Isocratic high-performance liquid chromatography of coenzyme A esters involved 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-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) in Catharanthus roseus plant cell cultures, an isocratic HPLC system was developed for the separation of coenzyme A (CoASH), acetyl-CoA, acetoacetyl-CoA, HMG-CoA, 3-methylglutaconyl-CoA (MG-CoA), and their respective 3'-dephospho-derivatives. Using an RP-18 column (250x4.6 mm, 5 mu m) retention in eluents containing 0.2 M sodium phosphate buffer (pH range 4.7-6.7) supplemented with methanol (12.5-20 mi per 100 mi of buffer) was studied. Baseline separation was obtained with an eluent consisting of buffer pH 5.0-methanol (100:17, v/v). The linearity for the quantitative determination 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 enzyme activities of HMG-CoA lyase (EC 4.1.3.4) and MG-CoA hydratase (HMG-CoA hydrolyase, EC 4.2.1.18) were detected.