Abstract
The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.
Highlights
The most prominent biochemicalconsequence of acyl-CoA dehydrogenase activities occurred earlier and was riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoAdehydrogenase (IVD)
Enzyme assays revealed that various acyl-CoA dehydrogenase activities, those of IVD and SCAD, in the riboflavindeficient rat liver mitochondria were severely reduced
In order to elucidate whether the decreased acyl-CoA dehydrogenase activities were caused by deficient conversion of apoproteintoholo-protein,or by adecreased amount of apoproteins, we estimated the amountof enzyme proteins in IVD a-electrontransfer flavoprotein (ETF) ornithine transcarbamylase (OTC)
Summary
Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. When rats were deprived of riboflavin, the first and most striking effect was the severe impairment of the mitochondrial oxidation of fatty acids and branched chain amiancoids This phenomenon was caused primarily by a drastic decrease in various acyl-CoA dehydrogenase activities [1,2,3,4,5]. Denhardt'ssolution, and 250 pg/ml of salmonsperm DNA.After dehydrogenases, electron transfer flavoprotein subunits, and prehybridization,[32P]RNA was addedinthesame buffer (total ornithine transcarbamylase in riboflauin-deficient rat liuer volume 0.5 ml), and hybridization was carried out at 42 "C for 36 h.
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