Preservation of the activity state of hepatic branched-chain 2-oxo acid dehydrogenase during the isolation of mitochondria.

A comparison was conducted of current methods for estimation of the activity states (proportion of enzyme in active, dephosphorylated, form) of hepatic branched-chain 2-oxo acid dehydrogenase. Practically all of the enzyme was active in freeze-clamped liver obtained from chow-fed and 48 h-starved rats, regardless of the presence of fluoride in the extraction and assay media to inhibit phosphatase activity. Likewise, the enzyme was almost completely active in mitochondria isolated by a conventional method from livers of chow-fed and starved rats. However, when fluoride and 4-methyl-2-oxopentanoate were included in the mitochondrial isolation medium the activity state was decreased to 73% and 47% in mitochondria isolated from chow-fed and starved rats respectively. Furthermore, branched-chain 2-oxo acid dehydrogenase became partially inactivated upon incubation of isolated mitochondria on ice in fluoride- and/or 4-methyl-2-oxopentanoate-supplemented media. The rate of inactivation was greater in mitochondria prepared from starved than from chow-fed rats, which correlated with the lower activity state found in mitochondria of starved rats isolated in the fluoride- and 4-methyl-2-oxopentanoate-supplemented media. Thus the activity state of branched-chain 2-oxo acid dehydrogenase is underestimated in mitochondria isolated in media supplemented with fluoride plus 4-methyl-2-oxopentanoate.