Solubilization, purification and kinetic properties of three membrane-bound long-chain acyl-coenzyme-A thioesterases from microsomes of photosynthetic tissue.
Microsomal membranes of developing pea (Pisum sativum) leaves contained almost one third of the total long-chain acyl-CoA thioesterase activity found in the leaf cell. Three distinct forms of long-chain acyl-CoA thioesterase were purified by a combination of cholate-solubilization, dialysis, ion-exchange, and gel-filtration chromatography. Purification factors of 4600, 100 and 280 were achieved for the thioesterase forms I, II and III, respectively. Apparent molecular masses were: form I, 28 kDa; form II, 140 kDa; form III, 139 kDa. All the three thioesterases showed overlapping specificities towards palmitoyl-CoA, stearoyl-CoA, and oleoyl-CoA but were inactive towards short-chain acyl-CoAs, such as acetyl-CoA and malonyl-CoA. Each thioesterase exhibited complex kinetic behavior, which was consistent with differential affinities of the enzymes for monomeric and micellar forms of their substrates. The significance of the kinetic behavior and possible regulatory role of these enzymes are discussed.