Document Detail


Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies.
MedLine Citation:
PMID:  2413806     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.
Authors:
D T Pang; B R Sharma; J A Shafer
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  242     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  1985 Oct 
Date Detail:
Created Date:  1985-11-08     Completed Date:  1985-11-08     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  176-86     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Antibodies / metabolism*
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
Female
Humans
Mathematics
Phosphotyrosine
Placenta / analysis
Pregnancy
Protein Kinases / isolation & purification*
Rabbits
Receptor, Insulin / metabolism*
Tyrosine / analogs & derivatives*,  metabolism
Grant Support
ID/Acronym/Agency:
AM 27659/AM/NIADDK NIH HHS; AM35249/AM/NIADDK NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies; 21820-51-9/Phosphotyrosine; 55520-40-6/Tyrosine; EC 2.7.-/Protein Kinases; EC 2.7.10.1/Receptor, Insulin

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


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