Conformation-specific precipitation of human alpha 2-macroglobulin by divalent zinc or calf thymus histone H3.
Highly purified native alpha 2-macroglobulin (alpha 2M), alpha 2M-trypsin, and alpha 2M-methylamine were compared in experiments designed to study protein precipitation. Significant turbidity developed within 30 min in solutions containing histone H3 and either alpha 2M-methylamine or alpha 2M-trypsin, as determined by absorbance at lambda = 550 nm. No turbidity was detected in solutions that contained histone H3 and native alpha 2M or histone H3 alone. Experiments with radioiodinated histone H3 or radioiodinated proteinase inhibitor confirmed that both the H3 and the alpha 2M "fast" forms (alpha 2M-methylamine, alpha 2M-trypsin) were present in the precipitates generated. As much as 70% of the 125I-alpha 2M-methylamine was recovered in the precipitate after incubation with a 120-fold molar excess of H3 (concentration of alpha 2M-methylamine, 0.28 microM). The ratio of histone to proteinase inhibitor by weight in the precipitate was approximately two. Under comparable conditions, somewhat less alpha 2M-trypsin precipitated from solutions containing H3 than did alpha 2M-methylamine; however, inactivation of the alpha 2M-trypsin with phenylmethylsulfonyl fluoride prior to incubation increased the level of precipitation significantly. Solutions containing poly-L-lysine (Mr approximately 13,000) instead of histone did not form precipitates with any of the forms of alpha 2M studied. In a second set of experiments, radioiodinated native alpha 2M, alpha 2M-trypsin, and alpha 2M-methylamine were incubated in solutions containing ZnCl2, BaCl2, CdCl2, CuSO4, MgCl2, or NiCl2 (concentration of divalent cation between 5 microM and 1.0 mM). Native alpha 2M was soluble in all of these salts. By contrast, alpha 2M-methylamine and alpha 2M-trypsin precipitated extensively from solutions containing greater than 100 microM ZnCl2. Precipitation was greater than 90% complete at 1 mM ZnCl2. A similar effect was not observed with any of the other divalent cations.
Gonias, SL; Feldman, SR; Pratt, CW; Pizzo, SV
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