Abstract
The effect of pyridoxal 5′-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5′-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5′-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [3H]pyridoxal 5′-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates 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 againstinhibition by pyridoxal 5′-phosphate. These results indicate that pyridoxal 5′-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.
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Natuzzi, D., Daddabbo, L., Stipani, V. et al. Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate. J Bioenerg Biomembr 31, 535–541 (1999). https://doi.org/10.1023/A:1026414826457
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DOI: https://doi.org/10.1023/A:1026414826457