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Purification and identification of the violaxanthin de-epoxidase as a 43 kDa protein

Arvidsson, Per-Ola; Bratt, Charlotte Eva; Carlsson, Marie and Åkerlund, Hans-Erik LU (1996) In Photosynthesis Research 49(2). p.119-129
Abstract
Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific... (More)
Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g–1 s–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase. (Less)
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organization
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type
Contribution to journal
publication status
published
subject
in
Photosynthesis Research
volume
49
issue
2
pages
119 - 129
publisher
Springer
ISSN
0166-8595
DOI
10.1007/BF00117662
language
English
LU publication?
yes
id
04b9ba5d-3f9b-46df-9b40-86ed1ac87302 (old id 1494400)
date added to LUP
2009-10-23 12:12:48
date last changed
2016-04-16 03:19:55
@article{04b9ba5d-3f9b-46df-9b40-86ed1ac87302,
  abstract     = {Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g–1 s–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase.},
  author       = {Arvidsson, Per-Ola and Bratt, Charlotte Eva and Carlsson, Marie and Åkerlund, Hans-Erik},
  issn         = {0166-8595},
  language     = {eng},
  number       = {2},
  pages        = {119--129},
  publisher    = {Springer},
  series       = {Photosynthesis Research},
  title        = {Purification and identification of the violaxanthin de-epoxidase as a 43 kDa protein},
  url          = {http://dx.doi.org/10.1007/BF00117662},
  volume       = {49},
  year         = {1996},
}