Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography
Department of Biochemistry and Molecular Pharmacology, Department of Neurobiology University of Massachusetts Medical School
Medical Subject Headings
Cell Line; Cell Membrane; Chromatography, Affinity; Humans; Kinetics; Melanoma; Molecular Weight; Nerve Growth Factors; Receptors, Cell Surface; Receptors, Nerve Growth Factor
The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.
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Citation: J Biol Chem. 1983 Mar 10;258(5):3370-5.
Puma, Patricia; Buxser, Stephen E.; Watson, Leslie; Kelleher, Daniel J.; and Johnson, Gary L., "Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography" (1983). Biochemistry and Molecular Pharmacology Publications and Presentations. Paper 198.