Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K(m) value of 1.5 microM and a V(max) of 286 pmol min(-1) (mg protein)(-1) and showed higher affinity for ATP and a lower V(max) value than intact RER (K(m) of 6.5 microM and V(max) of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.     

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of l-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of l-[(3)H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. K(m) and V(max) values for saturable uptake were 14.07 +/- 1.70 micro M and 26.3 +/- 0.80 pmol. mg protein(-1). 6 min(-1), respectively. l-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.     

Characterization of organic cation/carnitine transporter family in human sperm

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K(m) of 3.39+/-1.16 microM; V(max) of 0.23+/-0.14 pmol/min/mg sperm protein; and mean+/-SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K(m) of 25.9+/-14.7 microM; V(max) of 1.49+/-1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K(m) of 412.6+/-191 microM; V(max) of 32.7+/-20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to L-carnitine supplementation.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

Plasmodium falciparum: ATP/ADP transport across the parasitophorous vacuolar and plasma membranes

Previous studies have shown that ATP is required for the growth of the intracellular parasite, Plasmodium, outside its host cell, the erythrocyte, and that bongkrekic acid, an inhibitor of mitochondrial ATP/ADP transporter, inhibits intraerythrocytic Plasmodium maturation. We have characterized ATP/ADP transport of Plasmodium falciparum, isolated by either immune lysis or N2-cavitation. [3H]ATP uptake was due to ATP/ADP exchange since ADP efflux was dependent on exogenous ATP in an approximate 1:1 stoichiometry and both ATP influx and ADP efflux were equally inhibited by atractyloside (Ki = 100 nM). ATP uptake was not inhibited by the nucleoside transport inhibitor, nitrobenzylthioinosine. Conversely, adenosine and hypoxanthine transport were insensitive to atractyloside. ATP influx was characterized by a Km = 0.14 mM and Vmax = 1.2 nmol ATP/min/10(6) cells. Substrate specificity studies for nucleotide-induced ADP efflux indicated a preference for an adenosine ring and triphosphate, but transport did not require a hydrolyzable phosphate bond. Protein synthesis was measured with free parasites starved of glucose. Addition of 1.0 mM ATP resulted in a 40% recovery of total protein synthetic capacity in a process inhibited by 500 nM atractyloside, suggesting that uptake of erythrocyte-derived ATP by P. falciparum may be essential for maintaining maximal rates of protein synthesis during specific stages of intra-erythrocytic parasite maturation.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.     

ATPase activity and transport by a cGMP transporter in human erythrocyte ghosts and proteoliposome-reconstituted membrane extracts

We previously described the [(3)H]cGMP-binding characteristics of a CHAPS-solubilized protein that we proposed to be a cGMP transporter. We now report the ATPase activity of the membrane-bound, solubilized and reconstituted form of a cGMP transporter. The membrane-bound protein of unsealed ghosts had a linear ATPase activity over a 120 min incubation period with optimal activity of about 400 pmol/mg/min. The apparent K(m) and V(max) for ATP were about 0.5 mM and 300 pmol/mg/min, respectively. When solubilized with CHAPS the specific activity of the protein was reduced to about 70 pmol/mg/min. Reconstitution of the CHAPS preparation into phospholipid bilayer using rapid detergent removal by Extracti-gel column resulted in proteoliposomes which had ATPase activity similar to that found in the erythrocyte membranes. The proteoliposomes displayed a linear ATP-dependent uptake of [(3)H]cGMP with an apparent K(m) value of 1. 0 microM. This low K(m)-uptake of [(3)H]cGMP in proteoliposomes was not affected by 10 microM of AMP, cAMP and GMP, but was completely abolished in the presence of the non-hydrolyzable ATP analogue, ATP-gamma-S. Some ATPase activation was also observed in the presence of 2 microM cAMP, but it is unclear whether this activity was coupled to the cGMP transporter. Our results show that the membrane protein responsible for cGMP transport has an ATPase activity and transports the cyclic nucleotide in the presence of ATP.     

A unique multifunctional transporter translocates estradiol-17beta -glucuronide in rat liver microsomal vesicles

A wide array of drugs, xenobiotics, and endogenous compounds undergo detoxification by conjugation with glucuronic acid in the liver via the action of UDP-glucuronosyltransferases. The mechanism whereby glucuronides, generated by this enzyme system in the lumen of the endoplasmic reticulum (ER), are exported to the cytosol prior to excretion is unknown. We examined this process in purified rat liver microsomes using a rapid filtration technique and [(3)H]estradiol-17beta-d-glucuronide ([(3)H]E(2)17betaG) as model substrate. Time-dependent uptake of intact [(3)H]E(2)17betaG was observed and shrinkage of ER vesicles by raffinose lowered the steady-state level of [(3)H]E(2)17betaG accumulation. In addition, rapid efflux of [(3)H]E(2)17betaG from rat liver microsomal vesicles suggested that the transport process is bidirectional. Microsomal uptake was saturable with an apparent K(m) and V(max) of 3.29 +/- 0.58 microm and 0.19 +/- 0.02 nmol.min(-1).mg protein(-1), respectively. Transport of [(3)H]E(2)17betaG was inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and probenecid. Specificity of the transport process was investigated by studying the cis-inhibitory effect of anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on the uptake of [(3)H]E(2)17betaG. Collectively, these data are indicative of a novel multifunctional and bidirectional protein carrier for E(2)17betaG and other anionic compounds in the hepatic ER. This intracellular membrane transporter may contribute to the phenomenon of multidrug resistance.     

DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.     

Selective transport of adenosine into porcine coronary smooth muscle

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.     

Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis

Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain.     

Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342-557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 +/- 0.5 to 141 +/- 19 microM. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341-454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341-454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 +/- 5 microM). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 +/- 4.5 microm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition.     

Specificity of the Rat Vesicular Acetylcholine Transporter

The protein kinase A–deficient PC12 cell line PC12^A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.     

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: the mouse carnitine transporter OCTN3

pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni(2+)-chelating chromatography with a yield of 2mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [(3)H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K(m) for carnitine was 36 μM. [(3)H]carnitine transport was inhibited by carnitine analogues and some Cys and NH(2) reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.     

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 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM 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.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent K(m) of 2.8 mM and a V(max) of 260 nmol/min per mg protein. Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.     

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.     

大鼠脑线粒体NOS及L—Arg转运的生化特性

测定分离纯化的大鼠脑线粒体(mitochondria,Mt)L-精氨酸(L-arginine,L-Arg)/一氧化氮合酶(nitricoxidesynthase,NOS)/NO系统,L-Arg转运和NOS的活性。结果显示正常大鼠脑Mt膜上存在高亲和、低转运、可饱和的L-Arg转运体。最大转运速率Vmax为5.87±0.46nmol/mgpro·min