The action of carp complement on sensitized cells (EA) proceeds in two steps requiring Ca2+and Mg2+, 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 sensitized with carp antibody), carp complement, optimal Ca2+and optimal Mg2+, the hemolysis took place after a lag period of about 10 min, but when Ca2+was present at zero-time and Mg2+was added 20 min later, there was almost no lag. On the contrary, when Mg2+was present at zero-time and Ca2+was added 20 min later, there was a lag of about 20 min. These observations indicate that the step requiring Ca2+precedes that in which Mg2+functions. A hemolytic intermediate complex, which was provisionally designated EACx’, was prepared by treating EA with carp complement in the presence of Ca2+, but with the absence of Mg2+at 20°C for 10 min. 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 Ca2+but absence of Mg2+at 0°C for 3 h. It was confirmed that the cells in the state EACx’ were completely lysed when treated with a sufficient quantity of Cy’ and optimal Mg2+, and that Cy’ did not react with EA even in the presence of optimal Mg2+.
All Science Journal Classification (ASJC) codes
- Aquatic Science