Michael K. Pangburn** and Hans J. Müller-Eberhard*· 5 abstract: The substrate for the complement serine protease factor I (Mt 88 000) is the C3b portion of the bimolecular complex (Mr 326000) which is composed of C3b and factor H. This complex is in equilibrium with free C3b and factor H. C3b participates in the recognition function and the positive feedback of the alternative complement pathway, and therefore regulation of C3b activity by factors H and I is crucial for control of this pathway. Conversion of C3b to inactive C3b by factor I (EC 3.4. 21.45) was found to be ac-companied by a marked decrease in the fluorescence of the probe ANS (8-anilino-l-naphthalenesulfonate), and this change in fluorescence was used to monitor continuously the proteolytic reaction. The initial velocities of the reaction between factor I and various concentrations of the C3b, H complex were analyzed by applying the Michaelis-Menten equation. The analysis indicated that the reaction exhibited simple enzyme-substrate behavior, although the substrate is a bimolecular complex in equilibrium with its subunits. The

.^^. ctivation of the complement system leads to the generation of fluid-phase and particle-bound C3b (MT 176000) which fulfills various functions in complement reactions (Müller-Eberhard & Schreiber, 1980). Nascent C3b exhibits a metastable binding site (Müller-Eberhard et al., 1966) through which it may form an ester or amide bond with surface con-f From the Department of MolecularImmunology, Research Institute of Scripps Clinic, La Jolla, California 92037. Received June 9, 1982. This is Publication No. 2731 from the Research Institute of Scripps Clinic. This work was supported by US Public Health Service Grants Al 17354, CA 27489, HL 07195, and HL 16411. This work was presented inpart at the 66th Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, LA, April 16, 1982.* This work was done during thetenure of an Established Investigatorship from the American Heart Association and with funds contributed in part by the American Heart Association, California Affiliate. 8 Cecil H. and Ida M. Green Investigator in Medical Research, Research Institute of Scripps Clinic. association constant (Ka) for the complex of C3b and factor H was determined by measuring initial velocities at various concentrations of C3b above and below the estimated disso-ciation constant for the C3b, H complex. All of the initial velocity measurements were then used simultaneously to refine Km, VmíX, and K3 by using an iterative process which yielded the best-fit values of all three constants. The apparent activation energy of the rate-determining step was found to be

24900 cal-mol" 1, reflecting the strong temperature dependence of this proteolytic reaction. The Ka for the interaction between C3b and factor H was 1.6 X 106 M'1 at 37 C and 4.4 X 106 M" 1 at 20 C. The Ka for the fluid-phase C3b, H complex was compared to the Ka for the cell-boundC3b, H complex mea-sured in other studies. The comparison suggests that recog-nition of potentially pathogenic microorganisms by human C3b results in a decrease in affinity between factor H and C3b when C3b is bound to these organisms. stituents of biological particles (Law et al., 1979). C3b initiates the positive feedback system of the alternative pathway (Müller-Eberhard & Gótze, 1972) by forming a bimolecular, Mg-dependent complex with the proenzyme factor B (Mr 93 000) and by modulating factor B such that it can be cleaved by its activating enzyme factor D (EC 3.4. 21.46). The re-sulting C3 convertase (C3b, Bb)(EC 3.4. 21.47) acts on native C3 (Mr 185000) and by removing the …