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Isolation and identification of acetyl‐CoA carboxylase from rainbow trout ( Salmo gairdneri ) liver
Author(s) -
McKim J. M.,
Schaup H. W.,
Marien K.,
Selivonchick D. P.
Publication year - 1989
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02535233
Subject(s) - pyruvate carboxylase , biochemistry , biotin , avidin , acetyl coa carboxylase , rainbow trout , polyacrylamide gel electrophoresis , affinity chromatography , gel electrophoresis , biology , sodium dodecyl sulfate , trout , chemistry , microbiology and biotechnology , enzyme , fishery , fish <actinopterygii>
Abstract 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 (K d =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 [ 14 C]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 (M r =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 [ 14 C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl‐CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl‐CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl‐CoA carboxylase‐specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M r =230,000) purified from rainbow trout liver is acetyl‐CoA carboxylase with enzyme characteristics comparable to mammalian acetyl‐CoA carboxylase.