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Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane
- Source :
- Biochimica et Biophysica Acta (BBA) - Biomembranes. 1661(1):78-86
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.
- Subjects :
- Arginine
Organic Cation Transport Proteins
Antiporter
Proteolipids
Biophysics
Transport
Sodium Chloride
Tritium
Biochemistry
Reconstitution
chemistry.chemical_compound
Betaine
Carnitine
medicine
Animals
OCTN2
Solute Carrier Family 22 Member 5
Liposome
Tetraethylammonium
Microvilli
Sodium Radioisotopes
Membrane Proteins
Transporter
Biological Transport
Intracellular Membranes
Cell Biology
Hydrogen-Ion Concentration
Rats
Membrane
Kidney Tubules
chemistry
Liposomes
Carrier Proteins
Plasma membrane
medicine.drug
Subjects
Details
- ISSN :
- 00052736
- Volume :
- 1661
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Biomembranes
- Accession number :
- edsair.doi.dedup.....d1fe8578b53834bc871b962311e9b8ee
- Full Text :
- https://doi.org/10.1016/j.bbamem.2003.12.001