Hydroperoxide Isomerase

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577

304

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Jasmonic Acid by Several Plant Species

Vick

Zimmerman²

1984

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577

304

“…This enzyme converts the hydroperoxides resulting from the action of lipoxygenase with linoleic or linolenic acids to the corresponding a-ketol and has since been identified in many plant tissues (2,3,8,10,11,13). To determine the functional role of the hydroperoxide isomerase enzyme in plant metabolism, we have investigated the intracellular location of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…Substrate for the hydroperoxide isomerase assay was prepared by incubating 0.6 ml of linoleic acid (Hormel Institute)' solution prepared according to Surrey (9) (13). Cytochrome c oxidase was assayed by following the decrease in absorption at 550 nm of a reaction mixture containing 2.5 ml of 0.1 M potassium phosphate buffer, pH 7.0; 0.02 to 0.10 ml of enzyme; and 0.5 ml of cytochrome c solution.…”

mentioning

confidence: 99%

Intracellular Distribution of Hydroperoxide Isomerase

Zimmerman

Vick²,

Borg³

1974

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.The identification of a flaxseed hydroperoxide isomerase enzyme was first reported in 1966 (12). This enzyme converts the hydroperoxides resulting from the action of lipoxygenase with linoleic or linolenic acids to the corresponding a-ketol and has since been identified in many plant tissues (2,3,8,10,11,13). To determine the functional role of the hydroperoxide isomerase enzyme in plant metabolism, we have investigated the intracellular location of the enzyme. This paper reports the presence of hydroperoxide isomerase activity in mitochondria isolated from sunflower cotyledons and seeds by sucrose density gradient centrifugation. In addition, the paper reports the presence of this activity in the mitochondria of watermelon, flax, and cauliflower. MATERIALS AND METHODSContinuous, concave sucrose density gradients ranging from 33% (w/w) sucrose to 60% sucrose were prepared over a 3-ml base of 60% sucrose in 7.5-X 2.5-cm centrifuge tubes. The sucrose gradient contained 1 mm EDTA.Sunflower (Helianthus annuus L. var. Mingren) seeds were soaked overnight in water and germinated in moist paper toweling for 6 days in the dark. The cotyledons were prepared at 4 C under a nitrogen atmosphere by chopping 10 g of the cotyledons in an onion chopper with 20 ml of a medium containing 0.165 M Tricine buffer, pH 7.5; 10 mm EDTA; 10 mM dithioerythritol; 10 mm KCl; 10 mM MgCI2; and 1.0 M sucrose (7). The mixture was transferred to a mortar and ground gently under N, for 2 min with a pestle. The for 10 min to remove cellular debris, and the supernatant was centrifuged at 10,000g for 20 min. The resulting pellet was resuspended in 1 ml of the homogenizing medium. Large clumps of the pellet were broken by repeated withdrawal into a 2-ml volumetric pipette. The suspension was then layered on the sucrose density gradient and centrifuged 5 hr at 58,000g. For differential centrifugation, the 480g supernatant was centrifuged at 50,000g for 20 min, and the pellet was washed twice with homogenizing medium. The first 50,000g supernatant was centrifuged at 105,000g for 1 hr, and the pellet was washed twice with homogenizing medium.Mitochondria were isolated from ungerminated sunflower (Helianthus annuus L. var. Mingren) seeds by grinding 260 g of the seeds, with hulls removed, for 10 sec in a high speed grinder. The resulting meal was defatted by washing with 1300 ml of cold petroleum ether in a Buchner funnel at 4 C and placed in a vacuum desiccator to remove the petr...

“…Lipoxygenase was assayed spectrophotometrically at 234 nm in 50 mm K-phosphate (pH 6.0) by the method of Surrey (10). Hydroperoxide isomerase was measured in 50 mm K-phosphate (pH 6.5) as described previously by measuring the decrease in A at 234 nm caused by the disappearance of the conjugated 1 3-hydroperoxylinolenic acid (19 Figure 3 shows the formation of tOPC-8:0 from 12-oxo-tPDA over a period of 60 min. Except for a slight lag during the first 10 min, the reaction rate was linear during the period measured.…”

mentioning

confidence: 99%

Characterization of 12-Oxo-Phytodienoic Acid Reductase in Corn

Vick

Zimmerman²

1986

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12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a K,. of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a K. of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined.The conversion of linolenic acid to jasmonic acid by several plant species has recently been demonstrated with in vivo experiments (15, 16). The first enzyme of the jasmonic acid pathway, lipoxygenase, has been the subject of several reviews (1, 5, 1 1). The second enzyme, hydroperoxide cyclase, is present in many plant species and its properties in cell-free extracts have also been reported (12)(13)(14)17). The third enzyme, 12-oxo-phytodienoic acid reductase, has not previously been characterized. Its existence was inferred from the analysis of metabolites produced in vivo from 12-oxo-phytodienoic acid in various plant tissues (15,16). The enzyme, which catalyzes the reduction ofa double bond in the cyclopentenone ring of 12-oxo-PDA' (Fig. 1 Abbreviations: 12-oxo-PDA, 12-oxo-cis,cis-10, 15-phytodienoic acid; tOPC-8:0, (IR,2R)3-oxo-2-(2'-pentenyl)cyclopentaneoctanoic acid; tOPC-10:0, (IR,2R)3-oxo-2-(2'-pentenyl)cyclopentanedecanoic acid.The designation t or c indicates the orientation, trans or cis, of the side chains with respect to the plane of the ring.2 Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by the United States Department of Agriculture.plates were from Whatman, Inc., Clifton, NJ.Plant Materials. An acetone powder was prepared from corn kernels Zea mays L., var NK PX443 (Northrup King). First the kernels were rinsed with methanol and acetone to remove fungicide, then ground in a Wiley mill with a 2 mm screen. The resulting meal was ground to a flour in a Quad roller mill. The flour which passed through a 40 mesh sieve was homogenized in cold acetone (-20°C) with a Virtis 45 homogenizer and filtered through a Buchner funnel. Cold diethyl ether (-20°C) was used to wash the powder, and the solvents were then removed under vacuum. Enzymes were extracted from the powder by adding 2 ml of 50 mm K-phosphate (pH 7.2) for each g of powder and incubating the mixture for 30 min on ice. After centrifugation at 17,000g for 10 min, the supernatant was decanted and used as the enzyme source. The protein concentration was usually 10 to 12 mg/ml.For studies of enzyme activities in germinating corn seedlings, the corn kernels were rinsed with methanol and acetone to remove fungicide, then planted between two sheets of moist paper toweling 2 cm from the t...