Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor.
On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.
Carroscosa
J M
JM
Institut für Diabetesforschung, Krankenhaus München-Schwabing, München, F.R.G.
Schleicher
E
E
Maier
R
R
Hackenberg
C
C
Wieland
O H
OH
D019870:1-Phosphatidylinositol 4-Kinase; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D011972:Receptor, Insulin / Q000032:analysis*; D014018:Tissue Distribution; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014909:Wheat Germ Agglutinins; C027606:L-glutamic acid-L-tyrosine copolymer; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin