Effect of methylamine on the structure and function of the fourth component of human complement, C4.

The hemolytic activity of the fourth component of human complement, C4, was completely and irreversibly inhibited by the incubation of C4 in 2 m~ methylamine for 2 h at 23OC.The inactivation by methylamine was optimum at pH 8.5 to 10.5.When C4 was modified by ['4C]methylamine, 0.85 mol of amine was covalently incorporated/mol of protein.The label was found exclusively in the a subunit.In contrast, activation fragment C4b was completely unreactive with [''Clmethylamine.These observations indicate that the methylamine-reactive site of C4 has a direct role in its hemolytic function.If C4 was incubated at 75°C in pH 8.5 buffer with or without 0.1% (w/v) sodium dodecyl sulfate, the a subunit was p a r t i d y cleaved into M, = 59,500 and 43,500 fragments.Within 1 h, 49% of the subunit was converted to fragments.Incubation of C4 with 50 m~ methylamine for 2 h at 23"C, prior to heating at 75"C, prevented the autolytic cleavage.However, protease inhibitors, benzamidine hydrochloride, iodoacetamide, EDTA, soybean trypsin inhibitor, and eamino-n-caproic acid, had no effect on the cleavage.C4b did not undergo autolytic cleavage when incubated under identical conditions.Gigli et ul.(Gigli, I., Von Zabern, I., and Porter, R. R. (1977)