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פותח על ידי קלירמאש פתרונות בע"מ -
Wound-inducible pinene cyclase from grand fir: Purification, characterization, and renaturation after SDS-PAGE
Year:
1992
Authors :
לוינסון, אפרים
;
.
Volume :
293
Co-Authors:
Lewinsohn, E., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Gijzen, M., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Croteau, R., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Facilitators :
From page:
167
To page:
173
(
Total pages:
7
)
Abstract:
The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-α-pinene (40%) and (-)-β-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 μM) for activity, exhibited a Km value of 6 μm for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 °C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 μM), and α-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 mM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor. © 1992.
Note:
Related Files :
Abies grandis
Cations, Divalent
Detergents
enzyme purification
Species Specificity
tree
trees
עוד תגיות
תוכן קשור
More details
DOI :
10.1016/0003-9861(92)90380-F
Article number:
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
18906
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:25
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Scientific Publication
Wound-inducible pinene cyclase from grand fir: Purification, characterization, and renaturation after SDS-PAGE
293
Lewinsohn, E., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Gijzen, M., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Croteau, R., Institute of Biological Chemistry, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340, United States
Wound-inducible pinene cyclase from grand fir: Purification, characterization, and renaturation after SDS-PAGE
The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-α-pinene (40%) and (-)-β-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 μM) for activity, exhibited a Km value of 6 μm for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 °C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 μM), and α-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 mM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor. © 1992.
Scientific Publication
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