Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by pyridoxal 5 '-phosphate

The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has be en investigated. The inhibition of oxoglutarate/oxoglutarate exchange by py ridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohyd ride yielded an irreversible inactivation of the oxoglutarate carrier prote in. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependen t inactivation of oxoglutarate transport with an IC50 Of 0.5 mM, competed w ith the substrate for binding to the oxoglutarate carrier (K-i = 0.4 mM). K inetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosph ate indicated that modification of a single amino acid residue/carrier mole cule was sufficient for complete inhibition of oxoglutarate transport. Afte r reduction with sodium borohydride [H-3]pyridoxal 5'-phosphate bound coval ently to the oxoglutarate carrier. Incubation of the proteoliposomes with o xoglutarate or L-malate protected the carrier against inactivation and no r adioactivity was found associated with the carrier protein. In contrast, gl utarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/ or the amino-terminal glycine residue) which is essential for substrate tra nslocation and may be localized at or near the substrate-binding site.