alpha 2-Macroglobulin binding to cultured fibroblasts. Solubilization and partial purification of binding sites.
Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.
D000818:Animals; D002277:Carcinoma; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D009062:Mouth Neoplasms; D009374:Neoplasms, Experimental / Q000378:metabolism; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins