The action of carp complement on sensitized cells (EA) proceeds in two steps requiring Ca
2+ and Mg
2+, respectively. In order to decide which of these steps precedes in immune hemolysis, we carried out a kinetic experiment by use of a buffer and carp serum depleted of cations by passage through Amberlite IRC-50. In a reaction mixture composed of EA (sheep red blood cells sensi-tized with carp antibody), carp complement, optimal Ca
2+ and optimal Mg
2+, the hemolysis took place after a lag period of about 10min, but when Ca
2+ was present at zerotime and Mg
2+ was added 20min later, there was almost no lag. On the contrary, when Mg
2+ was present at zerotime and Ca
2+ was added 20min later, there was a lag of about 20min. These observations indicate that the step requiring Ca
2+ precedes that in which Mg
2+ functions. A hemolytic intermediate complex, which was provisionally designated EACx', was prepared by treating EA with carp complement in the presence of Ca
2+, but with the absence of Mg
2+ at 20°C for 10min. Furthermore, carp serum free from Cx', which was provisionally designated Cy', was also obtained by treating resin-passed carp serum with excessive EA in the presence of Ca
2+ but absence of Mg
2+ at 0°C for 3h. It was confirmed that the cells in the state EACx' were completely lysed when treated with a sufficient quantity of Cy' and optimal Mg
2+, and that Cy' did not react with EA even in the presence of optimal Mg
2+.
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