The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P450, cytochrome b5, and NADPH-cytochrome c (P450) reductase were measured in microsomes from aging artichoke tuber slices exposed to (24,25), and trans-cinnamic acid (1, 18) are hydroxylated, whereas p-chloro-N-methylaniline (27) was N-demethylated in Cyt P-450-dependent reactions. Involvment of Cyt P-450 is also suggested in the N-dealkylation of the herbicide Monuron2 (10).

Microsomes from apical buds of pea (Pisum saivnum L. var. Tdliphone a rames) seedlings hydroxylate lauric acid at the w-position. This oxidation is catalyzed by a cytochrome P450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent Km for lauric acid was 20 micromolar. NADPH-and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent Km values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 nucromolar for NADH.When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate w-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P450 species in pea microsomes.Several Cyt P450-dependent monooxygenases have been described in higher plants. These microsomal enzymes catalyze important oxidative steps in different biosynthetic pathways: hydroxylation of cinnamic acid (1, 13), a precursor of lignins and flavonoids, hydroxylation of fatty acids and cutin constituents (14,18), oxidation of kaurene and other intermediates in the biosynthesis of gibberellins (7), and hydroxylation of monoterpenic precursors of alkaloids (10). Cyt P450 may also be implicated in the oxidative N-demethylation of two foreign compounds, e.g. pchloro-N-methylaniline (20) In recent years, it has become evident that in the mammalian liver, Cyt P-450 exists in multiple molecular forms with broad and overlapping substrate specificities (9). Such evidence is not at hand concerning plant Cyt P450 enzymes.We have recently reported that aging Jerusalem artichoke tuber tissues contain an in-chain hydroxylating lauric acid monooxygenase (14) which, unlike the mammalian and microbial fatty acid hydroxylases, showed very strict substrate specificity (15). By ' Supported by the Centre National de la Recherche Scientifique, Equipe de Recherche Associ&e 104 and Grant ATP 4117.feeding to the tissues metabolic intermediates, the induction of this enzyme could be clearly dissociated from that of the cinnamic acid 4-hydroxylase, thus providing some evidence that the two monooxygenase activities were supported by distinct hemoproteins (15,16). In this paper, we describe a different lauric acid monooxygenase in pea seedlings. This enzyme has a higher apparent Km for laurate, yields w-hydroxylauric acid, and shows no NADPH-NADH synergism although NADH given alone sustains appreciable hydroxylation rates. Comparative induction studies confirm the existence of multiple forms of Cyt P-450 in higher plants. MATERIALS AND METHODSPlant Material. Pea seeds (Pisum sativum L. var Telephone a rames) were germinated and grown in moist vermiculite in darkness at 26°C for 7 d. The etiolated seedlings were then irradiated with far-red (730 ± 60 nm) for different times.Preparation of Microsomes. Apical buds were ground with a...

The substrate and product specifities of the (cytochrome P-450)-dependent laurate monooxygenase from tuber tissues of Jerusalem artichoke (Helianthus tuberosus L.) were investigated. The plant enzyme appeared strictly specific for the Clz free fatty acid and produced a mixture of C-8, C-9 and C-10 hydroxylated lauric acids, the C-9 derivative being predominant. No (2-12 or C-11 hydroxylated laurates were detected. The activity of the enzyme, which was not detectable in the intact tuber, was induced by slicing and aging the tissues on water, and strongly superinduced by the addition of manganese and phenobarbital to the aging medium. Regulation of laurate hydroxylase was clearly independent from that of cinnamic acid 4-hydroxylase, another plant cytochrome P-450 enzyme.

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