Detection of molecules with nerve growth factor binding activity in medium conditioned by L-929 fibroblasts.
L-929 fibroblasts (L cells) secrete a high molecular weight form of nerve growth factor (NGF) that is non-covalently bound and contains as part of its structure a molecule similar, if not identical, to beta-NGF in mouse submandibular glands. The other components of the NGF complex have not been characterized. In this study we used radiolabeled beta-NGF as a probe to detect molecules with NGF binding activity in L cell conditioned medium. The L cell NGF complex was dissociated at low pH, or with denaturants or detergents, and allowed to reassociate in the presence of 125I-beta-NGF. Radioactivity became associated with a complex that eluted in a high molecular weight volume on columns of Sephadex G-200 and Sephacryl S-500. Incorporation was saturable and did not occur under non-dissociating conditions. The complex was affinity cross-linked and studied by SDS gel electrophoresis. Radiolabeled molecules were observed with molecular weights of 151,000, 56,000 and 53,000. Labeling did not occur in the presence of excess unlabeled NGF or when cross-linking was done with fetal bovine serum, indicating that binding is specific and that binding activity is not derived from serum added to tissue culture medium. Solutions containing 7S NGF from mouse salivary glands were cross-linked by similar procedures but different banding patterns were observed. The data show that NGF binding molecules dissimilar from those in salivary glands are present in L cell conditioned medium.
Siminoski
K
K
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, MA 02115.
Murphy
R A
RA
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007739:L Cells / Q000502:physiology; D051379:Mice; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D003470:Culture Media; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D013388:Succinimides; C019358:disuccinimidyl suberate