Neurotrophin binding to human alpha 2-macroglobulin under apparent equilibrium conditions.
alpha 2-Macroglobulin (alpha 2M) is a broad-spectrum proteinase inhibitor and a carrier of certain growth factors. The purpose of this investigation was to characterize the interaction of alpha 2M with nerve growth factor-beta (NGF-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ciliary neurotrophic factor (CNTF) under apparent equilibrium conditions. Binding in solution was assessed using the cross-linking agent bis(sulfosuccinimidyl) suberate (BS3). Noncovalent binding of NGF-beta, NT-3, NT-4, and BDNF to native alpha 2M and alpha 2M-methylamine (a conformationally modified form of alpha 2M that is recognized by the alpha 2M receptor) reached apparent equilibrium in less than 20 min at 37 degrees C. Apparent KD values for the binding of NT-4, NGF-beta, NT-3, and BDNF to alpha 2M-methylamine were 61, 110, 120, and 150 nM, respectively. Native alpha 2M bound all four neurotrophins with decreased affinity. Unlabeled NGF-beta competed with the radioiodinated neurotrophins for binding to immobilized alpha 2M-methylamine. The K1 for unlabeled NGF-beta was 120 nM, in good agreement with the apparent KD determined by the BS3 method. The number of NGF-beta binding sites per immobilized alpha 2M-methylamine was 1.0. CNTF bound minimally, if at all, to native alpha 2M and alpha 2M-methylamine as determined using a number of techniques. The extent of binding was insufficient for the determination of an affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS)
Wolf
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BB
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Gonias
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SL
D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008744:Methylamines; D051379:Mice; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D020933:Neurotrophin 3; D011485:Protein Binding; D013388:Succinimides; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008744:Methylamines; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020933:Neurotrophin 3; D013388:Succinimides; D000511:alpha-Macroglobulins; C027451:methylamine; C035760:bis(sulfosuccinimidyl)suberate; C073922:neurotrophin 4