Radiation inactivation experiments predict that a large aggregate form of the insulin receptor is a highly active tyrosine-specific protein kinase.
The technique of radiation inactivation has been used on a highly purified insulin receptor in order to determine the functional molecular size responsible for tyrosine-specific protein kinase activity. When both insulin binding and kinase activities were analyzed with the same receptor preparations, the functional size for kinase activity was found to be larger than that for insulin binding activity. The radiation inactivation curve for kinase activity was multiphasic. This indicates that at least two components contribute to total kinase activity. The average minimal functional size for the kinase was 370,000 +/- 60,000 daltons (n = 7) which corresponds to the alpha 2 beta 2 form of the insulin receptor. The average functional size for larger forms was estimated to be approximately 4 X 10(6) daltons. (To minimize the complexity of the model used in this analysis, we have analyzed the radiation inactivation curves of the insulin receptor kinase activity with a two-component model. However, we believe that the larger component, greater than 1 X 10(6) daltons, is probably not a single molecular weight species but rather represents a continuum of sizes or aggregates of the alpha 2 beta 2 form of the receptor.) These larger forms contributed 93% of the total activity. Mild reduction of the insulin receptor preparation with dithiothreitol (DTT) activated the total kinase activity by 3.5-fold. Under this condition, the minimal functional kinase size was 380,000 +/- 30,000 daltons (n = 6) while the average functional size for the larger forms was approximately 3 X 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujita-Yamaguchi
Y
Y
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Harmon
J T
JT
Kathuria
S
S
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000528:radiation effects; D007328:Insulin / Q000032:analysis; D008433:Mathematics; D010316:Particle Size; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000528:radiation effects*; D011972:Receptor, Insulin / Q000378:metabolism / Q000528:radiation effects*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000494:pharmacology
D007328:Insulin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D004229:Dithiothreitol