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 been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate 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 translocation and may be localized at or near the substrate-binding site.
Natuzzi
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D
Department of Pharmaco-Biology, University of Bari, Italy.
Daddabbo
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L
Stipani
V
V
Cappello
A R
AR
Miniero
D V
DV
Capobianco
L
L
Stipani
I
I
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000737:chemistry*; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D014316:Tritium
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008081:Liposomes; D026901:Membrane Transport Proteins; C021723:oxoglutarate translocator; D014316:Tritium; D011732:Pyridoxal Phosphate; D008239:Lysine