[15] Bioluminescence-related acyltransferases from Photobacterium phosphoreum and Vibrio harveyi

This chapter discusses the bioluminescence-related acyltransferases from Photobacterium phosphoreum and Vibrio harveyi. The assay is based on the cleavage of [³H] tetradecanoyl-CoA to form a labeled hexane-soluble product. For the P. phosphoreum acyl- transferase, the enzyme preparation is incubated with 8μM [³H] tetradecanoyl-CoA (100 Ci/mol) in 1 M phosphate, 50 mM 2-mercaptoethanol, pH 7.0, in a total volume of 100 μl at room temperature (22°). The reaction is stopped by adding 10 μl of glacial acetic acid and the solution is extracted twice with 1 ml hexane. The hexane washes are combined and counted directly in 10 ml Econofluor (New England Nuclear). The assay for the V. harveyi enzyme is essentially identical, except that the solvent used is 50% ethylene glycol, 50 mM phosphate (pH 7). For acyltransferases, enzyme concentration and incubation time are chosen such that less than 60% of the substrate is cleaved. The 34 kDa acyltransferase can be isolated from the partially purified P. phosphoreum fatty acid reductase complex, with which it copurifies during ion-exchange, gel filtration, and aminohexyl Sepharose chromatography. Although a corresponding fatty acid reductase complex has not been isolated from extracts of V. harveyi, the 32 kDa acyltransferase can be separated from luciferase and the majority of the soluble protein by (NH₄)₂SO₄ fractionation.