C5 Convertase of the Alternative Pathway of Complement
KINETIC ANALYSIS OF THE FREE AND SURFACE-BOUND FORMS OF THE ENZYME*

Nenoo Rawal and Michael K. PangburnDagger

From the Department of Biochemistry, University of Texas Health Science Center, Tyler, Texas 75710

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (kcat). However, the surface-bound enzyme, ZymC3b,Bb had a Km (1.4 µM) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (Km = 24 µM). The kcat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s-1 and that for soluble C3b,Bb was 0.0110 s-1. Both forms of the enzyme had a low turnover number at Vmax (0.23 to 0.68 C5/min/enzyme). Substituting Mg2+ for Ni2+ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the Km determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

C5 convertases are serine proteases that cleave C5, the fifth component of complement. The cleavage of C5 is the last enzymatic step in the complement activation cascade resulting in the formation of two biologically important fragments, C5a and C5b.1 Both fragments play vital roles in killing microorganisms. C5a, the smaller fragment, is a potent chemotactic and spasmogenic anaphylatoxin. It mediates inflammatory responses by stimulating neutrophils and phagocytes. C5b, the larger fragment, initiates the formation of the membrane attack complex (C5b-9) which results in the lysis of bacteria and other pathogens. Although complement has important functions in host defense, it also contributes to the pathology in many inflammatory diseases (1), in xenotransplant rejection (2), and in reperfusion injury (3). This has prompted a search for inhibitors that can control activation of C5. Specific inhibition of C5 activation would preserve the immune clearance and opsonization functions of complement which depend on C3b, but it would prevent the generation of both C5a and C5b-9. Recent approaches in blocking complement activation include inhibitory anti-C5 antibodies (4), synthetic peptides (5, 6), synthetic protease inhibitors (7, 8), soluble constructs of complement membrane proteins (9-11), and transgenic animals that express proteins capable of inhibiting activation of human complement in xenotransplant models (12).

The C5 convertases of the complement system are complex enzymes. In their most common forms the enzymes appear to be made up of a C3 convertase and an additional C3b or C4b molecule (13-18). The C3 convertases are themselves bimolecular complexes. In the classical pathway the C3 convertase (C4b, C2a) consists of C2a, the proteolytic subunit, noncovalently bound to C4b. In the alternative pathway the C3 convertase has a similar structure with Bb, the proteolytic unit, noncovalently bound to a C3b molecule. Additional C3b molecules that are needed to allow efficient C5 cleavage by these C3 convertases are produced by the activation of C3 by the C3 convertases themselves (C4b, C2a, or C3b,Bb). The result of C3 activation is the covalent attachment of numerous C3b molecules within a few hundred angstroms of the C3 convertase. Originally it was thought that attachment of C3b molecules around the C3 convertase was sufficient to generate C5 convertase activity. Vogt et al. (19) revealed that the role of the additional C3b was to bind the substrate C5. Later a more defined structure was proposed in which the additional C3b binds covalently to a specific site on the C4b or C3b subunit of the C3 convertase resulting in the formation of the complexes C3b, C4b, C2a, or C3b2,Bb (16, 17, 20, 21). These structures are now thought to be the functional C5 convertases of the classical and alternative pathways, respectively. However, other forms of the enzyme have been shown to be capable of activating C5. Cobra venom factor forms a C5 convertase (CVF,Bb) that contains no additional C3b and is not cell-bound (22-24). The fluid phase alternative pathway C3 convertase (C3b,Bb) has been reported to cleave C5 in the presence of additional free or cell-bound C3b (19, 25). A covalent dimer of C4b that involves no C3b has been reported to express C5 convertase activity when complexed to C2a (18, 26). A number of studies have examined the affinity of C5 for the non-enzymatic subunits C3b or C4b in the fluid phase or on surfaces (16, 17, 19, 25, 27). In contrast, by examining the Km of the enzyme we have measured the interaction of C5 with only those C3b molecules that are part of functional C5 convertases.

Despite the amount of work directed toward discovering the subunit structure of the C5 convertase and the physiological consequences of C5 activation, no study has ever reported the enzymatic properties of the C5 convertase itself. In planning this study, we believed that a kinetic analysis of the C5 convertase would advance our understanding of the mechanism of action of this enzyme and that it might also reveal the functional role of the additional C3b molecule. The data show that the fluid phase monomeric C5 convertase is able to cleave C5 without a second C3b and that its catalytic rate is similar to that of the surface-bound C5 convertase. The surface-bound enzyme was found to have a 17-fold lower Km for C5 than the fluid phase C5 convertase. This increased affinity would lead to a proportional increase in the rate of C5 activation at physiological C5 concentrations. The catalytic rate of the natural surface-bound C5 convertase determined in this study indicates that the cleavage of C5 is one of the slowest enzymatic reactions known. This is most apparent in the turnover number which is one C5 molecule cleaved per 4 min per enzyme at Vmax.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents-- Nonidet P-40, a non-ionic detergent, EDTA, zymosan, trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated), methylamine (CH3NH2·HCl), and veronal were purchased from Sigma. Veronal-buffered saline (VBS) contained 5 mM barbital and 145 mM NaCl, pH 7.4. Gelatin veronal-buffered saline (GVB) was VBS containing 0.1% gelatin, and GVBE was GVB containing 10 mM EDTA. Chicken erythrocytes (Ec) were isolated from chicken blood purchased from Colorado Serum Co. (Denver, CO).

Purified Proteins-- Complement proteins used in the present studies were all purified from normal human plasma. C3 (28, 29), factor B (30), and factor D (31) were isolated as described in the references cited. C5 was isolated as described (32), except that ceramic hydroxylapatite (Bio-Rad) was used instead of hydroxylapatite. Purified C5b,6 was obtained from Advance Research Technologies (San Diego, CA). All proteins were homogeneous by polyacrylamide gel electrophoresis. Electrophoresis was performed in the presence of sodium dodecyl sulfate in 10% polyacrylamide gels under both reducing and non-reducing conditions. Any trace contaminants identified by immunodiffusion were removed by passing the protein preparations over a column containing the specific antisera coupled to CNBr-activated Sepharose. All proteins were treated with anti-factor H-Sepharose to remove traces of factor H. Protein concentrations of C3, C3b, C5, and factor D were determined spectrophotometrically using an E280 nm1% of 11.0. E280 nm1% values for C5b,6, C6, and factor B were 10.3, 10.8 and 12.7, respectively. All purified proteins were stored at -76 °C. Molecular weight values employed in the calculations were 176,000 for C3b, 190,000 for C5, 179,000 for C5b, 120,000 for C6, 299,000 for C5b,6, 93,000 for factor B, 63,000 for Bb, and 24,000 for factor D.

Preparation of ZymC3b-- C3b was first deposited on zymosan by resuspending 1 × 1010 zymosan particles in 0.2 ml of 10 mg/ml C3 and adding 5 µg of trypsin, followed by a 10-min incubation at 22 °C. The deposition of C3b by trypsin was repeated, and the cells were washed six times with 5 ml of GVB. The zymosan particles were resuspended in 100 µl of GVB and mixed with 50 µl of GVB containing factors B (35 µg) and D (0.5 µg) and 50 µl of 10 mM NiCl2. After 5 min incubation at 22 °C, 5 µl of 0.2 M EDTA were added. The bound C3b was amplified by adding 50 µl of C3 (500 µg) and incubating the cells for 1 h at 22 °C. The zymosan particles bearing C3b were washed, and the amplification procedure was repeated until the desired numbers of C3b/zymosan were obtained (33). The total number of C3b molecules bound to zymosan was estimated by a method utilizing 125I-C3 and radiolabeled factor H as described previously (34) and found to be approximately 150,000 C3b bound per zymosan particle.

Preparation of Monomeric C3b-- C3b was generated from greater than 99% native C3 by the action of factors B and D, present in amounts sufficient to cleave all the C3 to C3b. Briefly, 180 mg of C3 in 180 ml of VBS at 37 °C were incubated with 1 ml of 1 mg/ml factor B, 0.15 ml of 0.4 mg/ml of factor D, 2 ml of 50 mM NiCl2, and 3 ml of 5% phenol (included to prevent C3b dimer formation) (35) for 45 min at 37 °C. Trace amounts of C3b dimers and other components were separated from C3b by gel filtration on Bio-Gel A-0.5m. After storage at -76 °C, the purified C3b was again gel- filtered on Bio-Gel A-0.5m, and the C5 convertase activity of the C3b monomer was determined to be the same as the bulk frozen and thawed C3b, indicating that the C5 convertase activity of the fluid phase C3b used in this study was due to monomeric C3b. Furthermore, to make sure that no dimers could be formed during assays from traces of C3, C3b was treated with 0.2 M methylamine, pH 8.0, for 1 h at 37 °C to inactivate any C3 (36) that might be present but below the level of detection of all other methods of analysis. Activity of the treated C3b monomer was found to be similar to that of the untreated C3b.

Quantitation of Reaction Products-- C5b,6 was measured hemolytically by taking advantage of the sensitivity of chicken erythrocytes (Ec) to reactive lysis by human C5b,6. Tubes containing Ec (1.2 × 107) and 5 µl of pooled normal human serum as a source of complement proteins C7-C9 in a final volume of 225 µl of GVBE were kept ready on ice. An aliquot (25 µl) of the diluted sample from C5 convertase assays or purified C5b,6 was added, and the mixtures were incubated for 10 min at 37 °C. The unlysed cells were removed by centrifugation for 1 min at 10,000 × g. The amount of hemoglobin released was quantitated spectrophotometrically at 414 nm. To determine 100% lysis, Ec were lysed in 2% Nonidet P-40. Control enzyme assays containing C5 and C6, but no C5 convertase, were subtracted as the background. To ensure that no detectable C5b,6 was formed from the C5 and C6 in the pooled normal human serum used as a source of C7-9 during the lysis of Ec, reactions containing C5 convertase, but no purified C5 or C6, were used as controls. C5b,6 concentration was quantitated from a standard curve using purified C5b,6 (see "Results").

C5a, the other product of C5 cleavage, was quantitated using a commercial radioimmunoassay (RIA) from Amersham Pharmacia Biotech (Buckinghamshire, UK). The RIA was designed to measure C5a in serum, whereas the assays described herein employed purified proteins. In order to achieve complete precipitation of uncleaved C5 (which interferes with the RIA), 0.5% bovine serum albumin was added as a carrier protein after the addition of the RIA precipitating reagent. Addition of the carrier protein together with an overnight incubation at 0 °C resulted in complete precipitation of unreacted C5. Subsequent steps were performed as described by the manufacturer.

Demonstration of Constant C5 Convertase Enzyme Levels during Assays-- Due to the short half-life of C5 convertases, excess factors B and D were added to allow enzyme reformation during assays. Constant levels of enzyme during the 15-min assays at 37 °C were demonstrated by showing that the amount of C5b,6 produced was constant at different time intervals during the assay. This was done by incubating the C5 convertase for different lengths of time under the assay conditions described below and then determining the extent of C5 cleaved in the next 5 min at 37 °C. Surface-bound C5 convertase, ZymC3b,Bb, was incubated for 3, 6, 9, 12, or 15 min at 37 °C in assay mixtures containing factor B (1.0 µg, 430 nM), factor D (0.1 µg, 167 nM), 0.5 mM NiCl2 or 0.5 mM MgCl2, and ZymC3b bearing 27 ng of bound C3b (determined by binding of radiolabeled Bb and resulting in an enzyme concentration of 6.2 nM C5 convertase). Then, a mixture containing C5 (5 µg, 1053 nM) and excess C6 (2.5 µg, 833 nM) was added, and the amount of C5b,6 formed in the subsequent 5 min at 37 °C was determined as described above.

Kinetic Measurements of the Surface-bound C5 Convertase-- Enzyme velocities were determined under saturating concentrations of factors B and D and C6 in 0.5-ml siliconized microcentrifuge tubes. Assay mixtures contained varying concentrations of C5 (preincubated for 20 min at 37 °C to eliminate freeze/thaw-generated background C5b,6-like activity), factor B (0.6 µg, 258 nM), factor D (0.1 µg, 167 nM), C6 (2.5 µg, 833 nM), and 0.5 mM MgCl2 or 0.5 mM NiCl2. The reaction was started by the addition of zymosan particles bearing 27 ng of bound C3b resulting in 6.2 nM enzyme in a final volume of 25 µl of GVB. After 15 min incubation at 37 °C, further cleavage of C5 was prevented by transferring the assay tubes to an ice bath and adding 200 µl of ice-cold GVBE. Controls demonstrated that the EDTA, the cold temperature, and the dilution of the reaction mixture reduced the cleavage of C5 during subsequent steps to undetectable levels. C5b,6 was titrated by hemolytic assays using Ec and quantitated using standard curves generated with purified C5b,6 as described above.

Kinetic Measurements of the Fluid Phase C5 Convertase-- The assay conditions employed saturating amounts of factor B (3 µg, 1290 nM) and factor D (0.1 µg, 167 nM), as well as varying concentrations of C5, excess C6 (2.5 µg, 833 nM), 0.5 mM NiCl2, and GVB. Reactions were started with the addition of 100 ng of monomeric soluble C3b (23 nM final C3b concentration) and GVB to give a final volume of 25 µl. After 15 min incubation at 37 °C, reactions were stopped by dilution with 500 µl of ice-cold GVBE, and the amount of C5b,6 formed was quantitated as described above for the surface-bound C5 convertase.

Data Analysis-- The reaction velocity data was analyzed according to the Michaelis-Menten equation: v = Vmax [S]/(Km + [S]). The results were fit to this equation using nonlinear regression analysis, and the kinetic parameters, Km, Vmax and kcat, were determined using Grafit version 3.0 software (Erithacus software, Staines, Middlesex, London, UK).

Preparation of Labeled 125I-Factor B-- Factor B (100 µg) was radiolabeled with 125I for 30 min at 0 °C in a glass tube coated with IODO-GEN (Pierce). Free 125I was removed by centrifugal desalting (37). Specific activities of radiolabeled factor B ranged from 0.2 to 2.4 µCi/µg.

Determination of the Number of C5 Convertase Sites on Zymosan-- The number of cell-bound C5 convertase sites was determined by quantitating the number of C3b molecules capable of forming an enzyme. This was done by determining the binding of radiolabeled factor Bb to ZymC3b under the same saturating conditions used to maintain constant levels of C5 convertase during the assays. ZymC3b employed in the binding assays was preincubated for 15 min at 37 °C and washed 3 times with GVB to remove any free C3b. Various amounts of 125I-factor B were added to assay mixtures containing factor D (0.1 µg, 167 nM), C5 (5 µg, 1053 nM), C6 (2.5 µg, 833 nM), and 0.5 mM NiCl2. Formation of enzyme was started with the addition of 5 × 106 ZymC3b particles to give a final volume of 25 µl of GVB. After 5 min incubation at 37 °C, assays were diluted with 75 µl of GVB containing 0.5 mM NiCl2. Bound and free radiolabel were separated by layering 75 µl of the mixture on 250 µl of 20% sucrose in GVB containing 0.5 mM NiCl2 followed by centrifugation for 2 min at 10,000 × g at 22 °C. The amount of factor B bound to ZymC3b as Bb was determined by cutting the tube and counting the amount of radioactivity in the pellet (34). The radioactivity determined as cpm bound to ZymC3b is reported after subtracting nonspecific background binding to zymosan lacking C3b. Distribution of radioactivity in the 63-kDa Bb and the 30-kDa Ba fragments was determined by SDS-PAGE of cleaved 125I-factor B. The Bb fragment contained 71% of the label.

Measurement of the Rates of Formation and Decay of C5 Convertase-- The rate of formation of the surface-bound enzyme was determined by assembling the enzyme under saturating concentrations of factors B (1.0 µg, 717 nM) and D (0.1 µg, 278 nM), 0.5 mM NiCl2 or 0.5 mM MgCl2 and ZymC3b bearing 10 ng of C3b (based on Bb binding) in a final volume of 15 µl of GVB. The reaction was started with the addition of ZymC3b. Several such reaction mixtures were incubated at 37 °C for different time intervals (0.5, 1, 2, 3, 5, and 7 min) immediately after which 2.5 µl of 35 mM EDTA was added (to prevent additional formation of enzyme) followed by the addition of 7.5 µl of a mixture of C5 (5 µg) and C6 (2.5 µg). The assay mixture was incubated for 15 min at 37 °C, and the amount of C5b, 6 formed was quantitated hemolytically as described above.

The half-life of C5 convertases was measured by first premaking the enzyme for 2 min (both forms of the enzyme formed in less than a minute) at 37 °C in a reaction mixture containing factor B (9 µg, 717 nM), factor D (0.9 µg, 278 nM), 0.5 mM MgCl2, or 0.5 mM NiCl2 and ZymC3b bearing 242 ng of C3b in a final volume of 135 µl of GVB. At zero time additional formation of enzyme was stopped with 22.5 µl of GVB containing 35 mM EDTA. At various time intervals, aliquots (17.5 µl) of the enzyme preparation (kept at 37 °C) were withdrawn and assayed for remaining C5 convertase activity by mixing with 7.5 µl of GVB containing C5 (5.0 µg, 1053 nM) and C6 (2.5 µg, 833 nM). The amount of C5b,6 formed after 15 min at 37 °C was determined hemolytically, as described above.

The rates of formation and decay for the fluid phase C5 convertase (C3b,Bb) were measured under conditions similar to those used for the surface-bound enzyme. The assay mixtures contained saturating amounts of factors B and D, C5, C6, and divalent metal ion. Free monomeric C3b was used to form the fluid phase C5 convertase.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Quantitation of Reaction Product C5b,6-- To determine the extent of C5 cleavage, we chose to measure the amount of C5b formed. C5b is unstable unless complexed to C6, forming C5b,6. Therefore, in the present studies C5b was trapped as a stable complex using excess C6, and the C5b,6 generated was quantitated hemolytically using chicken erythrocytes (Ec). Fig. 1A shows the standard curve for purified C5b,6 in the range of 0-50 ng of the protein. C5b,6 determinations were read off standard curves in the range of 5-25 ng of the purified protein. Under certain conditions, C5 forms a C5b-like molecule without the cleavage of C5a (38). The C5b-like molecule can complex to C6, forming a functional C5b,6-like complex. Preincubation of C5 for 20 min at 37 °C reduced the background due to formation of freeze/thaw-generated C5b-like molecules.


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Fig. 1.   Characterization of the C5 convertase assay. A, quantitation of reaction product C5b,6. Various concentrations of purified C5b,6 (0-50 ng) in 25 µl of GVB were incubated for 10 min at 37 °C in a final volume of 250 µl of GVBE containing Ec (1.2 × 107) and 5 µl of pooled normal human serum as a source of C7-C9. Percent hemolysis was determined from the release of hemoglobin at 414 nm. B, rate of decay of surface-bound ZymC3b,Bb. The surface-bound C5 convertase was premade for 2 min at 37 °C. After adding GVBE to prevent additional formation of enzyme, the C5 convertase was allowed to decay at 37 °C. Aliquots were withdrawn after the indicated time intervals and assayed for remaining C5 convertase activity (for details see "Experimental Procedures"). bullet , Ni2+-containing ZymC3b,Bb; open circle , Mg2+-containing ZymC3b,Bb. C, demonstration that a constant amount of C5 convertase was maintained during the assay. ZymC3b was incubated for the indicated length of time with 0.5 mM NiCl2 or MgCl2 and saturating levels of factors B and D. C5 and C6 were then added, and C5b,6 formation was allowed to proceed for 5 min. C5b,6 formed was immediately quantitated as in A (details in text). bullet , Ni2+-containing ZymC3b,Bb; open circle , Mg2+-containing ZymC3b,Bb.

In order for the C5b,6 assays to quantitatively reflect the amount of C5b formed, C6 concentrations sufficient to capture all the C5b were determined. Preliminary studies in which C6 concentrations were varied demonstrated that 100 µg of C6/ml in assays was sufficient to maximize C5b,6 production. Because less than 1% of the C5 present was cleaved in any of the kinetic experiments reported here (except in the following controls) C6 concentrations occasionally below the concentration of C5 were still sufficient. In the first control, the functional activity of C5 was shown to be greater than 95% by cleavage with excess C5 convertase and analysis of the alpha - and alpha '-chains on SDS. In another control, the specific activity of commercial C5b,6 was verified by measuring its functional titer and comparing it to that of fresh C5b,6 formed under conditions that cleaved all the C5 in a reaction. The amount of fresh C5b,6 formed was found to be within 2-fold of the amount expected based on pure C5b,6 as the standard.

To validate that the quantitation of C5b,6 was a faithful measure of C5 cleavage, the second product C5a was also quantitated from the same assay mixture employed for C5b,6 determination. The anaphylatoxin was measured using a commercial C5a RIA kit. The amount of C5 cleaved, determined hemolytically as C5b,6, was found to be within 2-fold of the amount determined by C5a RIA. Comparison of the two methods indicated the hemolytic detection of C5b,6 to be approximately 5-fold more sensitive than the radioimmunoassay method used for C5a detection. Fifty percent lysis of Ec corresponded to 0.047 nmol of C5b,6 (14 ng), whereas 0.24 nmol of C5a caused 50% inhibition in the RIA. In addition, the hemolytic assays were fast, easy to perform, and relatively inexpensive.

Rates of Formation and Decay of C5 Convertases-- The rate of enzyme formation was examined by addition of C3b or ZymC3b to assays containing excess factor B, factor D, C5, C6, and metal ions. Formation was stopped by the addition of EDTA, and the amount of C5b,6 produced in the subsequent 15 min was determined. Formation of both the surface-bound and the free soluble form of the C5 convertase was found to be rapid with maximum enzyme levels achieved in less than a minute (data not shown). Fig. 1B shows the results of measurements of the decay rates of the surface-bound enzymes. The magnesium containing C5 convertase, ZymC3b,Bb(Mg2+), was found to be very unstable at 37 °C. The enzyme had a half-life of 1.5 min. Substituting Ni2+ for Mg2+ stabilized the enzyme approximately 5-fold resulting in a half-life of 8 min at 37 °C. The free forms of the C5 convertase decayed at rates similar to those of the surface-bound C5 convertase. C3b,Bb(Mg2+) exhibited a half-life of about 1.0 min at 37 °C, while the Ni2+ containing soluble enzyme had a half-life of 7 min at 37 °C (data not shown).

Effect of Factor H Contamination-- The control protein, factor H, was found to influence the C5 convertase activity significantly. The free form of the enzyme was in particular affected by the presence of factor H, whereas the surface-bound enzyme was much less affected due to restricted access of factor H to zymosan-bound C3b (34, 39). Concentrations as low as 10 ng of factor H/ml were sufficient to accelerate the decay of the fluid phase C5 convertase by 50% (data not shown). Therefore, as a precaution all purified proteins used in the assays were passed over anti-H-Sepharose in order to reduce the concentration of factor H to levels that were determined to have no detectable influence on the activity of the C5 convertase (less than 2 ng/ml in the assay).

Demonstration of Constant C5 Convertase Enzyme Levels during Assays-- Because the C5 convertases decay during analysis, it was important to prove that a constant level of enzyme was maintained during the assays. Of greatest concern was maintenance of the Mg2+-containing enzyme because of its 1-1.5 min half-life. Preliminary experiments were done to determine the concentrations of factors B and D required to yield a maximum amount of C5b,6 during a 15-min assay period at 37 °C using the surface-bound enzyme, ZymC3b,Bb(Mg2+). Employing these concentrations of factors B and D, the amount of C5 convertase present at various time points during the assay was determined by quantitating the C5b,6 formed in the 5-min period subsequent to sampling the enzyme. Fig. 1C shows that a constant amount of C5 convertase was present throughout the 15-min assay even though the Mg2+-containing C5 convertase was rapidly decaying and reforming during this period. Fig. 1C also shows that similar levels of activity of the Ni2+-stabilized enzyme were achieved and maintained during the 15-min assays. Although not shown, constant levels of enzyme activity were also demonstrated for the free forms of the C5 convertase, C3b,Bb(Mg2+) and C3b,Bb(Ni2+).

Determination of the Number of C5 Convertase Sites on ZymC3b-- To determine the number of C5 convertase sites on zymosan, only those C3b molecules capable of binding 125I-factor B as Bb were quantitated. Saturating concentrations of factors B and D, C6, C5, and Ni2+ were used to obtain the same amount of C5 convertase as would form during a normal assay. Ni2+ was used to minimize the decay of the Bb subunit of the enzyme during the 2-min spin through 20% sucrose, and centrifugation was done in the cold. As seen in Fig. 2 it was possible to saturate the C3b molecules on zymosan with radiolabeled factor B. The maximum amount of Bb bound was used to calculate the number of C5 convertase sites on zymosan. Because the number of Bb molecules bound to ZymC3b reflects only those C3b molecules capable of forming a C5 convertase, this analysis should give a better estimate of the number of C5 convertase sites present on zymosan than just counting the number of C3b/zymosan. The results presented in Fig. 2 indicate that the ZymC3b used in this study could form 83,500 C3b,Bb enzymes per particle under saturating concentrations of factors B and D. 


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Fig. 2.   Determination of the number of C5 convertase sites on ZymC3b. The amount of bound C3b capable of forming the convertase was measured by quantitating the amount of radiolabeled 125I-Bb bound to ZymC3b. The binding assays were performed using excess factor D and 0.5 mM NiCl2. C5 convertase (ZymC3b,Bb(Ni2+)) was formed on the surface of 5 × 106 ZymC3b particles for 5 min at 37 °C after which the particles were washed by centrifugation through 20% sucrose. Radiolabeled factor B had 203,739 cpm/µg. The amount of Bb bound was calculated from the radioactivity in the pellets and the specific activity of the Bb subunit.

Measurement of Kinetic Parameters of the Surface-bound C5 Convertase-- To measure the kinetic constants of the C5 convertase, preliminary experiments were conducted to determine the rate of C5 cleavage under saturating concentrations of factors B and D and C6. The results showed a linear increase in C5b,6 concentration over the entire 15-min assay period even at the lowest C5 concentration used in this study. Initial velocities were then measured at various substrate (C5) concentrations using a fixed enzyme concentration of 6.2 nM (calculated from the binding of radiolabeled Bb to ZymC3b). The velocity data obtained with ZymC3b,Bb(Mg2+) fit well to the Michaelis-Menten equation (v = Vmax [C5]/(Km + [C5])) as seen in Fig. 3A. Kinetic constants calculated from the data in Fig. 3A are summarized in Table I. The Km of ZymC3b,Bb(Mg2+) for C5 was 1.24 µM, and the kcat was 0.0038 s-1. For reasons explained under "Discussion," it was necessary to determine if in the present studies we had a subpopulation of surface-bound C5 convertases with very high Km values. If this was true then the Eadie-Hofstee plot would have a curvature at high C5 concentrations (low v/[S] values). However, this was not observed to be the case as our data showed an excellent fit to the linearized form of the Michaelis-Menten equation as shown in Fig. 3B. The velocity that was attained at about 11 µM C5 (Fig. 3A) did not increase more than 5% when the substrate concentration was raised to 60 µM (data not shown). Considered together these observations make it unlikely that surface-bound C5 convertases exist as a family of enzymes with a wide range of different substrate affinities as has been suggested by binding studies (16, 17, 25, 27, 40).


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Fig. 3.   Kinetic analysis of C5 convertase activity of ZymC3b,Bb(Mg2+) in the presence of varying concentrations of C5. A, initial rates of C5 cleavage by the surface-bound enzyme, ZymC3b,Bb(Mg2+), were determined by measuring the amount of C5b,6 formed in 15 min at 37 °C. Reactions contained saturating levels of factor B (0.6 µg, 258 nM), factor D (0.1 µg, 167 nM), C6 (2.5 µg, 833 nM), 0.5 mM MgCl2, and the indicated concentrations of C5 in a final volume of 25 µl of GVB. Reactions were initiated by adding ZymC3b particles bearing 27 ng of C3b (measured as in Fig. 2) which resulted in 6.2 nM C5 convertase in the assay. After 15 min at 37 °C the reactions were stopped by the addition of 200 µl of cold GVBE. Aliquots of 25 µl were analyzed for C5b,6 formation as described in Fig. 1A. The data were fitted by nonlinear regression according to the Michaelis-Menten equation to determine the Km and Vmax values using Grafit version 3.0 Erithacus software (Middlesex, Staines, London, UK). B, analysis of the C5 convertase initial velocity data using the Eadie-Hofstee function.

                              
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Table I
Kinetic parameters of the C5 convertases
Kinetic parameters of the free and the surface-bound C5 convertases of the alternative pathway were determined by nonlinear regression of data as shown in Figs. 3A, 4A, and 5A. Values given are mean ± S.D. from at least three independent experiments.

The kinetic parameters of the Ni2+-stabilized, surface-bound C5 convertase were also examined. The plot of velocity versus substrate concentration for ZymC3b,Bb(Ni2+) as shown in Fig. 4A fit well to the Michaelis-Menten equation. The Ni2+-containing enzyme exhibited a Km (1.4 µM) and a kcat (0.0048 s-1) similar to that of the Mg2+-containing surface-bound C5 convertase (Table I). The Eadie-Hofstee plot of the data for the Ni2+-containing C5 convertase shown in Fig. 4B was linear as observed for the Mg2+-containing surface-bound enzyme (Fig. 3B).


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Fig. 4.   Kinetic analysis of the Ni2+-containing surface-bound C5 convertase, ZymC3b,Bb(Ni2+). A, initial rates of cleavage of C5 by the surface-bound enzyme, ZymC3b,Bb(Ni2+), were determined by measuring the amount of C5b,6 formed in 15 min at 37 °C. Assay mixtures were identical to those in Fig. 3A containing saturating levels of factor B (0.6 µg, 258 nM), factor D (0.1 µg, 167 nM), C6 (2.5 µg, 833 nM), 0.5 mM NiCl2, and the indicated concentrations of C5 in a final volume of 25 µl GVB. Reactions were initiated by adding ZymC3b particles bearing 27 ng of C3b (measured as in Fig. 2) which resulted in 6.2 nM C5 convertase in the assay. After 15 min at 37 °C the reactions were stopped by the addition of 200 µl of cold GVBE. Aliquots of 25 µl were analyzed for C5b,6 formation as described in Fig. 1A. The data were fitted by nonlinear regression according to the Michaelis-Menten equation as in Fig. 3A to determine the Km and Vmax values. B, analysis of the C5 convertase initial velocity data using Eadie-Hofstee function.

Kinetic Analysis of the Free (Soluble) Form of the C5 Convertase-- To compare the kinetic parameters of the surface-bound C5 convertase with those of the free form of the enzyme, the rate of C5 cleavage by C3b,Bb(Ni2+) was measured. Monomeric C3b was used to make the free form of the enzyme (see "Experimental Procedures"). Fig. 5A shows that the enzyme, C3b,Bb(Ni2+), not only cleaved C5 but did so at a rate (kcat) slightly faster than that of the surface-bound C5 convertase (Table I). Moreover, the data, like that for the surface-bound C5 convertase, fit well to the Michaelis-Menten equation (Fig. 5A). However, analysis of the Km value revealed that the free form of the enzyme exhibited a 17-fold lower affinity for C5 (Km = 24 µM) than the surface-bound convertase (Km = 1.4 µM) (Table I). Due to the high Km observed for the free form of the enzyme, concentrations up to 12 mg C5/ml in the assays were required. This resulted in a high C5b,6 background due to C5b-like C5. It was therefore necessary to use higher concentrations of enzyme (23 nM) so that the background without enzyme was small compared with the C5b,6 formed with the enzyme present. Although the Eadie-Hofstee plot for C3b,Bb(Ni2+) was linear, considerable scatter was consistently observed in the data (Fig. 5B). A similar rate of C5b,6 formation was observed with the Mg2+-containing C5 convertase, C3b,Bb(Mg2+), but the scatter was far greater. Since no reliable results were obtained for the fluid phase Mg2+-containing C5 convertase, the kinetic constants of the enzyme have not been reported.


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Fig. 5.   Kinetic analysis of the C5 convertase activity of the soluble, monomeric form of the enzyme, C3b,Bb(Ni2+). A, the C5 convertase activity of the free form of the enzyme, C3b,Bb(Ni2+), was determined by measuring C5b,6 formation in the presence of varying C5 concentrations. Reactions were performed under saturating levels of factor B (3.0 µg, 1290 nM), factor D (0.1 µg, 167 nM), C6 (2.5 µg, 833 nM), 0.5 mM NiCl2, and the indicated concentrations of C5 in a final volume of 25 µl of GVB. Reactions were started with the addition of 100 ng of free monomeric C3b which resulted in 23 nM of the C5 convertase in the assay. After 15 min at 37 °C, reactions were terminated by the addition of 500 µl of cold GVBE. Aliquots of 10 µl were analyzed for C5b,6 formation as in Fig. 1A. The data were fitted by nonlinear regression according to the Michaelis-Menten equation as in Fig. 3A to determine the Km and Vmax values. B, analysis of the velocity data using the linear Eadie-Hofstee function.

To rule out the possible presence of C3b-C3b dimers or complexes in the assay of the fluid phase monomeric C5 convertase, purified C3b used for the formation of C3b,Bb(Ni2+), was again gel-filtered after storage at -76 °C. The C5 convertase activity of the gel-filtered C3b monomer was determined to be similar to the bulk frozen and thawed C3b, ruling out the possibility of C3b-C3b dimer formation due to freeze-thaw of C3b. In addition, all components used in the assay (C3b, C5, C6, and factors B and D) were treated with methylamine so as to inactivate any traces of C3 which might generate C3b dimers during the assays. The activity of fluid phase C5 convertase determined with the methylamine-treated components was found to be similar to that determined with reactants not treated with methylamine. These results indicate that the C5 convertase activity of the fluid phase enzyme in the present study was derived from enzymes containing monomeric C3b.

Effect of Increasing Concentrations of Monomeric C3b on the Activity of the Fluid Phase C5 Convertase-- A previous report has suggested an additional C3b molecule is required to bind the substrate C5 so that it can be cleaved by a fluid phase C5 convertase (25). We therefore determined the activity of the free form of the C5 convertase in the presence of increasing concentrations of monomeric C3b. The assay conditions used in the present study were such that the concentrations of factors B and D were saturating even at the highest concentration of C3b (200 ng) used in the assay. If the C5 binds to the C3b in the same enzyme complex that eventually cleaves it, then the mechanism of the reaction would be as shown in Reaction 1. 
  <UP>C3b,Bb*</UP>+<UP>C5</UP>→<UP>C3b,Bb*C5</UP>→<UP>C3b,Bb*</UP>+<UP>C5b</UP>+<UP>C5a</UP> (Reaction 1)
where * denotes the enzyme. In this case the rate of C5 cleavage should be directly proportional to the increase in C3b concentration as shown by the solid line in Fig. 6. But, if in addition to forming the enzyme, C3b is also required for substrate activation, i.e. C5 has to be held by another C3b in order to be cleaved, then both the substrate and enzyme concentration increase as the C3b concentration is increased. This mechanism is represented by Reaction 2. 
<UP>C3b,Bb*</UP>+<UP>C5,C3b,Bb</UP>→<UP>C3b,Bb*C5,C3b,Bb</UP>→<UP>C5b</UP>+<UP>C5a</UP>+<UP>C3b,Bb*</UP>+<UP>C3b,Bb</UP> (Reaction 2)
In this case the rate of C5 cleavage should be proportional to the square of the increase in C3b concentration as indicated by the dashed line in Fig. 6. The results suggest that little or no intermolecular cleavage of C5 involving two C3b molecules (i.e. reaction mechanism (2)) occurred in these assays. The data strongly favor a reaction mechanism (1) that involves a single trimolecular complex involving one C3b.


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Fig. 6.   Effect of increasing concentrations of C3b on the activity of the soluble form of the enzyme, C3b,Bb(Ni2+). C5 convertase activity of the free form of the enzyme, C3b,Bb(Ni2+), was determined by measuring the amount of C5b,6 formed in the presence of increasing concentrations of soluble, monomeric C3b. Reactions were performed under saturating concentrations of factor B (6.0 µg, 2581 nM), factor D (0.2 µg, 333 nM), C6 (2.5 µg, 833 nM), 0.5 mM NiCl2, the indicated concentration of C3b, and 20 µg of C5 (4211 nM) in a final volume of 25 µl of GVB. Reactions were started with the addition of free monomeric C3b to the assay. After 15 min at 37 °C, reactions were terminated by the addition of 500 µl of cold GVBE, and the amount of C5b,6 formed was determined as in Fig. 1A.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Studies of the C5 convertase have been difficult because in its most active form the enzyme is assembled as a multimeric complex on the surface of biological organisms. The complexity of the surface-bound enzyme led us to re-examine the ability of monomeric C3b to form a fluid phase C5 convertase. Although this enzyme is known as a C3 convertase, not a C5 convertase, C3b,Bb was found to cleave C5. A comparison of its C5 convertase activity (Table I) with the published C3 convertase parameters (41) revealed that at plasma concentrations of C3 and C5, and in the absence of control factors, this C3/C5 convertase would cleave approximately 9000 C3 for each C5 cleaved. Nevertheless, this weak C5 convertase activity was found to be significant and similar to that of the more biologically relevant surface-bound enzyme.

The use of monomeric C3b to form a more structurally defined C5 convertase was valuable for comparison with the surface-bound C5 convertase in which C3b was deposited in clusters. Determination of enzyme concentration was straightforward for the homogeneous fluid phase C3b but more difficult with ZymC3b. The concentration of surface-bound C5 convertase was estimated by measuring the number of Bb molecules bound (Fig. 2). The C3b concentration on zymosan was also measured by a less accurate but independent method utilizing factor H and radiolabeled C3b binding (34) and was found to be similar within the errors of these methods. In the fluid phase, every C3b was considered to form a C5 convertase, and saturating concentrations of factors B and D were used in all assays. Purified C3b was determined to be greater than 95% active in assays examining cleavage by factors H and I (not shown) and was electrophoretically and chromatographically homogeneous. For these reasons we are confident that the measured C5 convertase concentration accurately reflects the true concentration of the enzyme. Any error in these determinations would inversely affect the kcat values reported here, but the Km values would not be affected.

The alternative pathway C5 convertase is an unstable complex due to the rapid dissociation of its catalytic subunit, Bb. It has an extremely short half-life (t1/2 = 1.5 min, Fig. 1B). Substituting Ni2+ for Mg2+ stabilized the enzyme by 5-7-fold. Nevertheless, enzyme decay had the potential to significantly affect the assays. To overcome this difficulty preliminary experiments were done to determine the concentrations of factors B and D that would continuously replace the decaying Bb subunit of the enzyme. Fig. 1C shows that these concentrations were sufficient to maintain a constant level of enzyme throughout an assay. Other assays showed that no higher level of enzyme could be attained by increasing factors B and D further. An additional factor that could have affected the assays was the decay accelerating activity of trace concentrations of factor H. It was found that as little as 10 ng of factor H/ml in the assays caused a significant change in activity. As a result, all purified proteins were passed over anti-H-Sepharose in order to reduce the final concentration of factor H in the assays to less than 1/250,000 of the concentration in plasma.

Measurement of the kinetic constants of the fluid phase and surface-bound C5 convertases revealed that the major difference between the two forms of the enzyme was not in the catalytic rates of the enzyme but in the affinity for the substrate, C5 (Table I). The naturally occurring surface-bound C5 convertase had a 17-fold higher affinity for C5 than its free form as indicated by their Km values presented in Table I (1.4 µM for surface-bound and 24 µM for the soluble form of the enzyme). It may be pointed out that the two forms of the C5 convertase have identical catalytic subunits, Bb, that decay at similar rates. It was therefore not surprising to find that the two forms of the enzyme had catalytic rates that were approximately 2-fold apart.

Although no other studies have examined the interaction of C5 convertase with its substrate C5, several groups have measured the binding affinity of C5 for C3b and have reported binding constants (Kd) that vary from 0.005 to 2.1 µM (16, 17, 25, 27, 40). The highest affinity (0.005 µM) reported has been for the binding of C5 to C3b molecules deposited in clusters on sheep erythrocytes (16, 17). Although Kd and Km are not always comparable, our measurements of the Km for the surface-bound C5 convertase (ZymC3b,Bb) indicate that the majority of the enzymes have affinities for C5 that are 2 orders of magnitude weaker than the highest reported affinity. Binding constants reported by two other groups are in better agreement with our findings. DiScipio (27, 40) has described a Kd of 0.18 µM for C5 binding to C3b on zymosan, and Isenman et al. (25) have reported a Kd of 2.1 µM for C5 and free C3b. These binding constants were all determined at physiological ionic strength and indicate that surface-bound C3b has a 12-fold greater affinity for C5 than does soluble monomeric C3b. These results are in agreement with our findings of a 17-fold difference in the Km values of these two forms of the C5 convertases. An explanation for the difference between surface-bound and free C3b was provided by Hong and co-workers (17, 42) and Kinoshita and co-workers (16, 20, 21) who suggested that C3b-C3b and C4b-C3b dimers formed high affinity binding sites for C5 and that these were probably the true C5 convertase sites. Presumably the low Km value obtained for the surface-bound C5 convertase in this study was due to C3b-C3b complexes on the surface of zymosan, but nothing in this study allows us to draw any conclusions about the structures of surface-bound C5 convertases.

The effect of increasing C3b concentration on the activity of the free form of the C5 convertase (Fig. 6) suggests that a soluble monomeric C5 convertase can cleave a C5 bound to it without the help of a second C3b. Our data are in excellent agreement with the straight line shown in Fig. 6 that represents the reaction mechanism for an enzyme-substrate complex involving one C3b (Reaction 1). These findings are in contrast to the conclusion reached by Isenman et al. (25) that an extra C3b is required for C5 cleavage by a fluid phase enzyme. In their study it is possible that the supplemental C3b formed sufficient additional fluid phase enzyme to cleave enough C5 to be detected on SDS gels. Other workers (14, 15, 19, 25, 43) also had difficulty in measuring the C5 cleaving activity of the fluid phase enzyme. An explanation for this could be our observation that the fluid phase C3/C5 convertase cleaves only one C5 for every 9000 C3 cleaved at physiological concentrations of C3 and C5.

The normal plasma concentration of C5 (70 µg/ml, 0.37 µM) is only one-third the Km reported for the surface-bound C5 convertase (1.24 µM). This suggests that under physiological conditions the surface-bound C5 convertase will function at only one-fifth its maximum velocity (Vmax). However, many human cells synthesize the complement components C5-C9 (44, 45). Thus, C5 concentrations at the local site of injury or near stimulated immune cells may be considerably higher than the plasma concentration of C5. Because the surface-bound C5 convertase operates at one-fifth its Vmax with normal C5 concentrations, there exists the potential for a 5-fold increase in the rate of C5 activation if local C5 concentrations increase.

The natural, surface-bound C5 convertase exhibited an extremely low turnover number of 0.23 C5/min/enzyme at 37 °C (Table I). This turnover number of the enzyme is about 5 orders of magnitude slower than that of a typical enzyme such as ribonuclease A which has a turnover number of 8.4 × 104/min (46). The rate of catalysis of the convertase is approximately 10-fold less than that of the catalytic RNA enzymes called ribozymes which cleave RNA. These enzymes have turnover numbers of about 2/min (46). The catalytic rate of the C5 convertase also falls within the range of DNA enzymes that cleave RNA (turnover numbers from 3.4 to 0.03/min) (46) and engineered enzymes such as catalytic antibodies (turnover numbers from 5 to 0.0003/min) (47). This makes the C5 convertase one of the slowest natural protein enzymes reported. Apparently, the biological function of complement is well served by this level of activity which has been preserved during the evolution of the complement system.

The C3 convertase activity of the fluid phase enzyme C3b,Bb is well established, and its kinetic properties have been characterized (41). Now that we have demonstrated the ability of this fluid phase C3 convertase to cleave C5, the kinetic properties of the enzyme both as a C5 and a C3 convertase can be compared. As discussed earlier the fluid phase C3 convertase can cleave about 9000 C3 molecules for every C5 under physiological conditions. The surface-bound enzyme would be approximately 10 times more active at plasma concentrations of C3 and C5. The resulting ratio is within 5-fold of that found experimentally using model activators and serum (48). The biological relevance for this 900-fold difference in cleavage of C3 versus C5 on surfaces could be that a very large number of C3b molecules are required for opsonization of biological particles, although apparently many fewer C5b molecules are sufficient for cytolysis of pathogenic organisms. Likewise, C5a, the second product of the C5 convertase, is a potent biological effector at extremely small concentrations, whereas higher levels of production of the anaphylatoxin may cause unwanted systemic responses.

    ACKNOWLEDGEMENTS

We express our appreciation to Nicole S. Narlo and Kerry L. Wadey-Pangburn for excellent technical assistance.

    FOOTNOTES

* This research was supported by National Institutes of Health Research Grant DK-35081.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 903-877-7663; Fax: 903-877-5882; E-mail: pangburn{at}uthct.edu.

1 The abbreviations used are: C3b and C5b, the proteolytically activated form of C3 and C5, respectively; Ec, chicken erythrocytes; VBS, Veronal-buffered saline; GVB, gelatin-containing veronal-buffered saline; C3b,Bb, free form of the C5 convertase; ZymC3b, C3b bound to zymosan particles; ZymC3b,Bb, surface-bound C5 convertase; RIA, radioimmunoassay.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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