Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria.
The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.
D000818:Animals; D001692:Biological Transport; D002208:Caproates / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004058:Diffusion; D066298:In Vitro Techniques; D007651:Keto Acids / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D002208:Caproates; D002250:Carbon Radioisotopes; D007651:Keto Acids; C013082:alpha-ketoisocaproic acid