Related Experiment Videos
Molecular size of the epidermal growth factor receptor-kinase as determined by radiation inactivation
Abstract:
Radiation inactivation with high energy electrons from a linear accelerator was used to determine the functional molecular size of the epidermal growth factor (EGF) binding site and the tyrosine-specific protein kinase activity in A-431 membranes. The target size of the protein portion of the EGF receptor glycoprotein was 147,000 daltons when the radiation-dependent decrease in maximal binding capacity was measured. Since the target size is in good agreement with the molecular size of the protein portion of the EGF receptor determined by denaturing biochemical methods, it appears that the monomeric receptor is the functional binding site in situ. The target size of the EGF-stimulated kinase activity associated with the affinity-purified EGF receptor/kinase was 133,000 and 144,000 daltons when assayed for the ability to autophosphorylate or to phosphorylate a tyrosine-containing peptide, respectively. However, the target size of the kinase activity that did not adhere to an EGF-affinity column was 54,000 and 69,000 daltons when assayed for phosphorylation of endogenous and exogenous substrates, respectively. Intermediate target sizes were obtained when kinase assays were performed on membranes prior to fractionation by affinity chromatography. These results, taken with other biochemical data, indicate that A-431 membranes contain a kinase activity that is a domain of the glycoprotein that contains the EGF binding site and that the membranes also contain another tyrosine-specific kinase or kinases that have an average size of approximately 60,000 daltons.
Insights
Radiation inactivation determined the molecular size of the epidermal growth factor (EGF) binding site and associated kinase activity in A-431 membranes. Results suggest the EGF receptor is the functional binding site and other tyrosine kinases exist in the membranes.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Epidermal Growth Factor (EGF) receptor plays a crucial role in cell growth and differentiation.
- Understanding the molecular size of the EGF receptor and its associated kinase activity is vital for comprehending its function.
- A-431 cells are a well-established model system for studying EGF receptor signaling.
Purpose of the Study:
- To determine the functional molecular size of the EGF binding site using radiation inactivation.
- To characterize the tyrosine-specific protein kinase activity associated with the EGF receptor.
- To investigate the presence and size of other tyrosine kinases in A-431 membranes.
Main Methods:
- Radiation inactivation using high-energy electrons from a linear accelerator.
- Measurement of EGF binding capacity.
- Assay of tyrosine-specific protein kinase activity (autophosphorylation and substrate phosphorylation).
- Affinity chromatography for EGF receptor purification.
Main Results:
- The protein portion of the EGF receptor has a target size of 147,000 daltons, consistent with the monomeric receptor being the functional binding site.
- EGF-stimulated kinase activity associated with the purified EGF receptor showed target sizes of 133,000-144,000 daltons.
- Unbound kinase activity in membranes had target sizes of 54,000-69,000 daltons, indicating the presence of smaller tyrosine kinases.
Conclusions:
- The monomeric EGF receptor glycoprotein functions as the primary EGF binding site in situ.
- A-431 membranes contain a tyrosine-specific kinase activity that is a domain of the EGF receptor.
- A-431 membranes also harbor distinct tyrosine kinases of approximately 60,000 daltons.