Abstract. The uptake of [1 -14C]isopentenyl diphosphate by intact plastids purified from cell suspensions of Vitis vin~era L. cv. Muscat de Frontignan was investigated using vacuum-filtration and silicone-oil-filtering techniques. Transport across the plastid envelope which was stimulated by cations, such as Mg 2+ and Mn 2+, was characterized by a K m of approx. 0.5 mM and a Vm, X of 25 nmol.(mg protein)-~.h -l. The data showed that isopentenyl diphosphate apparently accumulated in the plastid against a concentration gradient. The involvement of a protein carrier was suggested by the strong inhibition of the uptake by compounds which are known to block SH groups. Thus, the saturation kinetics together with the pH optimum (7.5-8), the temperature dependence (maximum incorporation at 37 ~ and the competitive inhibition by a structural analogue of the substrate (aminophenylethyl diphosphate) provided evidence for a mechanism of uptake by facilitated diffusion. The carrier identified may thus play a major role in supplying the plastid compartment with isopentenyI diphosphate for isoprenoid biosynthesis.
Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.
A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera 1. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. l h e enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. l h e enzyme showed a native molecular mass of 68 f 5 kD as determined by gel permeation.O n sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 f 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 p~, respectively. The enzyme required MnZ+ and Mgz+ as cofactors and its activity was enhanced by Triton X-100. lnorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.
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