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.     

Molecular and functional characterization of organic cation/carnitine transporter family in mice

Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na⁺-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na⁺-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K_m=18.7 μM; V_max=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K_m=11.5 μM and V_max=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na⁺-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na⁺-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as ³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.     

Functional characterization of the carnitine transporter defective in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. Here we report the functional characterization of this transporter in human fibroblasts. Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake. Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency. In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+. Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine. Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters. A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range. These result indicate the presence of multiple types of carnitine transporters in human cells.     

Identification of two novel mutations in OCTN2 of three patients with systemic carnitine deficiency

Systemic carnitine deficiency is a potentially lethal, autosomal recessive disorder characterized by cardiomyopathy, myopathy, recurrent episodes of hypoketotic hypoglycemia, hyperammonemia, and failure to thrive. This form of carnitine deficiency is caused by a defect in the active cellular uptake of carnitine, and the gene encoding the high affinity carnitine transporter OCTN2 has recently been shown to be mutated in patients suffering from this disorder. Here, we report the underlying molecular defect in three unrelated patients. Two patients were homozygous for the same missense mutation 632A→G, which changes the tyrosine at amino acid position 211 into a cysteine (Y211C). The third patient was homozygous for a nonsense mutation, 844C→T, which converts the arginine at amino acid position 282 into a stop codon (R282X). Reintroduction of wild-type OCTN2 cDNA into fibroblasts of the three patients by transient transfection restored the cellular carnitine uptake, confirming that mutations in OCTN2 are the cause of systemic carnitine deficiency. Electronic Publication     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

Quaternary nitrogen compounds affect carnitine distribution in rats. Particular emphasis on edrophonium

Edrophonium (ethyl(m-hydroxyphenyl)dimethylamine) acutely modifies carnitine levels in different rat tissues, increasing hepatic and reducing blood and renal levels. After 2 h edrophonium treatment, the total serum carnitine levels were decreased by 16 (P < 0.001) and 33 (P < 0.001) percent in fed and fasted rats respectively compared to control, and in kidney the levels decreased by 11 (P < 0.05) and 34 (P < 0.001) percent, whereas in liver the edrophonium treatment increased the levels by 43 (P < 0.001) and 59 (P < 0.001) percent. The edrophonium action does not depend on the route of administration or on the nutritional state of the animal. Its activity on carnitine levels is neither accompanied by significant variation of serum parameters of carbohydrate, fat and protein metabolism nor of insulin levels. The edrophonium activity is not related to cholinergic action, as physostigmine and ambenonium at concentrations known to increase cholinergic activity do not modify carnitine distribution in tissues. Trimethylphenylammonium (TPA) and trimethyl(p-aminophenyl)ammonium (TPA · NH₂), compounds structurally similar to edrophonium, are on the contrary active on levels of carnitine and this effect is not related to their cholinergic potency. In 24 h fasted rats after the TPA and TPA. NH₂ treatment, the total serum carnitine levels were decreased by 32 (P < 0.001) and 13 (n.s.) percent respectively compared to control, and in kidney the levels decreased by 15 (P < 0.02) and 5 (n.s.) percent, whereas in liver the treatment increased the levels by 72 (P < 0.001) and 45 (P < 0.01) percent. Moreover atropine, an acetylcholine antagonist, affects carnitine distribution in a way similar to edrophonium. Edrophonium activity on carnitine distribution, probably affects (inter)cellular carnitine transport by direct action on plasma membrane. Effect on capillary endothelium may be responsible for its observed action on muscle contraction force in imminent ischemia.     

Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K_m of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a K_m for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a K_m for carnitine uptake of 1 to 3 μM and a V_max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.     

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist

In the brain β-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial β-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B^0,+ and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor α (PPARα), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter—OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARα activator–2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARα stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.     

Muscle contraction increases carnitine uptake via translocation of OCTN2

Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL(uptake)) of l-[(3)H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL(uptake) of [(14)C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL(uptake) of l-[(3)H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL(uptake) of [(14)C]IAP was much higher than that of l-[(3)H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane.     

Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat

P₂ fractions from brains of genetically seizure-susceptible (SS) rats as compared to seizure-resistant (SR) rats show decreased high affinity uptake of taurine. Uptakes of GABA and glutamate into P₂ fractions did not differ between the substrains. In neonatal SS rats that had never had a seizure, the uptake of taurine is decreased both into the whole brain in vivo and into P₂ fraction in vitro, as compared to age-matched SR rats. This indicates that decreased uptake is not a consequence of seizure activity per se. In non-seizure susceptible progeny of SS rats, the uptake of taurine into P₂ fraction did not differ significantly from that of SR rats. In kidney cortex slices from SS rats, taurine uptake is slightly greater than in slices from SR rats. We propose that the decreased taurine transport in the P₂ fraction of the brains of SS rats may reflect a defect in transport in vivo that contributes to seizure-susceptibility.     

Active-site-directed inhibition of carnitine acetyltransferase

Methoxycarbonyl-CoA disulfide has been used as an active-site-directed inhibitor of carnitine acetyltransferase. Stoichiometric addition of methoxycarbonyl-CoA disulfide to carnitine acetyltransferase showed the modification of one sulfhydryl group with concomitant loss of about 80% enzyme activity. The rate of modification of this sulfhydryl group is an order of magnitude faster than that of the remaining sulfhydryl groups in the enzyme. Methoxycarbonyl-CoA disulfide inactivation is biphasic: k₁ = 1.09 × 10²m^?1s^?1, k₂ = 1.1 × 10¹m^?1s^?1. This modification, Enz-SS-CoA is covalent; it can be reversed with either dithioerythritol or thiocholine. Acetyl-carnitine and acetyl-CoA protected the enzyme against methoxycarbonyl-CoA disulfide inactivation; however, carnitine did not. These results indicate the presence of a sulfhydryl group in carnitine acetyltransferase at the site of acetyl group transfer. Titration of carnitine acetyltransferase with nonspecific sulfhydryl reagents, DTNB, and ?-nitrophenoxycarbonyl methyl disulfide, revealed that four sulfhydryl groups were preferentially modified by these reagents. The results also show that seven other sulfhydryl groups are available for modification.     

Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent K_m of 18 μM) and carnitine and a low affinity for proline (apparent K_m of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in L. plantarum and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by trans substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.     

Lysine transport in two barley mutants with altered uptake of basic amino acids in the root

Amino acid uptake was examined in two barley (Hordeum vulgare L.) mutants R906 and R4402 which had been selected as resistant to the lysine analog S-(2-aminoethyl)-cysteine. The mutants were found to be allelic by crossing and examination of F₁ and F₂ progeny. The mutant genes were designated aec1a and aec1b, respectively. The uptake of the basic amino acids lysine, arginine, and ornithine from 50 micromolar solutions was strongly decreased in roots of the mutants, whereas uptake of neutral and acidic amino acids was unaffected. The pattern of uptake of lysine over the range 10⁻⁷ to 10⁻² molar was consistent with there being, principally, two uptake systems operating for basic amino acids in roots and that a low-concentration, high-affinity system is reduced or lacking in the mutants. The residual transport activity in the mutants had a different relative affinity for lysine and arginine to the wild-type system. Uptake of lysine by leaf slices was unimpaired in the mutants suggesting that the leaf uptake system is unaffected by the aec1 gene.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Effects of prostaglandin D2 on Na-dependent phosphate transport activity and its intracellular signaling mechanism in osteoblast-like cells

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D₂ (PGD₂) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD₂ stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD₂ on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD₂-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S₆ kinase reduced this effect of PGD₂, while Akt kinase inhibitor did not. These results suggest that PGD₂ stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S₆ kinase.     

Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytes

Xenopus laevis oocytes have been used for the expression of Na/P_i-cotransport activity by injections of poly(A)⁺ RNA (mRNA) isolated from an established renal cell line (OK cells). 3–5 days after mRNA injection, Na-dependent phosphate (P_i) uptake by oocytes was increased in a dose-dependent manner; there was no increase in Na-independent P_i uptake. Sucrose density-gradient fractionation indicated that the mRNA species encoding this activity is 2.4–2.8 kb in length. In Northern blots, using a cDNA probe related to human kidney-cortex Na/P_i-cotransport activity (NaPi-3), hybridization with a mRNA-species of 2.4–2.6 kb was obtained. Kinetic characterization ([P_i], [Na]) showed that expressed transport activity has properties similar to apical Na/P_i cotransport in OK cells.     

Single nucleotide polymorphism in CPT1B and CPT2 genes and its association with blood carnitine levels in acute myocardial infarction patients

Ischemic and reperfusion injuries in acute myocardial infarction (AMI) lead to mitochondrial dysfunction in heart cells. Lipid metabolism takes place in mitochondria where carnitine palmitoyltransferase (CPT) enzyme system facilitates the transport of long-chain fatty acids into matrix to provide substrates for beta-oxidation. We sequenced the coding regions of CPT1B and CPT2 genes to identify the single nucleotide polymorphism (SNP) in 23 AMI patients and 23 normal subjects. We also determined blood carnitine levels in these samples to study the impact of these SNPs on carnitine homeostasis. The sequencing of coding regions revealed 4 novel variants in CPT1B gene (G320D, S427C, E531K, and A627E) and 2 variants in CPT2 gene (V368I and M647V). There were significant increases in total carnitine (54.18 ± 3.11 versus 21.49 ± 1.03 μmol/l) and free carnitine (37.78 ± 1.87 versus 10.06 ± 0.80 μmol/l) levels in AMI patients as compared to normal subjects. CPT1B heterozygous variants of G320D and S427C among control subjects showed significantly higher levels of total and free carnitine in the blood. The homozygous genotype (AA) of CPT2 variant V368I had significantly less blood carnitine in AMI patients. Serum troponin T was significantly less in GG genotype of CPT1B variant S427C whereas the genotype AA of CPT2 variant V368I showed significantly higher serum troponin T levels. Further studies on large number of patients are necessary to confirm the role of CPT1B and CPT2 polymorphism in AMI.     

The uptake of fructose by Pseudomonas putida

Fructose transport was not apparently affected in a number of Pseudomonas putida strains with deranged activity of a common glucose-gluconate uptake system, indicating the existence of an independent fructose uptake system. Fructose uptake by glucose-gluconate uptake mutants was induced by fructose and obeyed saturation kinetics (apparent K _m =0.3 mM). The fructose uptake system serves to transport glucose in addition to fructose. The entry of fructose into P. putida cells appears to be mediated also by the glucose-gluconate uptake system, as shown by the ability to accumulate fructose of wild type cells grown on glucose, a substrate that induces the glucose-gluconate uptake system but not the fructose uptake system. In addition, fructose was found to be an inducer of the glucose-gluconate uptake system. The physiological significance of these observations is not clear because the fructose uptake system can provide the cell with a high enough internal concentration of fructose to support maximum growth rate on this hexose, as shown by following the growth course of glucose-gluconate uptake mutants on fructose.