NEUROTROPHIN BINDING TO HUMAN ALPHA(2)-MACROGLOBULIN UNDER APPARENT EQUILIBRIUM CONDITIONS

alpha(2)-Macroglobulin (alpha(2)M) 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(2)M 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(2)M and alpha(2)-methylamine (a conformationally modified form of alpha(2)M that is recognized by the alpha(2)M receptor) reached apparent equilibrium in less than 20 min at 37 degrees C. Apparent K-D values for the binding of NT-4, NGF-beta, NT-3, and BDNF to alpha(2)M-methylamine were 61, 110, 120, and 150 nM, respectively. Native alpha(2)M bound all four neurotrophins with decreased affinity. Unlabeled NGF-beta competed with the radioiodinated neurotrophins for binding to immobilized alpha(2)M-methylamine. The K-I for unlabeled NGF-beta was 120 nM, in good agreement with the apparent K-D determined by the BS3 method. The number of NGF-beta binding sites per immobilized alpha(2)M-methylamine was 1.0. CNTF bound minimally, if at all, to native alpha(2)M and alpha(2)M-methylamine as determined using a number of techniques. The extent of binding was insufficient for the determination of an affinity constant. The studies presented here indicate that all four neurotrophins bind with similar affinity to the same site in alpha(2)M which is available primarily after the alpha(2)M undergoes conformational change. Interaction of neurotrophins with alpha(2)M may be important in regulating neurotrophin activity and/or availability.