Amino acid esters of 9-(2′,3′-dihydroxypropyl-1′)-adenine are the specific inhibitors of protein synthesis on ribosomes

Peptide acceptor properties of phenylalanine and glycine esters of 9-(2,3-dihydroxypropyl-1)-adenine and 1-(2,3-dihydroxypropyl-1)-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[¹⁴C] Phe-tRNA in a ribosomal system charged with poly U. However, peptidyl esters of 9-(2,3-dihydroxypropyl-1)-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens.Abbreviations AcPhe acetyl-l-phenylalanine - HP-Ade 9-(2,3-dihydroxypropyl-1)-adenine - Phe-HP-Ade and Gly-HP-Ade l-phenylalanine and glycine esters of HP-Ade - Phe-HP-TUra l-phenylalanine ester 1-(2,3-dihydroxypropyl-1)-4-thiouracyl - AcPhePhe-HP-Ade and AcPheGly-HP-Ade acetyl-l-diphenylalanine and acetyl-l-phenylalanylglycine esters of HP-Ade respectively - AcPhe-puromycin acetyl-l-phenylalanyl-puromycin     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Beziehungen zwischen Carnitinstoffwechsel und Fettsäureassimilation bei Pseudomonas putida

Zusammenfassung Der Carnitinstoffwechsel und einige Beziehungen zum Fettsäurestoffwechsel wurden mittels der Wachstumskontrolle, der Bestimmung von Metaboliten und des Nachweises von Enzymaktivitäten in Pseudomonas putida untersucht. Der Stamm wuchs auf -Butyrobetain, D,L-und L-Carnitin, Glycinbetain, Cholin, D,L-Norcarnitin, D,L--Amino--hydroxybutyrat und D,L--Hydroxybutyrat. Obwohl der Stamm unverzweigte Fettsäuren von 2–16 C-Atomen zu untzen vermag, konnte er nur auf O-Acyl-L-carnitinen von 10 oder mehr C-Atomen in der Acylgruppe wachsen. Zugabe von Carnitin stimulierte das Wachstum auf langkettigen Fettsäuren.Die Bildung von Trimethylamin stieg, wenn Carnitin oder -Butyrobetain nur C-Quellen waren, und sank, wenn diese Trimethylammoniumverbindungen sowohl C-als auch N-Quellen waren. L-Carnitin induzierte sowohl die Carnitindehydrogenase als auch die -Hydroxybutyratdehydrogenase. -Butyrobetain als C-und N-Quelle induzierte ebenfalls die Carnitindehydrogenase. Im Rohextrakt betrug die spezifische Aktivität der -Hydroxybutyratdehydrogenase entsprechend dem Wachstum auf L-Carnitin oder D,L--Hydroxybutyrat 0,7 oder 1,6 Mol · min^-1 · mg^-1. Glycinbetain, Glucose und langkettige Fettsäuren reprimierten die Synthese beider Enzyme. Abhängig von der N-Quelle wird L-Carnitin offensichtlich auf zwei unterschiedlichen Stoffwechselwegen abgebaut.

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Interrelationships between carnitine metabolism and fatty acid assimilation in Pseudomonas putida

The carnitine metabolism and some relations to the fatty acid metabolism were studied in Pseudomonas putida by means of control of growth, analysis of metabolites, and determination of enzyme activities. The strain grew on -butyrobetaine, D,L-and L-carnitine, glycinebetaine, choline, D,L-norcarnitine, D,L--amino--hydroxybutyrate, and D,L--hydroxybutyrate. Although the strain used straight-chain fatty acids of 2–16 C-atoms, it was only able to grow on O-acyl-L-carnitines of 10 or more C-atoms in the acylgroup. Addition of carnitine stimulated the growth on long-chain fatty acids.The formation of trimethylamine increased, if L-carnitine or -butyrobetaine were the only carbon sources, and decreased, if these trimethylammonium compounds were carbon as well as nitrogen sources. L-Carnitine induced the carnitine dehydrogenase as well as the -hydroxybutyrate dehydrogenase. -Butyrobetaine as carbon and nitrogen source induced the carnitine dehydrogenase, too. In the crude extract the specific activities of -hydroxybutyrate dehydrogenase were 0.7 or 1.6 moles·min^-1·mg^-1 after growth on L-carnitine and D,L--hydroxybutyrate, respectively. The synthesis of both enzymes was repressed by glycinebetaine, glucose and long-chain fatty acids. Dependent on the nitrogen source L-carnitine was catabolized via two different pathways.

     

Glycosylation with thioglycosides activated by dimethyl(methylthio)sulfonium tetrafluoroborate: synthesis of two trisaccharide glycosides corresponding to the blood group A and B determinants

Two trisaccharide glycosides,p-trifluoroacetamidophenylethyl 3-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-2-O-(-l-fucopyranosyl)--d-galactopyranoside andp-trifluoroa-cetamidophenylethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group A and B determinants, were synthesized. A key fucosylgalactosyl disaccharide derivative was glycosylated with galactosaminyl or galactosyl donors, respectively. Dimethyl (thiomethyl)sulfonium tetrafluoroborate was used for thioglycoside activation in coupling reactions.     

Polycarnitine—a new biomaterial

The natural product l-carnitine is—due to its biotechnological accessibility and specific properties—on the way to becoming an attractive biobased bulk product. l-Carnitine is a natural betaine with vitamin properties. Carnitine is an essential part of the fatty acid metabolism of human beings and animals. Carnitine was first isolated in 1905 from meat extract and important recent developments include the biosyntheses of l-carnitine from l-lysine or -butyrobetaine. Our synthesis routes are designed to maintain the primary structure and specific properties of carnitine, such as hydrophilicity and stiffening effects for polymeric structures and applications. l-Carnitine is converted via lactonization or olefinization into polymerizable basic molecules. The properties and the applications of carnitine polymers are described.This revised version was published online in February 2005 with text corrections in the subsection Biotechnological production of L-carnitine of the Introduction section.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Preparation and properties of rainbow trout liver mitochondria

Summary Mitochondria prepared from rainbow trout liver consistently display high respiratory control and ADP/O ratios. These appear to possess a complete Krebs cycle, since pyruvate, palmitoyll-carnitine, citrate, and various Krebs cycle intermediates can all be oxidized. Rapid oxidation of pyruvate and palmitoyll-carnitine requires the pressence of malate. Oxidation of palmitoyll-carnitine appears to inhibit pyruvate oxidation. Malate stimulates -ketoglutarate oxidation while aspartate inhibits glutamate oxidation, indicating the presence of malate--ketoglutarate and glutamate-aspartate carriers. These properties are compared with those of liver mitochondria from other species of fish.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Ochrobactrum anthropi NCIMB 40321: a new biocatalyst with broad-spectrum l-specific amidase activity

Of 125 microorganisms that were able to use -hydroxy acid amides as sole nitrogen source, Ochrobactrum anthropi NCIMB 40321 was selected for its ability to hydrolyse racemic amides l-selectively. The substrate specificity of whole O. anthropi cells is remarkably wide and ranges from -H--amino-, -alkyl--amino, N-hydroxy--amino acid amides to -hydroxy-acid amides. After 50% conversion, both the l-acids formed and the remaining d-amides were present in >99% enantiomeric excess, and ammonia accumulated in stoichiometric amounts. Using mandelic acid amide as a model substrate, the hydrolysis was optimized. Optimal rates were observed at pH 8.5 at 50°C. At higher temperatures the initial rate was even higher; however, fairly rapid inactivation occurred.     

Effects of α-galactosidase digestion on lectin staining in human pancreas

Summary Effects of -galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B₄(GSAI-B₄) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B₄ positive acinar cells. -Galactosidase digestion following -galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B₄ positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple -galactosidase digestion as well as sequential digestion with - and -galactosidase. However, when -l-fucosidase digestion procedure was inserted between - and -galactosidase digestion, UEA-I staining imparted by -galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B₄ positive acinar cells. Furthermore, after sequential digestion with -galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B₄ positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion. These results confirmed that the -galactosidase induced GSA-II reactivity and the fucosidase induced PNA reactivity are due to precursors of different kinds of blood-group determinants and suggest that at least two kinds of B antigen determinants, i.e. Gal(1-3)[Fuc(1-2)]Gal(1-3,4)GlcNac and Gal(1-3)-[Fuc(1-2)]Gal(1-3)GalNAc are produced in GSAI-B₄ positive acinar cells. The synthesis of the latter type of B antigen is assumed to be controlled under the secretory gene in human pancreas.Abbreviation GalNAc N-acetyl-d-galactosamine - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - NeuNAc N-acetylneuraminic acid (sialic acid)     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Interaction of α2-macroglobulin with alkaline proteinase from an alkalophilicBacillus sp. grown in an extraordinarily alkaline environment

The interaction of ₂-macroglobulin (₂M) with an alkaline serine proteinase (ALPase I) from alkalophilicBacillus sp. grown in an extraordinarily alkaline environment was investigated. Stoichiometry of the reaction showed that ALPase I bound to ₂M in a molar ratio of about 21. The ₂M-ALPase I complex showed about 80% of the proteinase activity shown by ALPase I in the hydrolysis of succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and casein. The conformational changes in the ₂M molecule caused by the complex formation at pH 7.5 were determined from electron micrographs and difference spectra. The antigenic activity of the ₂M-ALPase I complex with the anti-ALPase I antiserum was found to be completely abolished. Immunoelectrophoresis of the complex incubated at pH 7.5 after 48 h showed no appreciable change, and the complex was recognized as exhibiting enhanced stability at pH 7.5.     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.