Subcellular fractionation of polyprenyl diphosphate synthase activities responsible for the syntheses of polyprenols and dolichols in spinach leaves.

Polyisoprenoid alcohols occurring in spinach leaves were analyzed by a two-plate TLC method. Z,E-mixed polyprenols (C(55-60)), glycinoprenols (C(50-55)), and solanesol (C(45)) were mainly found in chloroplasts, whereas dolichols (C(70-80)) were mainly found in microsomes. Analysis of enzymatic products derived from [1-(14)C]isopentenyl diphosphate and farnesyl diphosphate (FPP) with subcellular fractions revealed that chloroplasts and microsomes had the ability to synthesize Z,E-mixed polyprenyl (C(50-65)) and all E-polyprenyl (C(45-50)) diphosphates, and Z,E-mixed polyprenyl (C(70-85)) diphosphates, respectively. FPP and geranylgeranyl diphosphate (GGPP) were both accepted for these enzymatic reactions, the former being a better substrate than the latter. NMR analysis of naturally occurring spinach Z,E-mixed polyprenol (C(55)) and dolichol (C(75)) revealed that the number of internal trans isoprene residues in the former was three in comparison with two internal trans residues found for the latter. These results indicate that two kinds of polyprenyl diphosphate synthases occur in spinach: One is the chloroplast enzyme involved in the synthesis of the shorter-chain (C(50-65)) Z,E-mixed polyprenols and the other is the microsomal enzyme involved in the synthesis of longer-chain (C(70-85)) Z,E-mixed polyprenols, which is converted to dolichols.