PARTIAL-PURIFICATION AND RECONSTITUTION OF THE SARCOLEMMAL L-LACTATE CARRIER FROM RAT SKELETAL-MUSCLE

Purified sarcolemmal membranes from mixed rat hindlimb muscle were sol ubilized with octylglucoside and the extract subjected to hydroxylapat ite (HA) chromatography. Following protein elution with a sodium phosp hate gradient and detergent removal by dialysis, the HA eluate was rec onstituted into asolectin liposomes using a freeze-thaw procedure. Spe cific L-[C-14]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemma l vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carr ier exhibited Michaelis-Menten saturation kinetics with K-m and V-max values of 46.2+/-6.6 mM and 498.7+/-17.2 nmol/15 s per mg respectively . L-Lactate transport activity was inhibited 57 % by preincubation of proteoliposomes with 10 mM alpha-cyano-4-hydroxycinnamate, a known inh ibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE sh owed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from th e HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat ske letal muscle.