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S W May

Publications and source records attributed to S W May.

At least 37 records · Page 2Linked to original sources
PubMed1996

Virulence of feline Chlamydia psittaci in mice is not a function of the major outer membrane protein (MOMP).

The virulent strain of feline Chlamydia psittaci, the Cello strain, produces conjunctivitis and upper respiratory disease in cats. This same strain produces a lethal disease in mice when inoculated intraperitoneally (i.p.). In this study we have shown that the Baker strain of feline C. psittaci is attenuated in the mouse model system. Intraperitoneal inoculation of mice with the Baker strain produced no disease but did stimulate an immune response that protected the mice from subsequent produced i.p. inoculation with the virulent Cello strain. To determine if the difference between these two strains was in the major outer membrane protein (MOMP), the omp1 gene which codes for this protein was sequenced for both the Baker and Cello strains. The MOMP was chosen to study because in Chlamydia trachomatis this protein has been shown to contain neutralizing epitopes and has been shown to play a role in cell attachment. These functions make it a likely structural component capable of mutating and causing altered cell tropism and virulence. The DNA sequence of the omp1 was determined by amplifying the gene with PCR, cloning the PCR product into the pCR-II cloning vector and determining the DNA sequence of the inserted gene using primers to sites in the plasmid vector. From the DNA sequence, the deduced amino acid sequence of MOMP was determined for both the attenuated Baker and the virulent Cello strains of feline C. Psittaci. The results indicated that the omp1 gene of both strains contained 1179 base pairs which coded for a protein 392 amino acids. The DNA sequences of the omp1 gene of the two strains differed by only two base pairs which resulted in two amino acid changes in the MOMP. The Baker strain had a serine instead of a tryptophan at amino acid 7 and a tyrosine instead of an aspartic acid at amino acid 125 of the uncleaved protein. Neither amino acid change was in an area of the MOMP which could logically account for the difference in biological activity. Amino acid 7 was in the leader sequence which is cleaved from the authentic MOMP and is not present in the infectious elementary body. Amino acid 125 was in a conserved hydrophobic area of one of the constant regions of the protein. A change at this location was not likely surface exposed and thus could not affect cell adhesion, tissue tropism or neutralizing epitopes. Therefore, the differences in the primary structure of the MOMP from the Baker and Cello strains of feline C. psittaci could not account for the attenuation of the Baker strain for mice. The molecular basis of their difference is yet to be determined.

Amino Acid Sequence↗
PubMed1995

Reaction versus subsite stereospecificity of peptidylglycine alpha-monooxygenase and peptidylamidoglycolate lyase, the two enzymes involved in peptide amidation.

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine alpha-monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We have previously established that PAM and PGL exhibit tandem reaction stereospecificities, with PAM producing, and PGL being reactive toward, only alpha-hydroxyglycine derivatives of absolute configuration (S). We now demonstrate that PAM and PGL exhibit dramatically different subsite stereospecificities toward the residue at the penultimate position (the P2 residue) in both substrates and inhibitors. Incubation of Ac-L-Phe-Gly, Ac-L-Phe-L-Phe-Gly, or (S)-O-Ac-mandelyl-Gly with PAM results in complete conversion of these substrates to the corresponding alpha-hydroxylated products, whereas the corresponding X-D-Phe-Gly compounds undergo conversions of < 1%. The KI of Ac-D-Phe-Gly is at least 700-fold higher than that of Ac-L-Phe-Gly, and the same pattern holds for other substrate stereoisomers. This S2 subsite stereospecificity of PAM also holds for competitive inhibitors; thus, the KI of 45 microM for Ac-L-Phe-OCH2CO2H increases to 2,247 microM for the -D-Phe- enantiomer. In contrast, incubation of PGL with Ac-L-Phe-alpha-hydroxy-Gly, Ac-D-Phe-alpha-hydroxy-Gly, (S)-O-Ac-mandelyl-alpha-hydroxy-Gly, or (R)-O-Ac-mandelyl-alpha-hydroxy-Gly results in facile enzymatic conversion of each of these compounds to their corresponding amide products. The simultaneous expression of high reaction stereospecificity and low S2 subsite stereospecificity in the course of PGL catalysis was illustrated by a series of experiments in which enzymatic conversion of the diastereomers of Ac-L-Phe-alpha-hydroxy-Gly and Ac-D-Phe-alpha-hydroxy-Gly was monitored directly by HPLC. Kinetic parameters were determined for both substrates and potent competitive inhibitors of PGL, and the results confirm that, in sharp contrast to PAM, the configuration of the chiral moiety at the P2 position has virtually no effect on binding or catalysis. These results illustrate a case where catalytic domains, which must function sequentially (and with tandem reaction stereochemistry) in a given metabolic process, nevertheless exhibit sharply contrasting subsite stereospecificities toward the binding of substrates and inhibitors.

Amidine-Lyases↗
PubMed1995

Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.

The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abstraction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.

Adrenal Medulla↗
PubMed1995

Structural requirements for cocaine-sensitive and -insensitive uptake of phenethylamines into the adrenal chromaffin cell.

The adrenal medullary chromaffin cell is a commonly used model for the adrenergic neuron. Although much work has been done to study the transport system in the adrenal chromaffin vesicles, relatively little is known about cellular transport, especially with regard to structural features of phenethylamines required for intracellular accumulation. We have now investigated the structural requirements of phenethylamine-related compounds for their accumulation into cultured adrenal chromaffin cells. We find that two types of cellular uptake, previously described only for dopamine, norepinephrine, and epinephrine, are also present for [3H]tyramine. Although two types of accumulation occur, tyramine accumulation occurs mainly via a cocaine-insensitive process, whereas dopamine accumulation occurs predominantly via a cocaine-sensitive process. The accumulation of [14C]-phenethylamine and p-methoxyphenethylamine is not affected by cocaine, suggesting that a ring hydroxyl substituent is necessary for cocaine-sensitive accumulation. The compounds p-hydroxyphenylpropylamine and p-hydroxyphenyl-2-aminoethyl sulfide accumulate in the cell only via a cocaine-insensitive process, indicating that lengthening of the aminoalkyl side chain prevents cocaine-sensitive accumulation. We have performed conformational analyses of this series of compounds to determine whether the conformation of these compounds can be related to the kinetic data. For dopamine, tyramine, phenethylamine, and p-methoxyphenethylamine, two groups of energy-minimized conformers were found. We find that there is an approximately linear relationship between the Km values for these phenethylamines and the differences in minimized energies between the low- and highest energy conformer groups of each compound. A similar correlation was found for p-hydroxyphenyl-2-aminoethyl sulfide. These results are consistent with the hypothesis that these compounds undergo a conformational change from the low-energy conformer to the highest energy conformer before their cocaine-insensitive accumulation.

Adrenal Glands↗
PubMed1994

NN-dimethyl-1,4-phenylenediamine as an alternative reductant for peptidylglycine alpha-amidating mono-oxygenase catalysis.

C-terminal alpha-amidation is a structural feature essential to the biological activity of many peptide hormones. Peptidylglycine alpha-amidating mono-oxygenase (PAM; EC 1.14.17.3) catalyses conversion of glycine-extended peptide hormone precursors into their corresponding alpha-hydroxyglycine derivatives. This reaction is the first step in the C-terminal amidation process. We report here that in the presence of molecular O2, copper and PAM substrate, NN-dimethyl-1,4-phenylenediamine (DMPD) serves as the requisite electron donor for the mono-oxygenase, being oxidized in the process to a stable and highly chromophoric cation radical. By monitoring the rate of increase in absorbance at 515 nm, PAM activity can be easily followed. This provides a spectrophotometric assay for PAM, which represents the first continuous assay reported for this enzyme. DMPD-supported PAM-catalysed mono-oxygenation exhibits normal Michaelis-Menten kinetic behaviour. Steady-state kinetic studies established that both the ascorbate-supported and DMPD-supported PAM reactions exhibit apparent 'Ping Pong' kinetics. In addition, both electron donors give rise to similar pH profiles and identical inhibition patterns towards known competitive inhibitors of PAM. The stoichiometry between formation of the DMPD cation radical and the alpha-hydroxyglycine PAM product was determined to be 2:1, the value expected for a monooxygenase-catalysed reaction. The optimum pH for the DMPD-supported continuous PAM assay was found to be about 5.5. The major advantage of this assay over all previously reported methods is that it is continuous; thus accurate initial rates are easily obtained. Moreover, unlike previous assay methods, 125I-labelled or chromophorically modified substrates are not required. Kinetic parameters for a broad range of PAM substrates and inhibitors have been successfully obtained using this assay.

Amino Acid Sequence↗
PubMed1994

Microbial Production of Poly-beta-Hydroxybutyric Acid from d-Xylose and Lactose by Pseudomonas cepacia.

Poly-beta-hydroxybutyric acid (PHB) was produced from xylose and lactose by using Pseudomonas cepacia. Approximately 50% PHB (grams of PHB total/grams of biomass total) was produced. With a laser-based fluorescent probe, beta-galactosidase activity was shown to be induced in P. cepacia cells grown on lactose but not in those grown on glucose or xylose. P. cepacia has the potential to produce biodegradable thermoplastics from hemicellulosic hydrolysates and cheese whey.

Journal Article↗
PubMed1993

HPLC-based method for determination of absolute configuration of alpha-chiral amines.

We introduce a novel, HPLC-based method for facile determination of the absolute configuration of alpha-chiral amines. Our method is easily applied to a variety of compounds, including amino acid derivatives. The method involves initial derivatization of the chiral amine analyte with the chiral derivatizing reagent, N-succinimidyl alpha-methoxyphenylacetate (SMPA), to produce the corresponding diastereomeric adducts. Inspection of a particular rotomer of the SMPA adduct and application of simple rules correlates absolute configuration and HPLC elution order. A key aspect of our method is that it can be used to determine absolute configuration without using enantiomeric standards of the amine analytes. Furthermore, it is of utmost significance that our method can also be used to determine absolute configuration even when only one analyte enantiomer of unknown absolute configuration is present, as is often the case for enzymatic products, naturally derived compounds, or enantiomerically enriched compounds prepared via chiral syntheses. We have observed strict adherence between predicted and observed absolute configuration for a wide variety of alpha-chiral amines. The chromatographic method we present in this paper is very practical and has several important advantages over NMR-based approaches which have been previously developed. For example, microgram quantities of an analyte in a complex enzymatic mixture can be directly analyzed by our HPLC-based method while the impurities often preclude definitive proton assignments in the NMR approach.

Amines↗
PubMed1993

The oral antihypertensive activity of the methylated derivatives of phenyl-2-aminoethyl sulfide.

We have reported previously that phenyl-2-aminoethyl sulfide and its derivatives are excellent substrates for dopamine-beta-monooxygenase and produce an antihypertensive effect in spontaneously hypertensive rats after i.p. administration. In the studies reported herein, we demonstrate that alpha-methyl-phenyl-2-aminoethyl sulfide and 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl sulfide, the methylated and hydroxymethylated derivatives of phenyl-2-aminoethyl sulfide, respectively, decrease mean arterial pressure in conscious, unrestrained spontaneously hypertensive rats after p.o. administration. This antihypertensive effect after p.o. administration occurs without the undesirable transient rise in blood pressure observed previously after i.p. administration. Results using the methodology of food-reinforced operant conditioned behavior are consistent with the interpretation that the ring hydroxylated derivatives, 4-hydroxy-phenyl-2-aminoethyl sulfide and 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl sulfide, do not penetrate into the central nervous system. This finding supports our contention that the primary site of action for the antihypertensive activity of the sulfides may be the peripheral adrenergic nerve ending. In view of the current high degree of interest in chiral development, the enantiomeric specificity of the antihypertensive activity of alpha-methyl-phenyl-2-aminoethyl sulfide was also evaluated. Results from these studies demonstrate that the (S)-enantiomer of alpha-methyl-phenyl-2-aminoethyl sulfide is more effective in lowering blood pressure after p.o. administration than the (R)-enantiomer. The implications of our findings in terms of the mechanism of action of these compounds are discussed.

Administration, Oral↗
PubMed1992

Activation of an adrenergic pro-drug through sequential stereoselective action of tandem target enzymes.

The synthetic amino acid, 3,4-dihydroxyphenylserine (DOPS) has been of great interest for many years as an adrenergic pro-drug, since the L-threo diastereomer of DOPS can be a precursor of R-(-)-norepinephrine, the natural form of this neurotransmitter. We now report bioactivation of DOPS to the potent pharmacological agent, noradrenalone (arterenone), via sequential stereoselective action by two target enzymes--dopamine beta-monooxygenase (DBM) and L-aromatic amino acid decarboxylase (AADC)--acting in tandem. Enzymatic activation is stereospecific, with only the L-erythro DOPS diastereomer producing noradrenalone; this is consistent with the known stereospecificities of AADC and DBM. These results provide a heretofore unrecognized rationale for the bioactivity of L-erythro DOPS and provide a basis for the design of new adrenergic pro-drugs.

Adrenal Glands↗
PubMed1992

Peptide amidating enzymes are present in cultured endothelial cells.

Carboxy-terminal amidation is a prevalent post-translational modification necessary for the bioactivity of many peptides. We now report that the two enzymes essential for amidation, peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL), are present in both the cytosol and membrane fractions of cultured bovine aortic endothelial cells. Endothelial PAM exhibits ascorbate-dependent turnover and is inactivated by the mechanism-based inactivator, 4-phenyl-3-butenoic acid (PBA), whereas PGL activity is independent of ascorbate and is not affected by PBA. These enzymological characteristics correspond to those of amidating enzymes from other tissues. These results suggest a heretofore unrecognized role for alpha-amidated peptides in cardiovascular function.

Amidine-Lyases↗
PubMed1991

Functional and structural characterization of peptidylamidoglycolate lyase, the enzyme catalyzing the second step in peptide amidation.

Carboxy-terminal amidation is a prevalent posttranslational modification necessary for the bioactivity of many neurohormonal peptides. We recently reported that in addition to peptidylglycine alpha-monooxygenase (PAM), a second enzyme, which we now call peptidylamidoglycolate lyase (PGL), functions in the enzymatic formation of amides [Katopodis et al. (1990) Biochemistry 29, 4551]. The monooxygenase first catalyzes formation of the alpha-hydroxyglycine derivative of the glycine-extended precursor, and the lyase subsequently catalyzes breakdown of the PAM product to the amidated peptide and glyoxylate. We report here the first primary sequence data for PGL, which establish that it is part of the putative protein precursor which also contains PAM. We also show that PAM and PGL activities are colocalized in the secretory granular fraction of neurointermediate pituitary as would be expected for enzymes sharing the same precursor. Time course studies of the amidation reaction using purified soluble pituitary PAM and PGL indicate that both enzymes are essential for enzymatic amidation. Finally, PGL has no effect on the substrate or inhibition kinetics of PAM, and purified pituitary PAM has an acidic pH optimum consistent with its known localization in secretory granules.

Amidine-Lyases↗
PubMed1991

Kinetic analyses of peptidylglycine alpha-amidating monooxygenase from pancreatic islets.

Peptidylglycine alpha-amidating monooxygenase (PAM) plays an important role in the post-translational processing of bioactive neuropeptides by participating in C-terminal amidation. We have examined PAM activity in the pancreatic islets of the anglerfish (AF), Lophius americanus. It was previously demonstrated that the cofactor requirements and pH optimum for the fish PAM are essentially identical to PAM obtained from other tissues and species. The present study was performed to examine the enzymatic characteristics of the fish islet PAM in more detail. One of the questions addressed was the suitability of the AF islet neuropeptide Y-like peptide, aPY-Gly, as a substrate for the islet PAM. Partially purified PAM from AF islet secretory granules was incubated with [125I] aPY-Gly and the resulting products were analyzed by HPLC. The islet PAM readily mediated the formation of aPY-amide from aPY-Gly. PAM purified from bovine adrenal chromaffin granules also catalyzed the amidation of [125I] aPY-Gly. The kinetic parameters of the islet PAM were examined using trinitrophenylated-D-Tyr-Val-Gly (TNP-D-YVG) and 4-nitrohippuric acid (4-NHA). The Km of the islet PAM was 25 +/- 5 microM for TNP-D-YVG and 3.4 +/- 1 mM for 4-NHA. The competitive inhibitor of mammalian PAM activity, 4-methoxybenzoxyacetic acid, proved to be a potent inhibitor of the islet PAM as well, with an apparent KI of 0.06 mM. These results demonstrate that the AF islet PAM exhibits substrate compatibility, kinetic parameters, and inhibitor susceptibility quite similar to the characteristics of PAM from other tissues and species.

Adrenal Medulla↗
PubMed1991

The enantiomeric specificity of the antihypertensive activity of 1-(phenylthio)-2-aminopropane, a synthetic substrate analogue for dopamine beta-monooxygenase.

We have found that (R,S)-1-(phenylthio)-aminopropane (4a), a synthetic alternate substrate for the terminal enzyme of norepinephrine biosynthesis, dopamine beta-monooxygenase (DBM), is both an indirect sympathomimetic and a potent antihypertensive agent in spontaneously hypertensive rats. We demonstrate herein that there is a distinct enantiospecific difference in the activities of (R)-1-(phenylthio)-2-aminopropane (4b) and (S)-1-(phenylthio)-2-aminopropane (4c). We find that 4c, the more potent DBM substrate analogue, exhibits both the indirect sympathomimetic activity and the antihypertensive activity previously observed for the racemate and inhibits the active transport of catecholamines at the nerve terminal. In contrast, 4b, which is less potent as a DBM substrate or as an inhibitor of catecholamine uptake, does not exhibit an indirect sympathomimetic effect and is not an effective antihypertensive agent. These results suggest that the greater selectivity of the S enantiomer for both the catecholamine reuptake transporter and the target enzyme DBM accounts for its greater potency as an indirect-acting sympathomimetic agent as well as its activity as an antihypertensive agent. These results are also consistent with the hypothesized mechanism of action of this class of sulfur-containing DBM substrate analogues.

Animals↗
PubMed1990

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation.

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.

Alkylation↗
PubMed1990

Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

Alkenes↗
PubMed1989

Effects of dopamine beta-monooxygenase substrate analogs on ascorbate levels and norepinephrine synthesis in adrenal chromaffin granule ghosts.

Chromaffin granule ghosts from bovine adrenal medullae have been used to investigate the effects of prototypic dopamine beta-monooxygenase substrate analogs of two distinct classes on intravesicular reduced ascorbic acid (AscH2) levels and on norepinephrine synthesis. Phenyl-2-aminoethyl sulfide (PAES), a sulfur-containing substrate, was shown to concentrate within ghosts, a process that was time and ATP dependent, but reserpine insensitive. Dopamine beta-monooxygenase oxygenation of PAES resulted in accumulation of the oxygenation product, PAESO, without affecting intravesicular levels of AscH2. Similarly, incubations of ghosts with phenyl-2-aminoethyl selenide (PAESe) also resulted in rapid, time- and ATP-dependent, but reserpine-insensitive uptake. However, oxygenation of PAESe by dopamine beta-monooxygenase within ghosts was found to cause a marked decrease in intravesicular AscH2, without buildup of the oxygenated product, phenyl 2-aminoethyl selenoxide. These results illustrate two basic differences between the consequences of PAES and PAESe turnover: while PAES accumulation proceeds concomitant with PAESO production and without AscH2 depletion, PAESe accumulation proceeds with a marked lowering of internal AscH2 but without observable product formation. Both PAES and PAESe were capable of competing with dopamine, the physiological substrate, for enzymatic oxygenation and/or vesicular uptake, and were capable of significantly reducing norepinephrine synthesis. In experiments where ghosts were preincubated with either PAES or PAESe with delayed addition of dopamine, it was clear that neither compound nor their oxygenated products interfered with electron transport via cytochrome b561. These results are consistent with the hypothesis that the physiological activity observed with both PAES and PAESe may be related to their ability to gain entrance to adrenergic neurons and decrease norepinephrine synthesis within neurotransmitter storage vesicles.

Adrenal Medulla↗