β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Acetyl-l-carnitine attenuates neuronal damage in gerbils with transient forebrain ischeia only when givenBefore the insult

The underlying mechanisms leading to neuronal damage in cerebral ischemia are multifactoral. In this study, we evaluated the neuroprotective effects of acetyl-l-carnitine, a medication that may enhance metabolic recovery after cerebral ischemia. The 5-minute transient forebrain ischemia model in gerbils was used. Acetyl-l-carnitine was given 30 minutes before the insult in one set of animals and 30 minutes after the insult in a second set of animals with histological evaluation at 7 days (Group A) and 28 days (Group B). Damage assessment was done using a 4-point damage score and Mann-Whitney U test was used for statistical analysis. Compared to the controls, there was significant protection in the cerebral cortex, hippocampus and the striatum in animals treated with the medicationbefore the insult in Group A and Group B Post-ischemic therapy showed little evidence of neuronal protection in either group. Behavioral tests in the Group B animals showed no significant differences between the treated or the saline controls. Our study shows, that pre-ischemic treatment with acetyl-l-carnitine results in neuronal protection. This may have clinical significance in situations (such as bypass surgery) where treatment could be initiatedprior to the insult.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

D-penicillamine affects lipid peroxidation and iron content in the rat brain cortex

d-Penicillamine, a trifunctional aminoacid known for its ability to form metal complexes and for being a radical scavenger, has been investigated in vitro and in vivo in the rat brain cortex. At 50 M the drug facilitate lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of thed-penicillamine (25 and 50 mg/Kg i.p) was evaluated in vivo after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated withd-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe²⁺/Fe³⁺ ratio was significantly different from that of normal rats. On the contrary ratios obtained formd-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest thatd-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agents, can remove metal from action sites where lipid peroxidation may occur.     

What is happening to the monocotyledons?

An attempt is made to elucidate some of the more pronounced departures from traditional classifications in the book The families of the monocotyledons byDahlgren and collaborators, which embodies the latest opinions of the lateRolf Dahlgren on the subject. Consideration is given to the treatment of theLiliiflorae (especiallyLiliales andAsparagales) andBromeliiflorae, and to the theory offered for the origin of the monocots which identifies theDioscoreales as the most primitive order of the subclass.Dahlgren aimed at an eclectic classification (one based on a combination of similarity criteria and phylogenetic criteria) and the results of his use of cladistic methods (in association withF. N. Rasumssen) to supply the phylogenetic input are assessed. The resulting system itself is considered in the light of the distinction drawn byJ. S. L. Gilmour between natural and artificial classifications and their respective uses.Dedicated to the memory of JohnS. L. Gilmour.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Gonadal and adrenal steroids regulate neurochemical and structural plasticity of the hippocampus via cellular mechanisms involving NMDA receptors

Summary 1. The hippocampus is an important brain structure for working and spatial memory in animals and humans, and it is also a vulnerable as well as plastic brain structure as far as sensitivity to epilepsy, ischemia, head trauma, stress, and aging.2. The hippocampus is also a target brain area for the actions of hormones of the steroid/thyroid hormone family, which traditionally have been thought to work by regulating gene expression. Genomic actions of steroid hormones involve intracellular receptors, whereas nongenomic effects of steroids involve putative cell surface receptors. Although this distinction is valid, it does not go far enough in addressing the variety of mechanisms that steroid hormones use to produce their effects on cells. This is because cell surface receptors may signal changes in gene expression, while genomic actions sometimes affect neuronal excitability, often doing so quite rapidly.3. Moreover, steroid hormones and neurotransmitters may operate together to produce effects, and sometimes these effects involve collaborations between groups of neurons. For example, a number of steroid actions in the hippocampus involve the coparticipation of excitatory amino acids. These interactions are evident for the regulation of synaptogenesis by estradiol in the CA1 pyramidal neurons of hippocampus and for the induction of dendritic atrophy of CA3 neurons by repeated stress as well as by glucocorticoid injections. In addition, neurogenesis in the adult and developing dentate gyrus is contained by adrenal steroids as well as by excitatory amino acids. In each of these three examples, NMDA receptors are involved.4. These results not only point to a high degree of interdependency between certain neurotransmitters and the actions of steroid hormones, but also emphasize the degree to which structural plasticity is an important aspect of steroid hormone action in the adult as well as developing nervous system.     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl -cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R) reagents where R is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding -S-(-aminoethanethiol) and -S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)Glc, lacto-N-neodifucohexaose Gal1-4(Fuc1-3)Glc-NAc1-3Gal1-4(Fuc1-3)Glc, and lacto-N-neodifucooctaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)GlcNAc1-3Gal1-4(Fuc1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an -1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon.To prevent fucosylation at the glucosyl residue, lactulose Gal1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Endogenous sugar receptor pattern in human glioblastomas and gangliocytomas studied by histochemical application of biotinylated (neo)glycoproteins and affinity chromatography

Summary Biotinylation of chemically glycosylated bovine serum albumin, yielding a panel of neoglycoproteins, and of desialylated, naturally occurring glycoproteins allowed to systematically evaluate presence and distribution of various types of endogenous sugar receptors in the sections of human glioblastomas and gangliocytomas by a routine histochemical procedure. Pronounced cytoplasmic staining with markers, carrying constituents of natural glycoconjugates, e.g. for -galactoside-specific receptors, contrasted with the different intensities, noticed for - and -glucosidespecific receptors. Significant qualitative differences between the two tumor types were detected with N-acetyl-D-galactosamine-and sialic acid-carrying probes. Nuclear staining with only a part of the applied panel underscored the specificity of the protein-carbohydrate interaction. Fine structural features of the synthetic neoglycoproteins, e.g. the mode of coupling of the carbohydrate moiety to the protein, were found to exert a significant influence on their suitability as histochemical markers. On the basis of the histochemical results, exemplary biochemical analysis of certain classes of endogenous sugar receptors by affinity chromatography and subsequent gel electrophoresis, namely of -galactoside-, -fucoside-, -mannoside- and -glucoside-specific proteins, revealed presence and characteristics of respective sugar receptors that can contribute to the histochemical staining. Similar extent of histochemical staining with the respective probes notwithstanding, the different tumor types exhibited qualitative differences in the expression of individual endogenous sugar receptors. The combined histochemical and biochemical analysis is supposed to be of conspicuous value for biological and clinical investigations on endogenous sugar receptors.     

In vitro culture of switchgrass: Influence of 2,4-d and picloram in combination with benzyladenine on callus initiation and regeneration

The objective of this study was to determine the types and concentrations of auxins and cytokinins that would result in effective culture initiation and plant regeneration of switchgrass (Panicum virgatum L.). Whole caryopses and young seedling explants were cultured on MS medium containing different concentrations of picloram or 2,4-dichlorophenoxyacetic acid (2,4-d) in combination with benzyladenine. Best results from mature caryopses were obtained with 11.3–45.0 M 2,4-d in combination with 15.0 or 45.0 M benzyladenine (BA). More than 300 plants were obtained per embryogenic callus from some treatments after the second transfer to regeneration medium (90 days after initiation of the cultures). Regeneration was obtained from young seedling explants with both auxins. However, picloram was more effective over a wider range of BA concentrations than 2,4-d. Protocols developed during this study were used to regenerate hundreds of plants which could easily be established in the field.     

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.     

Alteration of Phosphoinositide Degradation by Cytosolic and Membrane-Bound Phospholipases after Forebrain Ischemia - Reperfussion in Gerbil: Effects of Amyloid Beta Peptide

The reperfusion of previously ischemic brain is associated with exacerbation of cellular injury. Reperfusion occasionally potentates release of intracellular enzymes, influx of Ca²⁺, breakdown of membrane phospholipids, accumulation of amyloid precursor protein or amyloid -(like) proteins, and apolipoprotein E. In this study, the effect of reperfusion injury on the activity of cerebral cortex enzymes acting on phosphatidyl [³H] inositol (PI) and [^l4C-arachidonoyl] PI was investigated. Moreover the effect of amyloid 25–35 on PI degradation by phospholipase(s) of normoxic brain and subjected to ichemia-reperfussion injury was determined. Brain ischemia in gerbils (Meriones unguiculatus) was induced by ligation of both common carotid arteries for 5 min and then brains were perfused for 15 min, 2 h and 7 days. Statistically significant activation of enzyme(s) involved in phosphatidylinositol degradation in gerbils subjected to ischemia-reperfusion injury was observed. Nearly all gerbils showed a higher activity of cytosolic PI phos-pholipase C (PLC) at 15 min after ischemia. Concomitantly, the significant enhancement of the level of DAG and AA radioactivity at this short reperfusion time confirmed the active PI degradation by phospholipase(s) in cerebral cortex and hippocampus. After a prolonged reperfusion time of 7 days after ischemia, both cytosolic and membrane-bound forms of PI-PLC were activated. The question arises if alteration of membranes by the degradation of phospholipids occurring after an ischemic episode potentates the effect of A on membrane-bound enzymes. A neuro-toxic fragment of amyloid, A 25–35, incubated in the presence of endogenous Ca²⁺, increased significantly the PI-PLC activity of normoxic brain. In its non-aggregated form, A 25–35 activates PI-PLC but in the aggregated form the enzymatic activity decreased. Thus, A 25–35 exerts a similar effect on the membrane-bound PI-PLC from normoxic brain or subjected to ischemia reperfussion injury. We conclude that the degradation of phosphatidylinositol by cytosolic phosphoinositide-phospholipase C may contribute to the pathophysiology of delayed neuronal death following cerebral ischemia. Thus, a specific inhibitor of this enzyme(s) may offer therapeutic strategies to protect the brain from damage triggered by ischemia. Ischemia-reperfusion injury had no effect on A-evoked alterations of synaptic plasma membrane-bound PI-PLC.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

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.