Effect of pyridoxal 5'-phosphate on the function of the purified mitochondrial tricarboxylate transport protein.
The effect of pyridoxal 5'-phosphate (PLP), a lysine-selective reagent, on the function of the purified and reconstituted mitochondrial tricarboxylate transport protein has been studied. PLP caused a concentration-dependent inhibition of citrate transport with an IC50 value of 0.09 mM. At 10 mM PLP virtually complete inhibition of transport was observed (i.e. 97%), thereby indicating that modification of a residue(s) whose participation is essential to the translocation mechanism had occurred. Substrate protection studies demonstrated that substrates for the tricarboxylate transporter (i.e., citrate, isocitrate, phosphoenolpyruvate, and malate) effectively protected against PLP inhibition, whereas other organic anions which are not transported by the tricarboxylate carrier (i.e., malonate, alpha-ketoglutarate, phosphate, and succinate) afforded considerably less protection. Kinetic studies in which the transport rate was measured at varying citrate and PLP concentrations indicated that PLP caused an increase in the apparent Km of transport with little change in the Vmax, thereby resulting in a primarily competitive inhibition pattern. In combination, the above findings indicate that PLP interacts with the tricarboxylate transporter at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal alanine residue) that is important in the translocation mechanism and may reside within or near the substrate binding site.
Gremse
D A
DA
Department of Pediatrics, College of Medicine, University of South Alabama, Mobile 36688.
Dean
B
B
Kaplan
R S
RS
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000187:drug effects*; D002474:Cell-Free System; D002951:Citrates / Q000378:metabolism*; D019343:Citric Acid; D007700:Kinetics; D008081:Liposomes; D008930:Mitochondria, Liver; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D051381:Rats
D002352:Carrier Proteins; D002951:Citrates; D008081:Liposomes; C020723:citrate-binding transport protein; D019343:Citric Acid; D011732:Pyridoxal Phosphate