Natural release of covalently bound C3b from cell surfaces and the study of this phenomenon in the fluid-phase system.
Covalently bound C3b is released from cell surfaces (EAC1423 and zymosan-C3b) on incubation under physiologic conditions. The release of C3b from cell surfaces occurs by the cleavage of the covalent bond. Sodium dodecyl sulfate (SDS) abolishes the release, thereby indicating the requirement of the native structure of C3b in this process. The phenomenon of release of C3b from cell surfaces has also been observed in the fluid-phase system by using C3b-[3H]glycerol. The kinetics of the release of [3H]glycerol from C3b-[3H]glycerol were studied at 37 degrees C in 0.15 M phosphate buffer, pH 7.4. The first-order rate constant was found to be 0.028 +/- 0.003 hr-1. The release does not take place in either 8 M urea or 6 M guanidine hydrochloride, at pH 7.4. Under alkaline conditions, the rate of release is unaffected in the presence of SDS, indicating that the release in this pH range is not dependent on the native structure of the protein. From the Arrhenius plot in the temperature range 18 to 37 degrees C, an apparent activation energy for the hydrolysis reaction of 21.2 kcal/mol was calculated. The release phenomenon is exclusive for ester-linked complexes, as inferred by the absence of release of [3H]threonine from C3b-[3H]threonine, wherein the linkage is of the amide type. The presence or absence of the C3a portion of the molecule has no effect on the rate of release. The modification of the -SH group of C3i-/C3b-[3H]glycerol alters the rate of hydrolysis of the ester bond between C3i/C3b and [3H]glycerol. Protease inhibitors (PMSF, benzamidine HCl, and DFP) do not alter the rate of release, indicating that the hydrolysis reaction is not due to trace amounts of contaminating proteases. Thus, it appears that some chemical group(s) of C3i/C3b is (are) involved in the intramolecular hydrolysis of the ester bond between C3i/C3b and small molecules. This phenomenon may play an important role in the release of C3b from receptive surfaces once the biologic functions that require covalently bound C3b have been mediated.
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D003176:Complement C3 / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D012756:Sheep; D012996:Solutions; D013696:Temperature
D003176:Complement C3; D012996:Solutions; D003179:Complement C3b; D005947:Glucose; D005990:Glycerol