[14] Preparation of Extracts from Plants

Gegenheimer, Peter

doi:10.1016/0076-6879(90)82016-u

Cited by 140 publications

(63 citation statements)

References 22 publications

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“…PEI was prepared according to the method of Gegenheimer (1990). The redissolved PEG precipitate was brought to 0.3% (w/v) PEI with gentle stirring, held on ice for 5 min, and centrifuged at 10, OOOg for 10 min.…”

Section: Pei Precipitationmentioning

confidence: 99%

“…A DEAE-trisacryl column eluted with a step gradient of NaCl worked well in small-scale trials (2-fold purification, 50% recovery) but not when used on a preparative scale, presumably because of the longer times required. A faster alternative was precipitation using PEI, a soluble anion exchanger more commonly used to remove nucleic acids (Gegenheimer, 1990). After the PEI ion-exchange step the sample was dialyzed to remove NaCl and to bring it to 1.6 M KAc for hydrophobic interaction chromatography on phenyl-agarose.…”

mentioning

confidence: 99%

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Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

1995

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l h e osmoprotectant glycine betaine is synthesized via the pathway choline -+ betaine aldehyde + glycine betaine. I n spinach (Spinacia oleracea), the first step is catalyzed by choline monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO.['4C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [l4C1betaine aldehyde to ['4Clglycine betaine, which is isolated by ion exchange. The assay was used i n the purification of CMO from leaves of salinized spinach. The 1 O-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein.

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Section: Pei Precipitationmentioning

confidence: 99%

mentioning

confidence: 99%

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

1995

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“…Cell fractionations were carried out with fresh, fungusinfected leaf material (48 h post-inoculation) using the protocol of Gegenheimer (1990). This protocol did not allow an enrichment of vacuolar proteins, which were probably present as contaminants in the cytosolic fraction.…”

Section: Cell Fractionationmentioning

confidence: 99%

Pathogen‐Defence Gene prp1–1 from Potato Encodes an Auxin‐Responsive Glutathione S‐Transferase

Hahn

Strittmatter

1994

European Journal of Biochemistry

110

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Genetic studies have previously implicated the prp1 gene family in the defence of potato against infection with the late blight fungus Phytophthora infestans. Here, we show that the concentrations of PRP1 mRNA as well as protein rapidly increase in potato leaves after fungal infection and stay at high levels during an extended period of the infection cycle. After separation of subcellular components by differential centrifugation, PRP1 protein was identified in the cytosolic fraction. Expression studies with chimeric promoter/β‐glucuronidase gene constructs in transgenic potato plants provided evidence that transcription of the prp1‐1 gene, representing one member of the prp1 gene family, is at least partly responsible for the accumulation of PRP1 mRNA and protein upon fungal infection. After expression of the prp1‐1 coding sequence in Escherichia coli, the corresponding 26–kDa protein exhibited glutathione S‐transferase activity with Km values of 9.8 mM and 0.11 mM for the artificial standard substrate 1‐chloro‐2,4–dinitrobenzene and glutathione, respectively. Photoaffinity labeling of the protein with tritiated 5–azido‐indole‐3–acetic acid suggested that the phytohormone indole‐3–acetic acid or a structurally related compound serve as a regulator or substrate of the prp1‐1 encoded glutathione S–transferase. This assumption was further supported by the inhibitory effect of the phytohormone on the enzyme activity in vitro. The implications of these findings for a potential involvement of indole‐3–acetic acid in the control of defence reactions are discussed.

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“…Fully expanded aspen leaves of various ages (5 g) were homogenized with a mortar and pestle using a modified procedure suggested by Gegenheimer (6). The PEB contained 50 mm Tris-HCl, 20 mM MgC92, 5% glycerol, pH 8.0.…”

Section: Partial Purification Of Isoprene Synthasementioning

confidence: 99%

Enzymatic Synthesis of Isoprene from Dimethylallyl Diphosphate in Aspen Leaf Extracts

Silver

Fall²

1991

Plant Physiol.

183

132

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Aspen (Populus tremuloides Michx.) leaf extracts contain a newly discovered enzyme activity that catalyzes the magnesium ion-dependent elimination of diphosphate from dimethylallyl diphosphate with rearrangement to form isoprene (2-methyl,1-3-butadiene). This isoprene synthase activity has been partially purified. The nonenzymatic reaction of dimethylallyl diphosphate to isoprene, known to be acid catalyzed, may be insignificant at physiological pH. In contrast, the enzymatic reaction may be responsible for the majority of light-dependent isoprene production by isoprene-emitting plants.that isoprene is formed nonenzymatically in plant chloroplasts. In the light, the thylakoid lumen is acidified to approximately pH 5 by proton pumping (1), and if DMAPP is contained in this compartment, the acid-catalyzed reactions described above could generate isoprene. Evidence for the presence of DMAPP in the thylakoid lumen is lacking.We have been working to investigate the biochemical and physiological basis for the light-dependent formation of isoprene in plants (8, 13, 14), and as an extension of this work initiated experiments to directly test for the enzymatic production of isoprene from DMAPP. This report describes the detection of a new enzyme activity in leaf extracts that catalyzes the formation of isoprene.

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[14] Preparation of Extracts from Plants

Cited by 140 publications

References 22 publications

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

Pathogen‐Defence Gene prp1–1 from Potato Encodes an Auxin‐Responsive Glutathione S‐Transferase

Enzymatic Synthesis of Isoprene from Dimethylallyl Diphosphate in Aspen Leaf Extracts

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