Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.
All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.
Fehling
E
E
Federal Centre for Cereal, Potato and Lipid Research, H.P. Kaufmann-Institute, Münster, Germany.
Lessire
R
R
Cassagne
C
C
Mukherjee
K D
KD
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D002569:Cerulenin / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D044943:Fatty Acid Desaturases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D007460:Iodoacetamide / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases; D012639:Seeds / Q000201:enzymology*; D012995:Solubility
D002569:Cerulenin; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D044943:Fatty Acid Desaturases; C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases; C036180:beta-hydroxyacyl-(acyl-carrier-protein)dehydrase; D007460:Iodoacetamide