Rate of acetate and mevalonate incorporation by extracts of Pinus pinaster seedlings

Abstract

The conditions for obtaining cell-free extracts of pine seedlings able to acetate and mevalonate incorporation have been investigated. The whole sequence of reactions which relate acetate to «active isoprene» in pine seems to be identical to the pathway in animal cells and microorganisms. Acetate is incorporated by the enzymes present in the extract and is transformed into MVA derivatives. Incorporation only takes place when CoA-SH and glutathion are added. In the absence of CoA-SH the reaction rate is very slow and its intensity is greatly reduced when NADPH is not supplied. The addition of malonate has no effect on the reaction. Incorporation of mevalonate by cell-free extracts of P. pinaster seedlings has been also demonstrated. The addition of fluoride to the reaction mixture as inhibitor of the phosphatases prevents its action on P-MVA and PP-MVA and allows a greater accu­ mulation of intermediates and a better visualization of the reactions of the metabolic system. Using mevalonate-l-,IC and 2-uC, the rate of its incorporation has been studied. The reaction is extremely rapid. Highest levels of P-MVA and PP-MVA are found after 15-30 minutes. As incubation time increases (0-10 hours) the production of further me­tabolites (geranyl-PP or neryl-PP?) also increases.