Document Detail

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions.
MedLine Citation:
PMID:  15248427     Owner:  NLM     Status:  MEDLINE    
This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.
Anna Maria Timperio; Christian G Huber; Lello Zolla
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of chromatography. A     Volume:  1040     ISSN:  0021-9673     ISO Abbreviation:  J Chromatogr A     Publication Date:  2004 Jun 
Date Detail:
Created Date:  2004-07-13     Completed Date:  2005-02-24     Revised Date:  2009-01-15    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  73-81     Citation Subset:  IM    
Department of Environmental Sciences, University of Tuscia, 01100 Viterbo, Italy.
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MeSH Terms
Chromatography, High Pressure Liquid
Photosystem I Protein Complex / chemistry*
Photosystem II Protein Complex / chemistry*
Plant Proteins / chemistry,  isolation & purification*
Spectrometry, Mass, Electrospray Ionization
Spinacia oleracea
Thylakoids / chemistry*
Reg. No./Substance:
0/Photosystem I Protein Complex; 0/Photosystem II Protein Complex; 0/Plant Proteins

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