Carnitine synthesis in rat tissue slices

The ability of rat liver, kidney, muscle, heart and testis tissue to carry out the $\text{in vitro}$ synthesis of carnitine via ε-N-trimethyllysine and γ-butyrobetaine was studied. All tissues formed γ-butyrobetaine from trimethyllysine, but liver and testis also formed carnitine in about 7% and 1% yield respectively. Liver slices formed trimethyllysine from lysine in about 6% yield. These $\text{in vitro}$ studies thus establish that liver has all the enzymes of the carnitine biosynthetic pathway. This tissue appears to be the primary site of carnitine synthesis in the rat as implied from whole animal studies in this and other laboratories.     

Metabolism and excretion of carnitine and acylcarnitines in the perfused rat kidney

Rat kidneys were perfused for 30 min with a Krebs-Henseleit bicarbonate buffer with 5 mM glucose. Albumin proved superior to pluronic polyols as oncotic agent with regard to carnitine reabsorption in the perfused kidney. The reabsorption of 30 μM (−)-[methyl-³H]carnitine was approx. 96% during the first 10 min. At 750 μM the reabsorption decreased to 40%. The tubular reabsorptive maximum (T_max) was approx. 170 nmol/min per kidney. The fractional reabsorption and clearance of (+)-carnitine, γ-butyrobetaine, and carnitine esters did not deviate significantly from that of (−)-carnitine. (+)-Carnitine was not metabolized by the perfused kidney. In perfusions with (−)-carnitine or (−)-carnitine plus 10 mM α-ketoisocaproate or α-ketoisovalerate increased amounts of acetylcarnitine, isovalerylcarnitine and isobutyrylcarnitine were found. Propionate (5 mM) inhibited acetylcarnitine formation. Isovalerylcarnitine, isobutyrylcarnitine and propionylcarnitine were actively degraded to free (−)-carnitine. In urine, we found a disproportionally high excretion of carnitine or carnitine esters formed in the kidney, compared to the same derivatives when ultrafiltrated. Leakage of metabolites formed in the kidney into preurine may explain this phenomenon.     

Uncoupling and isotope effects in γ-butyrobetaine hydroxylation

Replacement of unlabeled -butyrobetaine with -[2,3,4-²H₆]butyrobetaine has a profound effect on the stoichiometry between decarboxylation of 2-oxoglutarate and hydroxylation in the reaction catalyzed by human -butyrobetaine hydroxylase. The ratios between decarboxylation and hydroxylation are 1.16 with Unlabeled and 7.48 with deuterated -butyrobetaine as substrate. From these ratios an internal isotope effect of 41 has been calculated. DV in the overall reaction measured as 2- oxoglutarate decarboxylation is 2.5 and D_V/K is 1.0. For -butyrobetaine hydroxylase fromPseudomonas sp. AK 1, 2-oxoglutarate decarboxylation exceeds hydroxylation with 10% when deuterated -butyrobetaine is used. No excess was found with unlabeled substrate and no internal isotope effect could be calculated. D_V for the bacterial enzyme is 6.     

gamma-Butyrobetaine hydroxylation to carnitine in mammalian kidney.

Activity of γ-butyrobetaine hydroxylase (γ-butyrobetaine, 2-oxoglutarate dioxygenase; EC 1.14.11.1) in liver and kidney of several mammalian species was assayed by measurement of tritium release from γ-[2,3-³H]butyrobetaine. Crude extracts from cat, hamster, rabbit, and Rhesus monkey kidneys effectively converted γ-butyrobetaine to carnitine. In these species, the levels of hydroxylating activity in kidney exceeded or nearly equaled the level of γ-butyrobetaine hydroxylase activity in the corresponding liver. In contrast, dog, guinea pig, mouse, and rat kidney exhibited no or insignificant capacity to hydroxylate γ-butyrobetaine. The notion that the liver is the exclusive or primary site of carnitine synthesis must be reconsidered at least for some mammalian species.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Purification and characterization of d(+)-carnitine dehydrogenase from Agrobacterium sp. — a new enzyme of carnitine metabolism

d(+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of d(+)-carnitine to 3-dehydrocarnitine as initial step of d(+)-carnitine degradation. The NAD⁺-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7–5.0. The optimum temperature is 37°C and the optimum pH for the oxidation and the reduction reaction are 9.0–9.5 and 5.5–6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of d(+)-carnitine (l(−)-carnitine, crotonobetaine, γ-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of d(+)-carnitine oxidation. The equilibrium constant of the reaction of d(+)-carnitine dehydrogenase was determined to be 2.2 × 10⁻¹². The purified d(+)-carnitine dehydrogenase has similar kinetic properties to the l(−)-carnitine dehydrogenase from the same microorganism as well as to l(−)-carnitine dehydrogenases of other bacteria.     

Synthesis of l(-)-carnitine by hydration of crotonobetaine by enterobacteria

Summary Enterobacteria, especially Escherichia coli, Salmonella typhimurium and Proteus vulgaris, are capable of forming l(-)-carnitine by hydration of the double bond of crotonobetaine under anaerobic conditions. The carnitine hydrolyase is an inducible cytosolic enzyme which catalyses either the dehydration of l-carnitine or the hydration of crotonobetaine. In growing cultures, the addition of fumarate to a complex or minimal medium stimulated l-carnitine synthesis by diminishing the reduction of crotonobetaine to -butyrobetaine. However, l-carnitine synthesis was repressed after addition of nitrate or under aerobic conditions. If the carnitine hydrolyase was induced by l-carnitine or crotonobetaine, these respiratory chain electron acceptors did not impair carnitine formation by resting cells, indicating an epigenetical regulation of carnitine synthesis. Using this bacterial pathway for the biosynthesis of l-carnitine, conditions for producing a high yield are described. The method has some advantages in comparison with other biochemical or microbiological procedures for the production of l-carnitine.Dedicated to Professor Dr. H.-J. Rehm on the occasion of his 60th birthday     

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent K_m for γ-butyrobetaine and higher apparent V_max, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.     

Synthesis of carnitine from ε-N-trimethyllysine in post mitochondrial fractions of Neurospora crassa

An enzyme system in the post mitochondrial fraction of $\text{Neurospora}$$\text{crassa}$ when supplemented with appropriate cofactors formed carnitine from ε-N-trimethyllysine. These findings together with previous studies of ε-N-lysine methylation in this fungi, illustrate that carnitine synthesis in $\text{Neurospora}$ differs markedly in certain features from mammalian systems in that the entire synthesis is carried out employing free intermediates and cytosolic enzymes.     

Evidence for allosteric NADH regulation of Acinetobacter alpha-oxoglutarate dehydrogenase from multiple-inhibition studies.

Previous studies have suggested that the E1 component (α-oxoglutarate dehydrogenase) of the α-oxoglutarate dehydrogenase enzyme complex from $\text{Acinetobacter lwoffi}$ is inhibited by the end-product NADH. We have now carried out multiple-inhibition studies in the simultaneous presence of NADH and α-oxoadipate, both competitive inhibitors with respect to α-oxoglutarate. The results indicate that NADH acts at an allosteric site within the multi-enzyme complex.     

PPARα-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPARα−/− mice, indicating that PPARα is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPARα-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPARα−/− mice. Here we show that both enhancement of carnitine biosynthesis (due to increased γ-butyrobetaine dioxygenase activity), extra-hepatic γ-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPARα-dependent.     

Crotonobetaine reductase fromEscherichia coli — a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase fromEscherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, -butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, -butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40–45 °C). The K _m value for crotonobetaine was determined to be 1.1×10^–2M. -Butyrobetaine,^D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For -butyrobetaine (K _i =3×10^–5M) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.

     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Conversion of gamma-butyrobetaine to L-carnitine by Achromobacter cycloclast

L -Carnitine is an ubiquitous substance that plays a major role in the transportation of long-chain fatty acids. We investigated crucial factors that influence microbial conversion of γ-butyrobetaine to L-carnitine using an Achromobacter cycloclast strain. Two-stage culture results showed that γ-butyrobetaine induced enzymes essential for the conversion, which suggests that the precursor should be present in the initial cell growth stage. The addition of yeast extract enhanced L-carnitine production whereas inorganic nitrogen sources inhibited it. Under nitrogen-limiting conditions, the cells accumulated poly-β-hydroxybutyrate instead of L-carnitine. Among the trace elements tested, nickel addition enhanced L-carnitine production by almost twice that of the control and copper strongly inhibited the conversion. L-Carnitine production was reduced when the medium contained inorganic salts of sodium, potassium, and calcium at a concentration greater than 2 g l⁻¹. A higher L-carnitine yield was achieved when cells were incubated in a lower culture volume. The optimal pH for L-carnitine production was 5 to 5.5, whereas that of growth was 7.0, indicating that a pH shift was required. Under optimal conditions, L-carnitine concentrations as high as 15 g l⁻¹ were obtained in 62 h with a 45% molar conversion yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 309–315. Received 26 November 2000/ Accepted in revised form 27 February 2001     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.