Partial purification and reconstitution of the sarcolemmal L-lactate carrier from rat skeletal muscle.
Purified sarcolemmal membranes from mixed rat hindlimb muscle were solubilized with octylglucoside and the extract subjected to hydroxylapatite (HA) chromatography. Following protein elution with a sodium phosphate gradient and detergent removal by dialysis, the HA eluate was reconstituted into asolectin liposomes using a freeze-thaw procedure. Specific L-[14C]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemmal vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carrier exhibited Michaelis-Menten saturation kinetics with Km and Vmax. 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 inhibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE showed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from the HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat skeletal muscle.
Allen
P J
PJ
Department of Human Biodynamics, University of California, Berkeley 97420.
Brooks
G A
GA
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002845:Chromatography; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D027501:Monocarboxylic Acid Transporters; D009132:Muscles / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism*
D002352:Carrier Proteins; D007773:Lactates; D008081:Liposomes; D008565:Membrane Proteins; D027501:Monocarboxylic Acid Transporters; D011510:Proteolipids; C020485:proteoliposomes; D017886:Durapatite