Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of <i>Marah macrocarpus</i>

Hasson, E.; West, Charles A.

doi:10.1104/pp.58.4.473

Cited by 48 publications

(23 citation statements)

References 13 publications

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“…They are involved in a variety of catabolic and biosynthetic pathways and in the metabolism of drugs and xenobiotics. In plants, Cyt P-450 has been identified, for example, in the trans-cinnamic acid and kaurene hydroxylases (10,11). Others are suspected participants in a number of reactions associated with xenobiotic metabolism (6, 8) (for reviews see Refs.…”

Section: Introductionmentioning

confidence: 99%

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

O’Keefe

Leto²

1989

Plant Physiol.

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The microsomal fraction from the mesocarp of avocado (Persea amerkana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest pattems, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarites with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.Cytochrome P450 dependent monooxygenases are widespread in nature. They are involved in a variety of catabolic and biosynthetic pathways and in the metabolism of drugs and xenobiotics. In plants, Cyt P-450 has been identified, for example, in the trans-cinnamic acid and kaurene hydroxylases (10,11). Others are suspected participants in a number of reactions associated with xenobiotic metabolism (6, 8) (for reviews see Refs. 4 and 20). Within these monooxygenase systems, Cyt P-450 serves as a terminal oxidase, responsible for substrate recognition, binding, and oxygen redox chemistry (5, 29).The mechanistic and structural details of Cyt P450 dependent monooxygenases have been developed largely from studies of the bacterial (Pseudomonas putida) camphor hydroxylase system (23,29). Because of their importance in xenobiotic and drug metabolism, these enzymes have also been thoroughly studied in mammalian liver (3,5,31). In contrast, Cyt P-450 of higher plant origin has not been the subject of extensive biochemical characterization, primarily because of the low content in many plant tissues, difficulties with identification in Chl containing tissues, and supposed lability in homogenized plant extracts. ' (6,10,12,13,19,20). Cyt P-450 has been purified from tulip (Tulipa gesneriana) bulbs (12), and from tubers of Jerusalem artichoke (Helianthus tuberosus) (10). Additionally, an NADPH:Cyt P450 reductase has been purified from Jerusalem artichoke (2), and the related Cyt b5 and NADH:Cyt b5 reductase have been purified from Pisum sativum (16). Unfortunately, the tulip bulb Cyt P450 has not been associated with any enzymic activity, and polyclonal antibodies raised against it were not cross reactive with proteins from other species (1 1). The Jerusalem artichoke preparation can be reconstituted into its trans-cinnamate hydroxylase activity, but it has a low heme specific content and is not homogeneous (10). No primary sequence information has been reported for either o...

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Section: Introductionmentioning

confidence: 99%

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

O’Keefe

Leto²

1989

Plant Physiol.

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“…Since the P. hybrida F3',5'H could not be solubilized and remain active even though the procedure used here was similar, the F3',5'H must be extremely unstable when removed from its native lipid environment. Hasson and West (1976) found that solubilization of microsomal kaurene oxidase with deoxycholate or Triton X-100 inhibited enzyme activity. Some activity was restored by removal of deoxycholate but not by removal of Triton X-100.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

1994

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“…In plants, different isoforms of Cyt P-450 are implicated in essential biosynthetic pathways such as the production of lignin, at the level of cinnamate hydroxylation (1) and ferulate hydroxylation (11); the biosynthesis of cutin and suberin, at the level of fatty acid hydroxylation (15); and the biosynthesis of hormones (13), pigments (12), and phytoalexins (14). Cyt P-450s are also implicated in the metabolism of various herbicides in several plant species (8).…”

Section: Methodsmentioning

confidence: 99%

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Lesot

Benveniste²,

Hasenfratz³

et al. 1992

Plant Physiol.

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Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPHcytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPHCyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.markable by the presence of both FAD2 and FMN prosthetic groups, exists as a single form in animals (23). From Jerusalem artichoke (Helianthus tuberosus) tubers we purified a plant NADPH-Cyt P-450 reductase (2). This enzyme preparation, which migrated as a single broad band (82 kD) on 12.5% SDS-PAGE, was used to raise polyclonal antibodies (3). Western blotting of the reductase after 7.5% SDS-PAGE showed that the reductase preparation was composed of three proteins of very close molecular mass: 80, 82, and 84 kD. To get insight into the function and immunological relationships among these proteins, we set out to produce mAbs against the reductase preparation. A similar approach had produced mAbs specific for hepatic Cyt P-450 isoforms from rats treated with different inducers (21).Here we present the characterization of two families of mAbs we have obtained, and we discuss the relationship between the isoforms of Jerusalem artichoke NADPH-Cyt P-450 reductase. MATERIALS AND METHODSPurification of NADPH-Cyt P-450 Reductase NADPH-Cyt P-450 reductase (fpl) from Jerusalem artichoke (Helianthus tuberosus) was purified from solubilized microsomes as described (2).The microsomal Cyt P-450-dependent monooxygenases are multienzyme complexes consisting essentially of the hemoprotein Cyt P-450 and the flavoprotein NADPH-Cyt P-450 reductase (24). In plants, different isoforms of Cyt P-450 are implicated in essential biosynthetic pathways such as the production of lignin, at the level of cinnamate hydroxylation (1) and ferulate hydroxylation (11); the biosynthe...

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Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus

Abstract: The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-oi, ent-kaur-16-en-19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en7a-ol-19-oic The diterpene hydrocarbon ent-kaur-16-ene2 is oxidized via a series of irreversible steps in the sequence kaurene3 to kaurenol

Cited by 48 publications

References 13 publications

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

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