Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies.
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.
D T
DT
Pang
B R
BR
Sharma
J A
JA
Shafer
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008433:Mathematics; D019000:Phosphotyrosine; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification*; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000906:Antibodies; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D011972:Receptor, Insulin