Title
Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology, Department of Neurobiology University of Massachusetts Medical School
Publication Date
1983-03-10
Document Type
Article
Subjects
Cell Line; Cell Membrane; Chromatography, Affinity; Humans; Kinetics; Melanoma; Molecular Weight; Nerve Growth Factors; Receptors, Cell Surface; Receptors, Nerve Growth Factor
Disciplines
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Molecular Biology
Abstract
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.
Source
J Biol Chem. 1983 Mar 10;258(5):3370-5.
Journal/Book/Conference Title
The Journal of biological chemistry
Related Resources
PubMed ID
6298234
Repository Citation
Puma P, Buxser SE, Watson L, Kelleher DJ, Johnson GL. (1983). Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography. Biochemistry and Molecular Pharmacology Publications. Retrieved from https://escholarship.umassmed.edu/bmp_pp/198