Document Detail


The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins.
MedLine Citation:
PMID:  11680618     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.
Authors:
K Stroffekova; C Proenza; K G Beam
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Pflügers Archiv : European journal of physiology     Volume:  442     ISSN:  0031-6768     ISO Abbreviation:  Pflugers Arch.     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-10-26     Completed Date:  2002-02-21     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0154720     Medline TA:  Pflugers Arch     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  859-66     Citation Subset:  IM    
Affiliation:
Department of Anatomy and Neurobiology, Colorado State University, Fort Collins 80523, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Binding Sites
CHO Cells
Calcium Channels, L-Type / metabolism
Calmodulin / chemistry,  genetics
Cell Line
Cricetinae
Cysteine / metabolism*
Electric Conductivity
Fluoresceins / metabolism*
Fluorescent Dyes / metabolism*
Humans
Indicators and Reagents
Magnetic Resonance Spectroscopy
Mice
Muscle, Skeletal / metabolism
Mutagenesis
Oligopeptides / chemistry,  metabolism
Organometallic Compounds / metabolism*
Polymerase Chain Reaction
Proteins / metabolism*
Recombinant Proteins
Spectrometry, Fluorescence
Transfection
Grant Support
ID/Acronym/Agency:
NS24444/NS/NINDS NIH HHS
Chemical
Reg. No./Substance:
0/4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; 0/Calcium Channels, L-Type; 0/Calmodulin; 0/Fluoresceins; 0/Fluorescent Dyes; 0/Indicators and Reagents; 0/Oligopeptides; 0/Organometallic Compounds; 0/Proteins; 0/Recombinant Proteins; 52-90-4/Cysteine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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