FAD synthetase or ATP:FMN alenylyltransferase (FADS or FMNAT, EC 188.8.131.52) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coil showed apparent K(m) and V(max) values for FMN equal to 1.3 +/- 0.7 mu M and 4.4 +/- 1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 mu M. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
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|Titolo:||Mitochondrial localization of human FAD synthetase isoform 1|
|Data di pubblicazione:||2010|
|Appare nelle tipologie:||1.1 Articolo in rivista|