CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14a-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C.E. & Halkier, B.A. (1997) proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32±39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum±wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450´(L culture) 21 , with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14a-demethylation of obtusifoliol very actively (K m,app = 197 mm, k cat = 1.2 min 21) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (K s = 2.2 mm) and eburicol (K s = 2.5 mm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (K s = 289 mm), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and g-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The K s of plant CYP51 for LAB170250F (0.29 mm) and g-ketotriazole (0.40 mm) calculated from the type-II sp 2 nitrogenbinding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.

Cytochrome P450-dependent enzymes from wheat catalyze the oxidation of endogenous compounds (lauric and oleic acids) and of several herbicides (diclofop, chlortoluron, bentazon). Treatment of wheat seedlings with the safener, naphthalic anhydride and with phenobarbital increases dramatically several P450-dependent enzyme activities including diclofop and lauric acid hydroxylation. The parallel induction of lauric acid (omega-1)-hydroxylase and diclofop hydroxylase activities suggests that both compounds proceeds from the same or very similar forms of P450. To test whether either one or multiple P450 forms are involved in these oxidations, we have designed selective irreversible inhibitors of lauric acid (omega-1)-hydroxylase. Results of in vivo and in vitro experiments with acetylenic analogs of lauric acid (10- and 11-dodecynoic acids) strongly suggest that a single P450 catalyzes both laurate and diclofop hydroxylation. Treatment of wheat seedlings with these acetylenes results in a strong inhibition of the in vivo metabolism of diclofop although oxidation of chlortoluron and bentazon are not affected. Our results suggest that at least three distinct P450 forms are involved in the detoxification process of the three herbicides. Interestingly, we also demonstrate that herbicides themselves are potent inducers of the amount of total P450 and laurate/diclofop hydroxylase activies. This increased capacity of wheat to detoxify the herbicide through the induction of P450 enzymes seems to be for a large extend the mechanism which confers a tolerance on various herbicides.

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than alpha-tocopherol, alpha-tocopherylquinone, alpha-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol- 10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.

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