Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

Acid release,cytoplasmic alkalinization,and chromosome condensation during sea urchin fertilization and amine-lnduced parthenogenesis

We have studied the relationship between acid release, cytoplasmic alkalinization, and the extent of chromosome condensation during parthenogenetic activation of sea urchin eggs. The relative rate of acid release in Strongylocentrotus purpuratus eggs was determined from pH measurements of egg suspensions. Acid release in inseminated eggs began after a lag of 0.4 min and the relative rate increased 108-fold, declined, and release was essentially complete by 8-min postinsemination. An average of 3.8 ± 0.23 × 10^?12moles H⁺ cell^? was released as determined by backtitration with NaOH. Acid release characteristics of eggs parthenogenetically activated with either NH₄C1, methylamine ethylamine, n-propylamine, n-butylamine, or benzylamine were qualitatively similar. There was no detectable lag peroid and the increase in relative rate of acid release was directly proportional to the carbon number of the amine used, eg, from 8.3-fold methylamine to 470-fold with benzylamine. The total equivalents of acid released ranged from 0.50–8.2 × 10^?12 moles H⁺·cell^? in direct proportion to the concentration of amine used. The degree fo cytoplasmic alkalinization induced as a function of methylamine and benzylamine concentration was determined by pH measurements fo egg homogenates; egg cultures were also prepared for microscopic examination of chromosome condensation. None of the eggs had condensed chromosomes at 0.5-mM methylamine whereas a cytoplasmic alkalinization of 0.6 pH units was observed. Increased methylamine levels up to 10mM resulted in chromiosome condensation in only 20% of the eggs. A similar result was found with benzylamine. We conclude that acid release and cytoplasmic alkalinization during chemical parthenogenesis are insufficient to mimic sperm induction of chromiosome condensation and suggest that an additional factor(s) is required for chromosome condensation by low concentration of amines.     

Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [¹⁴C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [¹⁴C]SCN⁻ uptake and ³⁶Cl⁻ uptake activities coincided with [¹⁴C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.     

Amine transport at the plasmalemma of Riccia fluitans

Green thallus cells of the aquatic liverwort, Riccia fluitans, are rapidly depolarized in the presence of 1–20 μM NH₄Cl and 5–100 μM CH₃NH₃Cl, respectively. Simultaneously, the membrane conductance is increased from 0.41 to 1.2 S · m^?2. Uptake of [¹⁴C]methylamine is stimulated by increasing [K⁺]_o and inhibited by increasing [Na⁺]_o or [H⁺]_o, is highly voltage sensitive, and saturates at low amine concentrations.Double-reciprocal plots of (a) maximal membrane depolarization and (b) methylamine uptake vs. external amine concentration give apparent K_m values of 2 ± 1 μM ammonia and 25–50 μM methylamine; K_m values for changes in conductance and membrane current are greater and voltage dependent. Whereas the amine transport into the cell is strongly inhibited by CN^?, the amine efflux is stimulated.The current-voltage characteristics of the ammonia transport are represented by a sigmoid curve with an equilibrium potential of ?60 mV, and this is understood as a typical carrier curve with a saturation current of about 70 mA · m^?2. It is further concluded that the evidently carrier-mediated transport is competitive for the two amines tested, and that ammonia and methylamine are transported in the protonated form as NH₄⁺ and CH₃NH₃⁺ into the cytoplasm.     

Substrates for Sulfate Reduction and Methane Production in Intertidal Sediments

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [¹⁴C]methylamine was rapidly converted to ¹⁴CH₄ and ¹⁴CO₂ in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-¹⁴C]methionine and [2-¹⁴C]acetate were converted to ¹⁴CO₂ and not to ¹⁴CH₄ in fresh sediment. No reduction of ¹⁴CO₂ to ¹⁴CH₄ occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-¹⁴C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.     

Metabolism of Acetate, Methanol, and Methylated Amines in Intertidal Sediments of Lowes Cove, Maine

The fates and the rates of metabolism of acetate, trimethylamine, methylamine, and methanol were examined to determine the significance of these compounds as in situ methane precursors in surface sediments of an intertidal zone in Maine. Concentrations of these potential methane precursors were generally <3 μM, with the exception of sediments containing fragments of the seaweed Ascophyllum nodosum, in which acetate was 96 μM. [2-¹⁴C]acetate turnover in all samples was rapid (turnover time <2 h), with ¹⁴CO₂ as the primary product. [¹⁴C]trimethylamine and methylamine turnover times were slower (>8 h) and were characterized by formation of both ¹⁴CH₄ and ¹⁴CO₂. Ratios of ¹⁴CH₄/¹⁴CO₂ from [¹⁴C]trimethylamine and methylamine in uninhibited sediments indicated that a significant fraction of these substrates were catabolized via a non-methanogenic process. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [¹⁴C]methanol was oxidized relatively slowly compared with the other substrates and was catabolized mainly to ¹⁴CO₂. Results from experiments with molybdate and 2-bromoethanesulfonic acid suggested that methanol was oxidized primarily through sulfate reduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1% of total methane production.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Nucleophilic cleavage of the complex between human alpha-1-antitrypsin and porcine pancreatic elastase

Several nucleophilic amines were examined to determine their ability to split the alpha-1-antitrypsin-elastase complex. Hydrazine and hydroxylamine, their methyl derivatives, and methylamine were tested in the pH range of 8 to 10.6. Only hydrazine and methylamine produced complete and clean cleavage of the complex into its inhibitor and enzyme components. When [¹⁴C]-methylamine at pH 10.6 was used about 0.3 mol of the nucleophile was specifically bound per mol of the inhibitor component. An interfering reaction between methylamine and the native inhibitor was controlled by preincubation with unlabeled methylamine.     

Uptake and release of possible false transmitter amino acids by rat brain tissue

—The uptake of l [¹⁴C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm , partially inhibited by dinitrophenol and ouabain and elevated [K⁺], weakly Na⁺-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC₅₀= 0·5mm ) than by aspartic acid, GABA, glycine or methionine. The [¹⁴C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [³H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [³H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (K_i= 1 mm ). [¹⁴C]glutamine and [³H]GABA were released from brain slices by electrical stimulation or 50 mm K⁺, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [¹⁴C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Dynamic Storage of Dopamine in Rat Brain Synaptic Vesicles In Vitro

Abstract: The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [³H]dopamine ([³H]DA) for ～10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [³H]DA was lost with single exponential kinetics at a half-time of ～4 min at 30°C in 8 mM Cl^? medium, in which both voltage and H⁺ gradients are present in the vesicles. In 90 mM Cl^? medium, in which high H⁺ and Cl^? gradients but no voltage gradient are present, [³H]DA escaped at a half-time of ～7 min. In both high and low Cl^? media, ～40% of [³H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H⁺ gradient in the vesicles. The presence of both uptake-dependent and -independent efflux was observed in experiments using DA-free medium, instead of uptake inhibitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [¹⁴C]DA was lost about three times faster than [³H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first-order rate constant for uptake-independent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0°C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [¹⁴C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.     

On the estimation of proton gradient and osmotic volume in chloroplast membranes

The dependence of thylakoid osmotic volume on NH₄Cl uncoupling and on phosphorylation substrates is determined by the centrifuge filtration method. The values obtained are used to evaluate the transmembrane proton gradient in conjunction with either the 9-amino-acridine fluorescence quenching method or the [¹⁴C]methylamine uptake method. The pH values obtained with the two methods are compared and a linear relationship is demonstrated in the pH range from 1.4 to 2.7 ([¹⁴C]methylamine values). Different linear relationships are obtained depending on the presence or absence of electron acceptor. We conclude that the 9-aminoacridine method can be used for pH determination after calibration with other methods.     

Transport Properties of the Tomato Fruit Tonoplast : II. Citrate Transport

Citrate transport across the membrane of tomato fruit tonoplast vesicles was investigated. In the tonoplast vesicles, [¹⁴C]methylamine uptake was stimulated 10-fold by MgATP and strongly inhibited by NO₃⁻. Under identical experimental conditions, [¹⁴C]citrate uptake was inhibited by 5 millimolar free Mg²⁺, and this inhibition was reversed in the presence of ATP, presumably by ATP chelation of free Mg²⁺. No evidence was obtained in support of energy-linked ATP stimulation of citrate uptake. Citrate uptake showed saturation kinetics, and was inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid and by other organic acids. The pH-dependence of uptake suggested that citrate³⁻ was the transported species. Our results indicate that citrate transport across the tomato fruit tonoplast occurs by facilitated diffusion of citrate³⁻. The carrier shares some features in common with anion channels in that it is relatively nonspecific for organic acids and is inhibitable by 4,4′-diisothyocyano-2,2′-stilbenedisulfonic acid.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.