THE ROLE OF C4-BINDING PROTEIN AND f l lH IN PROTEOLYSIS OF C4b AND C3b* BY TEIZO FUJITA AND VICTOR NUSSENZWEIG

Clarification of the nature of the proteins involved in the cleavage of C3b and C4b is of great interest because these complement fragments are opsonins and, in addition, they modulate the activities of several key enzymes of the complement cascade. When these complement peptides are in solution, proteolysis by a serum enzyme, C3binactivator (C3bINA), 1 requires cofactors: f l lH in the case of C3b and C4-binding protein (C4-bp) or C3b-C4blNA cofactor in the case of C4b (1-4). Some uncertainties remain regarding the activities of the cofactors. The observations of Whaley and Ruddy (5) strongly suggest that only fl lH, among all serum proteins separated by molecular sieve chromatography, accelerates the inactivation of cellbound C3b by C3bINA, whereas the results of Nagasawa and Stroud (4) indicate that, in addition to f l lH, the C3b-C4bINA cofactor can also function in the cleavage of fluid phase C3b by C3bINA. Other sources of complication are that preparations of the cofactors are frequently cross-contaminated and that C4-bp contains two proteins that are antigenically identical but display slight differences in molecular weight and net charges (6). In the present paper we report the isolation of both forms of C4-bp and compare their activities, as well as that o f f l lH , on cell-bound and fluid phase C3b and C4b. In addition, we studied the relationship between C4-bp and the C3b-C4bINA co factor. Materials and Methods Materials. N,N'-methylene-bis-acrylamide, acrylamide, N,N,N' ,N' , tetramethylene diamine, ammonium persulfate, sodium dodecyl sulfate (SDS), Bio-Rad Laboratories, Richmond, Calif.; diisopropylfluorophosphate (DFP), Calbiochem-Behring Corp., American Hoechst Corp., San Diego, Calif.; bovine serum albumin (BSA), Miles Laboratories, Inc., Miles Research Products, Elkhart, Ind.; Sepharose CL-6B, CNBr-activated Sepharose 4B, Pharmacia Fine Chemicals, Division of Pharmacia, Inc., Piscataway, N.J.; trypsin, soybean trypsin inhibitor, Worthington Biochemical Corp., Freehold, N. J.; DEAE-cellulose (DE-52), Whatman Chemicals, Division of W & R Balston, Maidstone, Kent, England.