To study the kinetics of lactate transport in an isolated, nonmetabolizing system, skeletal muscle sarcolemmal membrane vesicles were purified from 22 female Sprague-Dawley rats. l(+)-[U-14C] Lactate at 10 concentrations demonstrated saturation kinetics with a Vmax of 139.4 nmol/mg/min, and an apparent Km of 40.1 mm. Threefold higher initial rates of l(+)-lactate uptake were seen at 37 °C than at 25 °C, indicating temperature sensitivity. Transport was stereospecific for the l(+) isomer: isotopic d(-) uptake rates remained linear at concentrations from 1 to 200 mm, and 1 mm d(-) remained 6-fold lower in net uptake after 60 min than the l(+) isomer. Furthermore, unlabeled 10 mm d(-)-lactate in the external medium could only inhibit 1 mm isotopic (l(+) uptake by 12%, whereas unlabeled 10 mm l(+)-lactate and pyruvate inhibited 82 and 71%, respectively. Additionally, 10 mm β-hydroxybutyrate and acetoacetate could moderately inhibit (27 and 32%, respectively) 1 mm l(+)-lactate transport, but the unsubstituted aliphatic monocarboxylates (formate, acetate, propionate), tricarboxylic acid cycle intermediates (malate, succinate, oxaloacetate, α-ketoglutyrate, citrate), amino acids (alanine, aspartate, glutamate), and palmitate or adenosine in 10-fold excess could not effectively inhibit 1 mm l(+)lactate uptake under cistransport conditions. 4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid could inhibit l(+)-lactate transport by only 13%, so that lactate transport does not appear to be affected directly by Cl- or HCO3- fluxes. It was demonstrated that KCl could not evoke a membrane potential-induced overshoot of lactate uptake in the presence or absence of valinomycin. Moreover, gluconate could substitute for Cl-, indicating that Cl- flux does not contribute to a membrane potential-dependent component of the transport mechanism, suggesting an electroneutral translocation process. Protein-modifying reagents significantly inhibited 1 mml(+)-lactate transport during pH-stimulated conditions (p-chloromercuriphenyl-sulfonic acid, 83%; N-ethylmaleimide, 86%; HgCl2, 56%; mersalyl, 63% inhibition). We conclude that the skeletal muscle lactate transporter is a membranebound protein, specifically associated with the sarcolemma, that demonstrates saturation kinetics, competition, stereospecificity, and sensitivity to temperature as well as various ionic cis-inhibitors. The lactate transporter is a potentially important regulator of lactate flux across skeletal muscle, and may help to regulate intracellular pH and intermediary metabolism during lactic acidosis. © 1990.
