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.
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
B
B
Zhang
R
R
Paxton
G W
GW
Goodwin
Y
Y
Shimomura
R A
RA
Harris
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D005615:Freezing; D007651:Keto Acids / Q000494:pharmacology; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D008933:Mitochondrial Swelling / Q000187:drug effects; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007651:Keto Acids; D009097:Multienzyme Complexes; C013082:alpha-ketoisocaproic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002950:Citrate (si)-Synthase; D005459:Fluorides