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Biochem J. 1994 Dec 15;304 ( Pt 3):751-60.

The kinetics, substrate and inhibitor specificity of the lactate transporter of Ehrlich-Lettre tumour cells studied with the intracellular pH indicator BCECF.

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  • 1Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.

Abstract

1. Suspensions of cultured Ehrlich-Lettre tumour cells were loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and changes in intracellular pH upon addition of L-lactate and other monocarboxylates were continuously monitored by fluorimetry using dual-wavelength excitation (450/500 nm) and single-wavelength emission (> 520 nm). 2. The rapid fluorescence changes were analysed by first-order regression analysis, and with suitable calibration procedures this enabled calculation of initial rates of proton uptake associated with monocarboxylate transport. 3. The stoichiometry was shown to be one proton per lactate molecule transported. 4. The kinetics of carrier-mediated transport of a wide range of monocarboxylates were determined at 25 degrees C. The Km values for L-lactate, pyruvate and D-lactate were found to be 4.54, 0.72 and 27.5 mM respectively, similar to values found previously for rat erythrocytes. This similarity was shared with a wide range of variously substituted C2, C3 and C4 monocarboxylates, all of which were transported with similar Vmax. No stereoselectivity was found in the Km values for D- and L-2-chloropropionate (0.75 mM) or D- and L-3-hydroxybutyrate (11 mM), but in the latter case the Vmax. of the D-isomer was twice that of the L-isomer. 5. The temperature-dependence of L-lactate transport demonstrated a transition point, with activation energies of 60 and 109 kJ.mol-1 above and below 19 degrees C respectively The Km for L-lactate below the transition temperature was about half that above it. 6. Inhibition of lactate transport into tumour cells by a wide range of compounds known to inhibit the erythrocyte monocarboxylate carrier was analysed. Patterns of inhibition were similar to those seen in the erythrocyte, but the Ki values were 2-4-fold higher in the tumour cells. 7. It is concluded that tumour cells contain an isoform of the monocarboxylate carrier with functional properties almost identical with that found in erythrocytes. This is probably identical with MCT1, which was recently cloned and sequenced from Chinese Hamster Ovary cells [Kim Garcia, Goldstein, Pathak, Anderson and Brown (1994) Cell 76, 865-873].

PMID:
7818477
[PubMed - indexed for MEDLINE]
PMCID:
PMC1137398
Free PMC Article
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