Carotene cell-free systems

Studies with cell-free systems have now confirmed the proposed desaturation of phytoene to the more unsaturated carotenes. Cell-free systems obtained from tomato plastids and spinach chloroplasts will convert [ C]phy-toene (the 15-cis isomer) into lycopene and cyclic carotenes (Kushwaha et al., 1970 Subbarayan et al., 1970), as will an extract from P. blakesleeanus (Davies, 1973). Extracts ofH. cutirubrum (Kushwaha et al., 1976) incorporate radioactivity from tran.s-phytoene into the more unsaturated carotenes. With Flavobacterium extracts [ CJphytoene (the 15-cis isomer) was also converted to the more unsaturated carotenes (Brown et al., 1975). Later studies with tomato plastid extracts have demonstrated the conversion of c/i-[ C]phytofluene, rra/ij-[ C]phytofluene, and /ra/i5- -[ C]carotene to neurosporene and lycopene (Qureshiet al., 1974a,b). Cell-free extracts from H. cutirubrum (Kushwaha et al., 1976) also convert tran -phytofluene to fra/is- -carotene, -carotene to neurosporene, and neurosporene to lycopene. The conversion of neurosporene to lycopene has also been demonstrated using extracts from the Cl 15 mutant of P. blakesleeanus (Davies, 1973). 

A great diversity in molecular structure is observed among herbicides which inhibit carotene biosynthesis as is exemplified by the structures of norflurazon, fluridone and difunone (shown below). Nonetheless, many of these compounds, which comprise a subset of the larger group known as bleaching herbicides, appear to inhibit the same step in the biosynthetic pathway to the carotenoids (1 ). The inhibited step is the desaturation of 15-cis phytoene to 15- cis phytofluene (Figure 1) and the build-up of phytoene in plants and in cell-free systems which have been treated with these herbicides is well documented (2-4). 

Cell-free Systems. More direct evidence can be obtained by investigating cell-free carotene biosynthesis. From Phycomyces blakesleeanus, a fungus in which P-carotene formation is inhibited by norflurazon... 

A cell-free system from photosynthetically active species has to be developed that is able to produce substantial amounts of intermediates and desaturated carotenes. Noteworthy a recent report using isolated chromoplasts from daffodil flowers. Narcissus pseudonarcissus > and isopentenyl pyrophosphate as substrate noted increased levels of phytoene and geranylgeraniol vs. control in the presence of high concentration (50 j,M) of SAN 6706 (27). These cell-free assays should be investigated further with regard to possible different sensitivity against inhibitors because of the species used. 

Phytoene has been incorporated into carotenoids in several cell-free systems (Kushwaha et al., 1970, 1976 Subbarayanet al., 1970 Davies, 1973 Brown et al., 1975), and its position as an intermediate in the biosynthesis of more unsaturated carotenes is now well accepted. Previously some workers had concluded that phytoene did not behave as a precursor of carotenoids (for a review, see Britton, 1976a). This conclusion appears to have resulted from the accumulation of a metabolically inactive pool of phytoene in studies with inhibitors, and a failure of phytoene in this pool to be converted under certain conditions to carotenes on removal of the inhibitor. 

The direct conversion of lycopene to cyclic carotenes has also been demonstrated with a number of cell-free systems. Extracts from tomato plastids and spinach chloroplasts have been shown to incorporate radioactivity from lycopene into 8-, y-, a-, and jS-carotene (Kushwaha r al., 1%9 1970 Papa-stephanou, 1973). Incorporation of radioactivity from lycopene into -caro-tene and other carotenes has also been reported with bean leaf chloroplasts (Decker and Uehleke, 1%1 Hill et al., 1971) and a cell-free system from P. blakesleeanus (Davies, 1973). The occurrence of a-zeacarotene (7, 8 -dihy-dro- ,il -carotene) and /3-zeacarotene (7, 8 -dihydro-/8,il -carotene) in some organisms suggests that neurosporene can also be cyclized. Whether or not this pathway is of significance in the biosynthesis of y-, 8-, a-, and /3-caro-tenes is not clear, however (Britton, 1976b). Possibly, the enzyme that cy-clizes lycopene also cyclizes neurosporene, but at a much slower rate. Label from neurosporene has also been incorporated into /3-carotene by cell-free... 

Extensive studies in vitro from many groups have confirmed that exposure of LDL to a variety of pro-oxidant systems, both cell-free and cell-mediated, results in the formation of lipid hydroperoxides and peroxidation products, fragmentation of apoprotein Bioo, hydrolysis of phospholipids, oxidation of cholesterol and cholesterylesters, formation of oxysterols, preceded by consumption of a-tocopherol and accompanied by consumption of 8-carotene, the minor carotenoids and 7-tocopherol. 

Enzyme Systems. Carotenoid biosynthesis by crude cell-free preparations from Halobacterium cutirubrum 0-carotene), Phycomyces blakesleeanus mutants (/8-carotene), and a Neurospora crassa mutant (phytoene) has been demonstrated. Detailed studies of carotenogenic enzymes from tomato fruit... 

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