Dissociation of Prelycopersene Pyrophosphate Synthetase from Phytoene Synthetase Complex of Tomato Fruit Plastids*

The partially purified phytoene synthetase enzyme complex obtained from tomato fruit plastids dissociates into two or more subunit species on chromatography in low ionic strength buffer on DEAE-cellulose. One of these subunits prelycopersene pyrophosphate synthetase, has a molecular weight of approximately 40,000, whereas the phytoene synthetase complex has a molecular weight of 200,000. The prelycopersene pyrophosphate synthetase catalyzes the conversion of isopentenyl pyrophosphate to geranylgeranyl and prelycopersene pyrophosphates. The identities of these substances were established by thin layer chromatography in several solvent systems. The formation of both geranylgeranyl and prelycopersene pyrophosphates by this enzyme supports earlier results with cruder enzyme systems which suggested that these compounds are intermediates in the synthesis of phytoene.


SUMMARY
The partially purified phytoene synthetase enzyme complex obtained from tomato fruit plastids dissociates into two or more subunit species on chromatography in low ionic strength buffer on DEAE-cellulose.
One of these subunits, prelycopersene pyrophosphate synthetase, has a molecular weight of approximately 40,000, whereas the phytoene synthetase complex has a molecular weight of 200,000. The prelycopersene pyrophosphate synthetase catalyzes the conversion of isopentenyl pyrophosphate to geranylgeranyl and prelycopersene pyrophosphates.
The identities of these substances were established by thin layer chromatography in several solvent systems. The formation of both geranylger-any1 and prelycopersene pyrophosphates by this enzyme supports earlier results with cruder enzyme systems which suggested that these compounds are intermediates in the synthesis of phytoene.

Determination of Molecular Weight of the Enzyme System Forming Acid-Labile
Compounds -Assays were carried out for the molecular weight of the enzyme system forming acid labiles in order to determine whether the loss of phytoene synthetase activity was due to selective inactivation of one activity of the complex or to dissociation of the enzyme system. The protein fractions eluted from the DEAE-cellulose column with 0.2 M KC1 were pooled and concentrated in an Amicon concentrator equipped with a PM-10 filter. This protein fraction was subjected to Sephadex G-200 gel filtration and the elution volume of the acid-labile synthesizing enzyme was compared with those of other proteins of known molecular weight (Fig.  2). The molecular weight determined by this procedure was approximately 40,000, thus indicating that the phytoene synthetase complex had dissociated into two or more subunits on DEAE-cellulose chromatography.
A molecular weight of approximately 40,000 for the acid-labile synthesizing enzyme was also obtained on sucrose density gradient centrifugation.
Identification of Acid-labile Products -Preliminary experiments indicated that at least two acid-labile compounds were synthesized by the above enzyme system. Extraction of these compounds from an incubation mixture was effected with three 3-ml aliquots of H,O-washed n-butyl alcohol and one 6ml aliquot of benzene. The combined extracts were concentrated under reduced pressure at a temperature of 35" or lower. The concentrated residue was chromatographed on a 0.1 M ammonium phosphate-buffered, pH 6.8, silica gel plate prepared according to the method of Rilling (11). Small sections of the silica gel chromatogram were scraped into counting vials and then assayed for radioactivity by liquid scintillation spectrometry. An RF value of 0.4 to 0.5 was obtained. This R, value is characteristic of both geranylgeranyl and prelycopersene pyrophosphates.
To identify the allylic pyrophosphates further, the compounds were hydrolyzed with HCl as reported under "Experimental Procedures," and the liberated terpenols were extracted with silica gel-purified petroleum ether (boiling point 40-60"). The extract was concentrated under nitrogen and then subjected to thin layer chromatography in two systems. In one The incubation conditions and the extraction and hydrolysis methods for the geranylgeranyl and prelycopersene pyrophosphates are also described under "Experimental Procedures." The chromatographic plate was coated with Silica Gel G (0.5 mm thick; particle size, 10 to 40 +nj and then impregnated with paraffin oil. The solvent system used for chromatography was CH,OH:H,O (88:12, v/v) saturated with white oil. One-centimeter sections were scraped from the chromatographic plates directly into vials and then assayed for radioactivity. of these, chromatography was performed on Silica Gel G in a solvent system of benzene:ethyl acetate (80~20, v/v) (System IV of Qureshi et al. (12)). The radioactive terpenols had RF values of 0.4 to 0.5 (geranyl linalool) and 0.78 to 0.80 (prelycopersene alcohol). In the second system, reverse phase thin layer chromatography was carried out on Silica Gel G plates impregnated with paraffin oil (System VI of Qureshi et al. (1211, (Fig.  3). After development of the chromatogram in methanol:H,O (88:12, v/v), l-cm sections of the gel were scraped from the chromatographic plate into counting vials. Radioactivity was determined by liquid scintillation spectrometry. In the reverse phase chromatographic system (Fig. 3)