CHARACTERIZATION OF BOVINE SERUM FACTOR TRIGGERING THE LYSIS OF LIPOSOMES VIA COMPLEMENT ACTIVATION

Our previous studies have shown that the degree of damage to a liposom e corresponds to the variability of the animal species from which the serum comes, and that a complement activating factor (CAF) play's an i mportant role in inducing the activation of the complement system, ult imately leading to the lysis of the liposomes. In this study, our atte ntion focused on the characterization of the bovine serum factor (bCAF ) that is involved in complement-mediated immune lysis of the liposome . The active fraction containing CAF partially purified with PEG and a mmonium sulfate results in marked activation of the complement system via the alternative pathway when interacted with CAF-depleted serum, w hereas the active fraction or CAF-depleted serum alone does not activa te the complement. The interaction between lipopolysaccharide (LPS), h eparin, zymosan or their mixture in place of CAF and CAF-depleted seru m does not result in any significant activation of the complement syst em. Results from pretreatment with rabbit anti-bovine IgM IgG and rabb it anti-bovine Ige Ige indicate that activation of the complement syst em is not attributable to the antibody which is generally involved in activation of complement via the classical pathway. The results have f urther been proven by pretreatment with Concanavalin A (Con A) sepharo se and protein G sepharose ruling out the possibility of antibody-medi ated activation of complement. Our studies on collagenase and trypsin digestion demonstrate that the relative activity of CAF does not dimin ish with increase in collagenase concentration, and decreases with inc rease in trypsin concentration, strongly indicating that CAF does not have a collagen-like domain in its structure. The relative activity of CAF is dramatically inhibited after reduction with 2-mercaptoethanol (2-ME), clearly demonstrating that CAF is sensitive to reduction with 2-ME and confirming a sulhydryl-dependent protein. The optimal activit y of CAF is observed in the range of 35-45 degrees C and its half-life at 37 degrees C is about 105 h. Further mow, the relative activity of CAF increases and gradually approaches a plateau level with the incre ase of Mg2+ concentration. Obviously, complement activation induced by CAF depends on adequate Mg2+ concentration, confirming that this depe ndence is characteristic of the alternative pathway.