Carotenogenic enzymes from phycomyces

Cell extracts from car mutants of Phycomyces blakesleeanus, which can convert '4C—labelled mevalonic acid into unsaturated carotenes, were treated with high ionic strength buffer and a range of detergents in an attempt to solubilise the carotenogenic enzymes. Phytoene synthetase was dislodged from the membrane with high molarity Tris-HC1 buffer, but the enzymes which convert phytoene into s—carotene required detergent treatment for solubilisation. Of the 18 detergents used, only Tweens 40 and 60 and Zwittergent 3-08 solubilised these proteins whilst retaining their enzymic activities. INTRODUCTION Since the late 1960s a range of car strains of Phycomyces have been isolated which have deletions in genes involved in carotene biosynthesis (Ref.1 & 2). Cell—free systems from these mutants have been used to investigate discrete steps in the carotene pathway (Ref.3—6). Subcellular fractionation of crude cell extracts has shown that a membrane fraction is required for the formation of s-carotene prior to its deposition in lipid globules (Ref.7 & 8). Since most protein purification techniques are not applicable to membrane—bound enzymes, it is necessary to solubilise such proteins, whilst retaining their enzymic activities, before purification steps can be attempted. This report presents the results of attempts to solubilise carotenogenic enzymes from the C5 (phytoene-accumulating) and C115 (s-caroteneaccumulating) strains of Phycomyces. The routine preparation of cell extracts and the purification and radioassay of 4C—labelled carotenes have been described in detail in earlier publications (Ref.3 & 5). TREATMENT WITh HIGH IONIC STRENGTH BUFFER Cell extracts of C5 and C115 were prepared in Tris-HC1 buffer, pH 8.0, containing 5mM dithiothreitol, of molarity 25 to 400mM. Enzymic activities of the resultant cytosolic fractions showed that increasing ionic strength resulted in a parallel rise in phytoene formation from [2—14C}mevalonic acid. However, the enzymes catalysing the phytoene to s—carotene steps were not solubilised under these conditions (Table 1). TABLE 1. The effect of buffer concentration on carotenogenic activities of cytosolic fractions from CS and CilS Buffer concentration (mM) CS Phytoene % Control CliS Phytoene activity a s-Carotene 400 96.7 95.1 3.1 200 47.3 31.6 1.9 100 41.5 15.7 N.A. 25 7.5 10.5 N.A. N.A., not assayed; a, control activity 'refers to the incorporation of [2-14C]mevalonic acid (1iCi) into carotenes with crude cell extracts (10000 x g supernatant)