Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

How does l-glutamate transport relate to selection of mixed nitrogen sources in Rhizobium leguminosarum biovar trifolii MNF1000 and cowpea Rhizobium MNF2030?

When growing on a mixture of ammonia and l-glutamate as nitrogen sources, Rhizobium leguminosarum biovar trifolii MNF1000 utilizes ammonia exclusively, while cowpea Rhizobium MNF2030 utilizes both compounds at similar rates. l-Glutamate transport in both strain MNF1000 and MNF2030 is active, giving rise to a 60-fold concentration gradient across the membrane of cells of strain MNF2030. Both strains produce two kinetically distinguishable glutamate transport systems under all conditions of growth — a high affinity system with an apparent K _m of 0.06–0.17 M but of relatively low V _max, and a low affinity system with a K _m of 1.2–6.7\ M, but of higher overall capacity. l-Glutamate transport activity in cells of MNF2030 was relatively insensitive to the presence of ammonia in the growth medium. By contrast, ammonia in the growth medium resulted in low activities of glutamate transport in cells of MNF1000 which were provided with a carbon source, offering one explanation for the failure of this strain to use glutamate in the presence of ammonia. However, in cells of MNF1000 growing on glutamate as sole source of carbon and nitrogen, the glutamate transport system is synthesized, even in the presence of accumulated or added ammonia. This suggests that the regulation of the glutamate permease also depends on availability of carbon source.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Lactic acid excretion via carrier-mediated facilitated diffusion in Lactobacillus helveticus

Summary During growth on a complex medium containing 2% (w/v) lactose, Lactobacillus helveticus produced about 180 mm lactate. Due to the acidification, the external pH decreased to 3.7. The pH remained constant at a level of 0.5–0.7 units (40 mV), and µ_Lac decreased gradually from –60 to 0 mV. The mechanism of lactate extrusion was studied with resting cells. Upon dilution of lactate-loaded cells in a buffer containing [¹⁴C]-lactate, a typical counterflow was observed, suggesting that a carrier system was employed in lactate excretion. Influx of lactate could not be driven by an artificial membrane potential, indicating that lactate was electroneutrally transported. By examining efflux under various lactate anion and lactic acid concentrations, the undissociated form of the acid was shown to influence the velocity of the transport process. A pH-dependent apparent K _m value of the carrier system was observed in efflux experiments with increasing internal lactate concentrations. It was concluded that the mode of end-product excretion can be defined as a carrier-mediated facilitated diffusion with the undissociated lactic acid or the lactate anion in symport with one proton, respectively, as the object of transport.Abbreviations L _tota total lactate - L _undissb free lactic acid - L _dissc lactate anion - pH_ed external pH - pH_ie internal pH - pH transmembrane H⁺ gradient - µ_Lacf transmembrane gradient of total lactate - µ_HLg transmembrane gradient of the free lactic acid - µ_Lh transmembrane gradient of the lactate anion - V _Effii efflux velocity Offprint requests to: G. Gottschalk     

Obtaining functional membrane vesicles from Leuconostoc oenos to study l-malate transport mechanisms

Membrane vesicles from the malolactic bacterium Leuconostoc oenos were obtained by a modified version of the procedure of Kaback [Methods Enzymol 22:99–120 (1971)]. Protoplasts were produced at frequencies greater than 95% by a method entailing mutanolysis digestion and osmotic shock. Glycerol or polyethyleneglycol 600 was required as an osmotic stabilizer while the use of sucrose prevented closed vesicle formation during osmotic shock. The membrane vesicles retained their functional properties and accumulated l-malic acid in response to an ATPase-induced proton gradient across the membrane of ATP-loaded vesicles. l-Malate uptake was strongly inhibited by dicyclohexylcarbodiimide, a specific inhibitor of membrane-bound ATPase. These data support the possibility of a pH-dependent transport of l-malate. Vesicles not loaded with ATP were slightly permeable to malic acid with an initial uptake rate (0.5 nmol·l^–1·s^–1) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These results confirm two simultaneous uptake mechanisms in L. oenos, a permease-mediated transport and a passive diffusion for the anionic and the undissociated forms of l-malic acid respectively.     

Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Transport of lactate and other short-chain monocarboxylates in the yeast Candida utilis

Summary Lactic acid grown cells of the yeast Candida utilis transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoicheiometry of 1:1. The accumulation ratio at pH 5.5 was about twenty. The symport accepted the following monocarboxylates (K _svalues at 25°C, pH 5.5 in brackets): d-lactate (0.06 mM), l-lactate (0.06 mM), pyruvate (0.03 mM), propionate (0.05 mM) and acetate (0.1 mM). The system was inducible and was subject to glucose repression. The affinity of the symport for lactate was not affected by pH over the range 3–6, while the maximum transport velocity was strongly pH dependent, its optimum pH being around pH 5. Undissociated lactic acid entered the cells by simple diffusion. The permeability for the undissociated acid increased exponentially with pH, the diffusion constant increasing 35-fold when the pH was increased from 3 to 5.5.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Mode of action of furosemide on the chloride-dependent short-circuit current across the ciliary body epithelium of toad eyes

Summary The effects of furosemide on the chloride-dependent short-circuit current across the toad ciliary epithelium were examined. Under control conditions, the short-circuit current obeyed Michaelis-Menten kinetics against medium chloride concentration, the Michaelis constant (K _m ) for chloride being 90mm and the maximal short-circuit current (V _max) 128 A/cm². Furosemide added to the aqueous side of the epithelium rapidly reduced the short-circuit current; the effect was reversible. The effect of furosemide addition to the stromal side was much smaller and slower than that from the aqueous side. The dose-dependent range of furosemide action was from 0.1 m to 1mm with 50% inhibition occurring at about 3 m. Line-weaver-Burk plot of the short-circuit current against the chloride concentration showed that furosemide decreased the value ofV _max and increased theK _m ; the inhibition being of mixed type. A Hill plot of the dose-response curve yielding a slope of unity suggested one furosemide molecule combines with one chloride transport site. Probenecid, a competitive inhibitor of organic acid transport, reduced the effects of furosemide significantly when added simultaneously. The involvement of organic acid transport system in the mechanism of furosemide action on chloride transport was suggested.Department of Ophthalmology.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Zum Abbau derl-Äpfelsäure durch Milchsäurebakterien

Zusammenfassung Es wird die Aktivität von intaktenL. plantarum-Zellen verschiedener Arten gegenüberl-Äpfelsäure, Oxalessigsäure und Brenztraubensäure getestet. Bei der Dissimilation vonl-Äpfelsäure lassen sich zwei pH-Optima unterscheiden, 2,6–3,0 für eine MDH-Aktivität und 3,6–4,0 für eine Malic-Enzym-Aktivität. Stoffwechselprodukte der Brenztraubensäure-Decarboxylierung sind außer Kohlendioxid Äthylalkohol und Acetoin bzw. Diacetyl.L. plantarum ist außerdem zur Oxydation der Brenztraubensäure befähigt. Ausl-Äpfelsäure entsteht kein Acetoin (Stamm L).

---

The dissimilation ofl-malic acid by lactic acid bacteriaIV. The activity of intact cells ofLactobacillus plantarum particularly considering the decarboxylation of pyruvic acid

Summary The decomposition ofl-malic, oxaloacetic and pyruvic acids by intact cells of three strains ofL. plantarum is investigated. The dissimilation ofl-malic acid shows two pH-optima, at pH2.6–3.0 for a malatedehy drogenase activity and at pH 3.6–4.0 for a malic enzyme activity. The decarboxylation of pyruvic acid yields CO₂, ethyl alcohol, acetoin and diacetyl.L. plantarum is also able to oxidize pyruvic acid. The acetoin produced byL. plantarum Strain L does not originate froml-malic acid.

     

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.     

Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of l-cysteine to l-cystine. The pH-optimum of this reaction was determined to be around 8–8.5 and a stoichiometry of 4 SH-groups oxidized for one O₂ consumed was obtained. This thiol-oxidation system was specific for D-and l-cysteine; Dl-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent K_m for l-cysteine was determined as 3.3 mmol l^-1. Rates of 200 mol cysteine oxidized mg^-1 chlorophyll h^-1 were normally obtained. Extremely high rates of oxygen uptake were measured using l-cysteine methyl ester and l-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N-disalicyliden-1,3-diaminopropane 5,5-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O₂ (dark inactivation).Abbreviation DTNB 5,5-dithio-bis(-2-nitrobenzoic acid). Ellmann reagent     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

The immune response of allophenic mice to the synthetic polymer GLΦ

The immune response of allophenic mice of type C57BL/6(A × SJL) F₁ to GL administered in complete Freund's adjuvant was tested. Control mice of the three strains C57BL/6, A, and SJL are all nonresponders to this antigen. However, the F₁ generations of C57BL/6 × A, C57BL/6 × SJL, and A × SJL were all responders to the antigen, so that the complementarity of at least two genes is confirmed. The allophenic mice showed no further complementation beyond the F₁ generation, a result which may argue against the possibility that more than two genes control the response to GL in these mouse strains. Characterization of the allophenic mice over several months showed that they exhibit chimeric drift, both in their coat color and in peripheral white blood cell population. There is no apparent correlation of coat color to the lymphocyte composition of the mice at any one time. The mice are true chimeras, since killing of the two populations of white blood cells with two different anti-H-2 sera produced a 100 percent killing. The immune response of individual allophenic mice to GL showed a good correlation to the number of A × SJL lympho-cytes in the animal.Abbreviations used in this paper are GL an amino acid polymer of 57 %l-glutamic acid, 38%l-lysine, and 5%l-phenylalanine - GLT¹⁵ an amino acid polymer ofl-glutamic acid,l-lysine, and 15 %l-tyrosine - (T,G)-A-L an amino acid polymer having a polylysine backbone with side chains of polyd-l-alanine, terminating in short sequences of tyrosine and glutamic acid - GAT₁₀ an amino acid polymer of 60%l-glutamic acid, 30%l-alanine, and 10%l-tyrosine - GLA₅ an amino acid polymer of 57%l-glutamic acid, 38%l-lysine, and 5%l-alanine - DNP 2,4 dinitrophenyl - BGG bovine gamma globulin - FCS fetal calf serum - PWBC peripheral white blood cell - SWBC spleen white blood cell - T cell thymus-derived lymphocyte - B cell bone marrow-derived lymphocyte