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K A Nałecz

Publications and source records attributed to K A Nałecz.

At least 19 recordsLinked to original sources
PubMed2001

Transport of L-carnitine in isolated cerebral cortex neurons.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nałecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nałecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

Animals↗
PubMed2000

Transport of alpha-ketoisocaproate in rat cerebral cortical neurons.

Transport of alpha-ketoisocaproic acid (KIC), the product of leucine transamination, was studied in the cerebral cortex cells isolated from the adult rat brain. The process of [(14)C]KIC accumulation was followed in the presence of aminooxyacetate, an inhibitor of transaminases. Accumulation of KIC was not affected by Na(+) replacement, its initial velocity was observed to be higher upon lowering of external pH. Addition of KIC promoted acidification of cytoplasmic pH, monitored with 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The detected inhibition of KIC accumulation by alpha-cyano-4(OH)cinnamate pointed to an involvement of one of monocarboxylate transporters (MCT), although 4,4'-diisothiocyano-2,2'-stilbenedisulphonate was without effect. Accumulation of KIC was inhibited by lactate; the effect of pyruvate was detected to be much weaker. Other branched-chain alpha-ketoacids (ketoisovalerate, keto-methylvalerate), as well as beta-hydroxybutyrate and valproate decreased the transport of KIC by 30, 60, and 80%, respectively. The observed characteristics of KIC accumulation in the cortical neurons indicate an involvement of one of the MCT transporters. A crucial role of SH group(s) in the process of KIC accumulation, excluding MCT2, indicates the MCT1, although an involvement of another isoform of MCT in the process of KIC transport in neurons from cerebral cortex of adult brain has not been definitely excluded.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Transport of alpha-ketoisocaproate in neuroblastoma NB-2a cells.

Transport of alpha-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free keto-acid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and beta-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyano-2,2'-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na+].

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Effect of palmitoylcarnitine on the cellular differentiation, proliferation and protein kinase C activity in neuroblastoma nb-2a cells.

Palmitoylcarnitine is synthesized through the action of palmitoylcarnitine transferase I--an enzyme specifically inhibited by etomoxir. An increase of the intracellular content of palmitoylcarnitine in neuroblastoma NB-2a cells after administration of carnitine was correlated with an inhibition of cell proliferation and a concomitant promotion of differentiation processes. The activity of protein kinase C was measured in vivo, with cells permeabilized through the use of streptolysin O and a peptide substrate. Palmitoylcarnitine inhibited the phorbol ester stimulated reaction of the peptide phosphorylation in a concentration dependent way. The degree of protein kinase C inhibition was correlated with intracellular increase of the palmitoylcarnitine content, pointing to this compound as a natural modulator of protein kinase C activity.

Animals↗
PubMed1996

Carnitine--a known compound, a novel function in neural cells.

Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) seems to fulfill in the brain a different role than in peripheral tissues. Carnitine is accumulated by neural cells in a sodium-dependent way. The existence of a novel transporter in plasma membrane, specific to compounds with a polar group in the beta-position with respect to carboxyl group, has been postulated. The presence of a carnitine carrier in the inner mitochondrial membrane has been proven and the protein has been purified. It is postulated that its major role in adult brain would be translocation of acetyl moieties from mitochondria into the cytoplasm for acetylcholine synthesis. The latter process is stimulated by carnitine and choline in a synergistic way in cells utilizing glucose as the main energetic substrate. Carnitine metabolism in neural cells leads to accumulation of different acyl derivatives of carnitine. Palmitoylcarnitine can influence directly the activity of protein kinase C. An involvement of carnitine in a decrease of palmitate pool used for palmitoylation of regulatory proteins has been postulated.

Acetylcholine↗
PubMed1995

Transport of carnitine in neuroblastoma NB-2a cells.

Carnitine accumulation was measured in cultured neuroblastoma NB-2a cells. This process was found partially sodium dependent and its kinetics to be a sum of a saturable transport (Km = 123 +/- 13 microM) and diffusion (D = 63 +/- 7 pmol/mg protein/min/mM). On the contrary to previous reports on neural cells, the accumulation of carnitine was found insensitive to gamma-aminobutyric acid (GABA). Measurements of carnitine accumulation in the presence of different compounds resulted in the conclusion that carnitine transport does not occur through the known systems specific toward choline and/or amino acids. For instance, an observed inhibition of carnitine transport by serine and cysteine, without any effect of alanine, excluded a possible role of ASC amino acid transport system. An involvement of a new transporter is thus postulated, specific toward compounds with a polar group in the beta position with respect to the carboxylic group.

Animals↗
PubMed1995

Effect of gamma-aminobutyric acid on the carnitine metabolism in neural cells.

Isolated rat cerebral cortex cells were able to accumulate L-carnitine and this process was competitively inhibited by 1 mM gamma-aminobutyric acid (GABA) with a shift of Km from 7.8 +/- 1.9 mM to 14.6 +/- 4.0 mM. Addition of GABA also affected distribution of carnitine derivatives. The decrease of acetylcarnitine level by 1.6 fold was correlated with the inhibition of carnitine acetyltransferase (1.77 times). A postulated involvement of this enzyme in delivering acetyl moieties for acetylcholine synthesis would suggest a negative feedback between GABA and the level of acetylcholine.

Acetylcarnitine↗
PubMed1995

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells.

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 microM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of beta-oxidation might be correlated with the lower level of acetylcholine synthesis.

Acetylcholine↗
PubMed1995

Mechanism of carnitine transport catalyzed by carnitine carrier from rat brain mitochondria.

The transport mechanism of reconstituted carnitine carrier purified from rat brain mitochondria was studied kinetically. Short and medium chain acyl carnitine derivatives had much higher affinity to the carnitine carrier in comparison with long chain acyl carnitine derivatives, therefore both homologous (carnitine/carnitine) and heterologous (carnitine/acetylcarnitine) antiports were analysed. A complete set of half-saturation constants was established for various substrate concentrations on both the external and the internal side of the membrane. Bisubstrate initial velocity analyses of the exchange reaction resulted in a kinetic pattern which is consistent with a sequential antiport mechanism. This type of mechanism implies formation of a ternary complex of the carrier with one internal and one external substrate molecule before the transport reaction occurs.

Animals↗
PubMed1994

Synergistic effect of choline and carnitine on acetylcholine synthesis in neuroblastoma NB-2a cells.

An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 microM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.

Acetylcholine↗
PubMed1993

Purification of carnitine carrier from rat brain mitochondria.

Brain mitochondria isolated from rats of different age were solubilized with Triton X-100 and the detergent extract was subjected to chromatography on dry hydroxyapatite and celite. The highest specific activity (110 mumol/10 min per g protein) measured after reconstitution of isolated proteins into phosphatidylcholine vesicles correlated with the appearance of a polypeptide with a molecular mass of 33,000. Activity of the carnitine carrier, both in isolated mitochondria and in the reconstituted system, varied with animal age, being twice higher in suckling rats than in adults. After reconstitution, the carnitine exchange showed sensitivity to SH groups modifying reagents, N-ethylmaleimide and mersalyl. Acetyl, propionyl and palmitoyl esters of carnitine decreased carnitine/carnitine exchange. Short and medium chain acyl derivatives were more potent inhibitors, pointing to a different substrate specificity of carnitine carrier in brain, in comparison with other tissues.

Aging↗
PubMed1992

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.

Amino Acid Sequence↗
PubMed1992

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

Acetoacetates↗
PubMed1991

Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nałecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

Animals↗
PubMed1990

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.

Animals↗
PubMed1990

Monocarboxylate carrier from bovine heart mitochondria: reconstitution in a functionally active state.

A method has been developed for functional reconstitution of the isolated pyruvate carrier. Optimal conditions were reached after removal of deoxycholate with 27 mg Triton X-100 per gram of moist Amberlite XAD-2 beads and 7 micrograms protein/mg of phospholipid. Uptake of pyruvate into proteoliposomes and sensitivity of the carrier towards inhibitors resemble those in intact mitochondria.

Animals↗