. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 666 K. SODERHALL ET -6 -5 -4 -3 logC (mg/ml) -2 FIGURE 1. Effects of polymyxin B treatment of lipopolysaccharide (LPS) or laminarin (Lam) upon induction of clotting enzyme activity in Limulus amebocyte lysate. Various concentrations (C) of LPS or Lam were pretreated with polymyxin B (PmB) (100 U/ml) for 30 min before they were assayed for the induction of clotting enzyme activity, using the chromogenic peptide assay (see Materials and Methods). system is a carboxymethylated curdlan (CMPS), with a degree of polymerizatio
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 666 K. SODERHALL ET -6 -5 -4 -3 logC (mg/ml) -2 FIGURE 1. Effects of polymyxin B treatment of lipopolysaccharide (LPS) or laminarin (Lam) upon induction of clotting enzyme activity in Limulus amebocyte lysate. Various concentrations (C) of LPS or Lam were pretreated with polymyxin B (PmB) (100 U/ml) for 30 min before they were assayed for the induction of clotting enzyme activity, using the chromogenic peptide assay (see Materials and Methods). system is a carboxymethylated curdlan (CMPS), with a degree of polymerization of approximately 500. In our hands, this CMPS also activated proclotting enzyme and generated clotting enzyme activity (Table I), which resulted in subsequent gelation of Limulus amebocyte lysate. Polymyxin B (final concentration, 1000 U/ml) did not affect CMPS-induced activation of proclotting enzyme activity, suggesting that acti- vation was not due to endotoxin contamination (data not shown). Activation showed a curious dependence upon dosage, with inhibition occurring at CMPS concentrations above 10~2 mg/ml (data not shown) as also has been reported by Kakinuma et al. (1981). At these high concentrations (greater than 10~2 mg/ml), the CMPS preparation caused the immediate formation of a precipitate in Limulus lysate which could not be redissolved in 1% NaCl or distilled water. The supernatant prepared by removing this precipitate still contained endotoxin-inducible hydrolyzing activity towards Ac-Ile-Glu-Gly-Arg-pNA and coagulogen (Table III). This indicates that high concen- trations of CMPS did not inactivate or precipitate critical components in the "endotoxin pathway" of the Limulus lysate clotting system or precipitate the coagulogen. Thus, based on these observations, it is suggested that there are two separate pathways in the clotting system of Limulidae, one activated by endotoxin and the other by CMPS. Consistent with this suggestion, different lysate p
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