Kinetic measurement of the membranolytic activity of serum complement using bioluminescent bacteria.
A method for studying the kinetics of serum complement activity is presented. The assay utilises Escherichia coli and Bacillus subtilis cells which have been made bioluminescent by expressing an insect luciferase gene from Pyrophorus plagiophthalamus. The diffusion of the luciferase enzyme substrate through the cell membranes is very slow, and therefore a change in membrane permeability is seen as a change of the in vivo luminescence of the cells. Treatment of the bacteria with human serum resulted in a bell-shaped curve of in vivo luminescence since this procedure facilitates passage of the substrate to the cytoplasm. The time point of maximum light emission was dependent on serum dilution. In vivo luminescence also proved to be a good indicator of the viability of bacterial cells. Using C1q deficient serum, or following treatment of normal serum with EGTA and Mg2+ it was possible to separate the membranolytic activities of alternative and classical pathways.
Virta
M
M
Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland. marko.virta@abo.fi
Karp
M
M
Rönnemaa
S
S
Lilius
E M
EM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis; D002470:Cell Survival; D003165:Complement System Proteins / Q000032:analysis*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008163:Luminescent Measurements; D011994:Recombinant Proteins; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D003165:Complement System Proteins; D008156:Luciferases