Isolation and mapping of the beta-hydroxyacyl dehydratase activity of chicken liver fatty acid synthase.
Chicken liver fatty acid synthase is cleaved by kallikrein into polypeptides ranging in molecular weight from 10,000 to 100,000. Fractionation of the digest by ammonium sulfate and chromatography on a Matrix Red A affinity column resulted in the isolation of a polypeptide (Mr = 26,000) containing the beta-hydroxyacyl dehydratase activity, but no other partial activities normally associated with the fatty acid synthase. The specific activity of the dehydratase increased 9 to 12 times in this fraction, an increase that is within the expected range based on relative molecular weight. Kinetic parameters of the purified dehydratase toward the model substrate, crotonyl-CoA, showed no change in apparent Km values and a 12-fold increase in Vmax values as compared to dehydratase activity of the intact synthase. However, the purified fragment did not catalyze the hydration of the crotonyl-N-acetylcysteamine derivative, a substrate that is readily hydrated by the intact synthase. Antibodies against the purified 26-kDa fragment cross-react with the intact synthase and the hydratase-containing fragments produced at all stages of digestion with kallikrein or trypsin as shown by Western blot analyses. The results show that the beta-hydroxyl dehydratase activity of the fatty acid synthase is located in the reduction Domain II (Tsukamoto, Y., Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15312-15322) of the synthase subunit.
Tsukamoto
Y
Y
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Texas Medical Center, Houston 77030.
Wakil
S J
SJ
D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D004746:Enoyl-CoA Hydratase / Q000302:isolation & purification* / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000302:isolation & purification* / Q000378:metabolism; D006836:Hydro-Lyases / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight
D064429:Fatty Acid Synthases; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase