associated enzymatic activity that converts mono-[125I]-D-Tyr-Val-Gly into mono-[125I]-D-Tyr-Val-NH2 has been studied. The activity is primarily soluble and shows optimal activity at pH 7 to pH 8. Amidation activity was stimulated 9-fold by addition of optimal amounts of copper (3 microM). In the presence of optimal copper, ascorbate stimulated the reaction 7-fold; none of the other reduced or oxidized cofactors tested was as effective. Taking into account the dependence of the reaction on ascorbate and molecular oxygen and the production of glyoxylate [2], it is suggested that the alpha-amidation enzyme is a monooxygenase. Lineweaver Burk plots with D-Tyr-Val-Gly as the varied substrate demonstrated Michelis-Menten type kinetics with the values of Km and Vmax increasing with the addition of ascorbate to the assay. A variety of peptides ending with a COOH-terminal Gly residue act as inhibitors of the reaction. Two synthetic peptides, gamma 2MSH and ACTH(1-14), with carboxyl termini similar to the presumed physiological substrates for the enzyme, act as competitive inhibitors with similar K1 values. It is likely that this secretory granule alpha-amidation activity is involved in the physiological biosynthetic alpha-amidation of a wide range of bioactive peptides.

Animals↗
PubMedDate not supplied

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase.

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.

Acetylation↗