|Plank, David - UNIVERSITY OF MINNESOTA|
|Plaisance, Kathryn - UNIVERSITY OF MINNESOTA|
|Gengenbach, Burle - UNIVERSITY OF MINNESOTA|
Submitted to: Plant Lipids International Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: July 8, 1996
Publication Date: N/A
Technical Abstract: Acetyl coenzyme A carboxylase (ACCase) is a biotin containing enzyme that catalyzes the ATP dependent carboxylation of acetyl CoA to form malonyl CoA. There is increasing evidence that dicots contain both a multi subunit, "prokaryotic type" ACCase as well as multi functional, "eukaryotic type" ACCases. We investigated whether both types of ACCases were present in soybean seeds and leaves. Crude extracts were obtained from separated embryos and seed coats of soybean seeds 40 days after flowering. Extracts were applied to a Q Sepharose column and fractionated with a KCl (0 to 500 mM) gradient. For embryos, two peaks of ACCase activity were detected. One peak, which eluted at approximately 300 mM KCl, contained the multi subunit ACCase (38 kDa biotinylated subunit). The second peak, which eluted at approximately 400 mM KCl, contained a multi functional ACCase (210 kDa subunit). For extracts from the seed coat, a single peak of ACCase activity ywas detected. This peak contained a multi functional ACCase (210 kDa subunit) that eluted at approximately 300 mM KCl. In an effort to further characterize the multi subunit ACCase, chloroplasts were isolated from 10 to 12 day old soybean leaves using a continuous Percoll (10 to 80%) gradient. Purified chloroplasts contained the 38 kDa biotinylated subunit of the multi subunit ACCase as well as a low concentration of a 220 kDa biotinylated polypeptide. Attempts at further purification of the multi subunit ACCase from isolated chloroplasts resulted in dissociation of the enzyme and loss of activity. When dissociation of the multi subunit ACCase was induced by ammonium sulfate, the activity could be reconstituted by combining the 0 to 15% and 35 to 50% ammonium sulfate fractions. The results of further studies of the multi subunit ACCase will be reported.