Changes in the binding of "fast"-form .alpha.2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes

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Abstract
Human .alpha.2-macroglobulin (.alpha.2M).sbd.CH3NH2 specifically binds to 3T3-L1 [mouse] fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4.degree. C. Binding to fibroblasts follows 1st-order kinetics only for the first 20-30 min of reaction, k1 = 160 .mu.M-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of .alpha.2M.sbd.CH3NH2/mg of cell protein or 60,000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows 1st-order kinetics, k1 = 720 .mu.M-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of .alpha.2M.sbd.CH3NH2/mg of cell protein or 60,000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for fast-form .alpha.2M but not for native .alpha.2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade .alpha.2M.sbd.CH3NH2 at 37.degree. C. For both cell types, the concentration of .alpha.2M.sbd.CH3NH2 needed for half-maximal uptake is 65 nM. Adipocytes take up more ligand and degrade less on a per cell basis than fibroblasts, a result that is consistent with inhibition of intracellular proteolysis by insulin; treatment of fibroblasts with insulin for several hours results in decreased degradation and increased uptake of ligand.

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