Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver.
Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (Kd = 10(-15) M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures--polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [14C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons +/- 3.3%. This protein was shown to bind avidin (Mr = 16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [14C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)
McKim
J M
JM
Department of Food Science, Oregon State University, Corvallis 97331.
Schaup
H W
HW
Marien
K
K
Selivonchick
D P
DP
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification*; D000345:Affinity Labels; D000818:Animals; D001710:Biotin; D002645:Chickens; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005777:Geese; D008025:Ligases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013045:Species Specificity; D014337:Trout
D000345:Affinity Labels; D001710:Biotin; D008025:Ligases; D000103:Acetyl-CoA Carboxylase