ISOCRATIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF COENZYME-A ESTERSINVOLVED IN THE METABOLISM OF 3S-HYDROXY-3-METHYLGLUTARYL-COENZYME-A - DETECTION OF RELATED ENZYME-ACTIVITIES IN CATHARANTHUS-ROSEUS PLANT CELL CULTURES

For studies on enzyme activities involved in the metabolism of 3S-hydr oxy-3-methylglutaryl-coenzyme A (HMG-CoA) in Catharanthus roseus plant cell cultures, an isocratic HPLC system was developed for the separat ion of coenzyme A (CoASH), acetyl-CoA, acetoacetyl-CoA, HMG-CoA, 3-met hylglutaconyl-CoA (MG-CoA), and their respective 3'-dephospho-derivati ves. Using an RP-18 column (250x4.6 mm, 5 mu m) retention in eluents c ontaining 0.2 M sodium phosphate buffer (pH range 4.7-6.7) supplemente d with methanol (12.5-20 mi per 100 mi of buffer) was studied. Baselin e separation was obtained with an eluent consisting of buffer pH 5.0-m ethanol (100:17, v/v). The linearity for the quantitative determinatio n of the esters (range 0.5-10 nmol) was tested. Detection limits were found to be between 2.5 and 6 pmol. Guanosine was used as an internal standard. In cell-free preparations of C. roseus cell cultures, the en zyme activities of HMG-CoA lyase (EC 4.1.3.4) and MG-CoA hydratase (HM G-CoA hydrolyase, EC 4.2.1.18) were detected.