Molecular Immunology, Vol. 16. pp. 821427. Pergamon Press Ltd. 1979. Printed in Great Britain.

AMYLOID P-COMPONENT(Clt) AND COMPLEMENT: LACK

OF PHYSICAL OR FUNCTIONAL RELATIONSHIP*

NEIL R. COOPER and ROBERT J. ZICCARDI

Department of Molecular Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037, U.S.A.

(Received 25 July 1978; received for publication 3 April 1979)

Abstract--Studies were carried out to determine if Cl (, which is identical to the P-component of amyloid (AP), plays a role in Cl structure or function, or modulates the activity of Cl or its sub-units. Analyses of Cl in serum by a direct immunodiffusion method showed conclusively that AP was not a constituent of C I Further, AP did not interact physically with Cl or with Clq, Clr or Cls individually or in any combination, nor did it activate precursor C 1. Although some preparations contained C 1 r-like hemolytic activity, this was due to the variable presence of a contaminating serine esterase, with properties suggesting that the contaminant was Clr. AP was also completely devoid of ability to provide Clr-like activity to sera genetically deficient in Clr. Immunodepletion studies showed that AP was not essential for hemolytic activity. As reported by others, Ap formed a calcium-dependent bond with agarose. In addition, AP interacted with IgG and aggregated in the presence of calcium.

These studies cumulatively demonstrate that AP is not a sub-component of Cl and further show that AP does not interact with, or modulate the activity of Cl or any of its sub-units. The present experiments also furnish alternative explanations for earlier studies which led to the conclusion that AP was a fourth Cl sub- component

INTRODUCTION

In 1974 Assimeh, Bing and Painter described a novel plasma protein which was detected in EDTA eluates of Sepharose-IgG columns through which human serum had been passed (Assimeh et al., 1974). This protein was termed Clt because of multiple properties enumerated below which suggested that it was a fourth sub-component of Cl (Assimeh & Painter, 1975~; Assimeh & Painter, 19756). Clt, as well as Clq, Clr and Cls, was found in the euglobulin fraction of serum. Ultracentrifugal analyses of serum in the presence of calcium showed Clt to be present in the 19 S region of density gradients along with C 1 q, C 1 r and Cls, while comparable studies in EDTA, which dissociates Cl, showed all 4 proteins in fraction corresponding to 11 S. Cl t and also Cls could be eluted from Sepharose-IgG columns through which serum had been passed with EDTA and Clt could rebind to Sepharose-IgG columns only if Clq was first attached. Similar studies indicated that Clt served as a bridge to attach Cls to Clq via a calcium-dependent bond. Studies with EAClq,4 also showed that Clt could mediate the attachment of Cl s to cells. Clt provided Cl hemolytic activity in a test system containing EAC4, limited Clq and excess Cls. Finally, in ultracentrifugal studies Cl t increased the sedimentation rate of Clq, Cls and Clq-Clr-Cls mixtures.

Other studies (Pinteric et a/., 1976) indicate that Cl t, which has a molecular weight of 223,000 daltons, is

*This is publication number 1512 from the Research Institute of Scripps Clinic. This work was supported by USPHS Grants CA 14692, Al 14502, I SO7 RR-05514 and Program Project Grant AI 07007 from the NIH and Grant- in-Aid No. 77-941 from the American Heart Association.

composed of apparently identical non-covalently associated sub-units of 22.000 daltons which have a characteristic pentagonal arrangement when visualized in the electron microscope which is very similar to that of C-reactive protein (CRP) and to the P-component of amyloid (Bladen et al., 1966; Skinner et al., 1974). Amino acid sequence studies show CRP to be closely related, but not identical to Cl t (Osmand et al., 1977). Although initial amino acid sequence studies (Skinner et ul., 1974) suggested that Cl t was distinct from P-component of amyloid (AP), later studies indicate identical amino-terminal sequences (Skinner et al., 1976) and immunologic identity for the two proteins (Osmand et al., 1977; Skinner et al., 1976; Pepys et al., 1977; Katz et al., 1977). Thus C 1 t and AP are identical proteins.

Our earlier studies pdiled to show an essential role for AP in Cl activity and indicated that it did not sediment with the Cl containing fractions of normal serum (Ziccardi & Cooper, 1977). Gigli et al. also were unable to find evidence for a role for Ap in Cl activity (Gigli et al., 1976). The demonstration by Pepys et ul. that Ap binds to a number of matrixes including Sepharose in a calcium-dependent fashion (Pepys et al., 1977~; Pepys et al., 19776) suggested to Painter (Painter, 1977) the possibility that the presence of AP in an EDTA eluate of Sepharose IgG columns through which serum had been passed might be adventitious and not indicative of the presence of Clt in Cl. This appears to be the case, as Painter’s studies (Painter, 1977) show that removal of amyloid P-component from serum has no effect on the hemolytic or molecular properties of C 1 in serum. Recent work also indicates that earlier studies showing enhancement of the sedimentation rate of Clq-Cls mixtures by amyloid P-component were the result of the

MI”” 16/l&~ 821

822 NEIL R. COOPER and ROBERT J. ZICCARDI

inadvertent addition of Clr to the reaction mixture techniques. AP ws not tmmunologically detectable in highly

(Painter, 1977). purified preparations of Clq, Clr and Cls. Cl was formed

Still unexplatned, however, are several observations from the purified components ofcomplement and isolated by

relating to the possible role of AP in Cl structure and sucrose density gradient ultracentrifugation (SDG) as has

function raised by the earlier studies of Assimeh and been described (Cooper & Ziccardi, 1977).

Painter (Assimeh & Painter, 1975~; Assimeh & Radio/&&e

Painter, 1975h). These include the finding that AP

binds to Sepharose IgG-Clq but not to Sepharose AP and Cls were radiolabeled with izsl through the use of

solid phase lactoperoxidase or with chloramine T as IgG (Assimeh 6i Painter, 1975a; Assimeh & Painter, previously described (Cooper & Ziccardi, 1977).

1975/~). the Clr-like hemolytic activity of amyloid P-

component (Assimeh & Painter. 1975~; Assimeh &

Painter, 197% Taylor c’f ul., 1977), and the

observation that AP is present in the 19 S fractions of serum (Assimch & Painter, 1975a). The possibilities that AP interacts with Cl or modulates its function have not been addressed. The results of our studies dealing with the possible role of AP in Cl structure

and activity are presented here.

MATERIAL. AND METHODS

I.\olcrriofr D/ .?I P

AP was purilied from fresh human serum by either of two methods. First. AP was eluted with EDTA* from a Sepharose-IgG (Pharmacia Fine Chemicals, Piscataway, NJ) column through which human serum had been passed exactly as described by Assimeh ef cl/. (Assimeh or 01.. 1974). AP in this pool was separated from Cls and other impurities by pevikon block electrophnresis Lit pH 8.6. In the second method. human serum euglobulin was fractionated on DE32 cellulose exactly as described for the isolation of C 1 s (Valet & Cooper, 1974). AP eluted from such columns in a broad peak located before but overlapping with Cls. Fractions containing AP (average conductance 14 mR-i/cm at 22°C) were pooled and passed through an ACA 34 column (LKB Instruments. Rockvillc. MD) in phosphate buffered NaCl containing I mM EDTA. Ap eluted from the column as a discrete peak. The puritied preparations gave one band with a mol. wt of 25.000 daltons when analyzed by SDS polyacrylamide gel clcctrophoresis (SDS-PAGE) and were unreactive with antisera to IgA, IgG. IgM. Cls, C3, C4, C5, C6. C8. factor B, Cl inactivator, tibronogen. /j lipoprotein, fi,-glycoprotein I. b,-glycoprotein III, haptoglobin, transferrine, ceruloplasmin, r,-glycoprotein, a,/? glyco- protein, z,-lipoprotein, r,-antitrypsin, a,,-acid glycoprotein, xzHS glycoprotein. z,-macroglobulm. albumin and prenlbumin. They did. however. yield one line with antiserum to human serum.

Antisera to AP was raised in rabbits by Injection of 100 pg of purified AP in complete Fruend‘s adJuvant into the poplitcal lymph nodes. Four weeks later intramuscular and subcutaneous injections of 100 /Ig of AP in complete Freund’s adjutants were given and a week later the rabbits were bled. The antisera were monospecific for AP without absorption as they generated one precipitin line when analyzed in Ouchterlony or immunoelectrophoretic analyses wjith whole serum. This precipitin line showed identity with isolated AP.

Sepharose, Sepharose-IgG and Sepharose-IgG-CIq resins were prepared exactly as described by Assimeh et (I/. (1974) and loaded into small columns (Assimeh & Painter, 1975~). Highly purified unlabeled and radiolabeled AP were passed through the columns in the presence of IO mM calcium after which the columns were eluted with IO mM EDTA. The effluents and eluants of the columns were examined for the presence of AP by Ouchterlony analysis and by radio- chemical techniques.

Depletion of AP from human .wrum

The lgG fraction ofantiserum to AP wascoupled at a ratio of 40 mg/g to CNBr Sepharose (Pharmacia Fine Chemicals, Piscataway. NJ). EDTA was added to a concentration of IO mA4 to normal human serum which was then passed through the immune adsorbent column. Protein containing fractions were pooled, concentrated to the original serum volume, dialyzed into buffer containing 0.15 mMcalcium and 0.5 mM magnesium and the absence of AP verified by Ouchterlony analysis.

SDS pal_vacr_vtamrd~~ gel c~lr~ctrophoresis (SDS-PAGE).

Experiments were performed using a modification (Ziccardi & Cooper, 19766) of the method of Weber and Osborn (Weber & Osborn. 1969). Following electrophoresis the gels wereeither stained or sectioned at 2-mm intervals and analyzed for radioactivity.

Sucrose den.virJ gradrent (SDG) uliracmrriJugarion.

SDG studies were carried out essentially as previously published (Ziccardi & Cooper. 1977). In brief, linear 9-31:‘” sucrose gradients were formed in barbital buffered saline containing 0.15 mM calcium chloride (Mayer, 1961). The gradients were centrifuged at 4’C in an SW 50.1 rotor at 40,000 rev/min for I6 hr in a Beckman L2-65B ultracentrifuge (Beckman Instruments. Palo Alto, CA). Gradient fractions were collected from the bottom of the tubes and assayed for radioactivity or functional activity. Radioactivity remaining in the empty centrifuge tube was also determined.

Whole complement activity (CH50) was measured as previously described (Mayer, 1961). Cl hemolytic activity was also assessed by standard hemolytic techniques (Ziccardi & Cooper, 1977). Clr-like hemolytic activity of AP was appraised in mixtures containing Clq and Cls and varying amounts of AP or Clr, as specified in the text, which were incubated for 15 min at 30°C in the presence of 1 mMcalcium in low ionic strength buffer as previously described (Ziccardi

C‘ompl~wwn1 rc’qycw t.5 & Cooper, 1977). Cells in each sample were washed twice

Cla (Calcott & Muller-Eberhard. 1972; Yonemasu & before being assayed for Cl activity.

Stroud, 1971). Clr (Ziccardi & Cooper, 19766) and Cls (Valet & Cooper. 1974) were isolated by published

C.1 crcl;,,crt;on a,ssu~,

Reconstituted gradient purified precursor macromolecular Cl, having a radiolabel in the Cls sub-component, was

*Abbreviations used: AP, amyloid P-component or Clt; incubated with samples for 20 min at 30°C after which SDS

DTT. dithiothrcitol: EDTA. ethylenediaminetetraacetic urea and DTT were added and the mixtures analyzed by

acid: SDG. sucrose density gradient ultracentrifugation: SDS-PAGE. Activation was quantitated by determining the

SDS. sodium dodecyl sulfate; SDS-PAGE. polyacrylamide proportion of radioactivity shifted from the 87,000 dalton

gel electrophoresis in the presence of sodium dodecyl sulfate. peak of proenzyme Cls to the 59,000 dalton peak of the

C I t and Complement 823

heavy chain of Cls. Exact details of this method have been published (Cooper & Ziccardi. 1977).

RESULTS

Isolation und properties oj A P

Either of the two methods of purification yielded 2-4 mg of AP from 500 ml of serum, i.e. 2030% yield. SDS polyacrylamide gel analyses of the isolated AP showed typical preparations to exhibit a single band under reducing or non-reducing conditions with a mol. wt of 25,000 daltons (Fig. 1). The elution position of AP from gel filtration (G-200 and ACA 34) columns and the sedimentation rate of 9.5 in sucrose density gradients indicate a mol. wt in excess of 200,000. These findings are consistent with the reported structure of Clt or AP of 10 non-covalently bonded sub-units (Pinteric et a/., 1976; Osmand et al., 1977). The preparations gave one line with antisera to human serum in immunodiffusion studies but were not reactive with a spectrum of antisera to specific plasma proteins. Monospecific antisera to AP, which was elicited in rabbits, revealed the proteins to have a 1 - mobility in immunoelectrophoretic separations of serum or isolated AP, in either agar or agarose in buffers containing calcium. In the presence of EDTA, AP had mobility comparable to that of albumin. Also, double immunodiffusion studies with sera containing C-reactive protein (CRP) and antLAP and anti-CRP indicated that the proteins were immunologically unrelated, confirming earlier studies by others (Pepys et al., 1977~).

The following experiments showed that isolated AP in labeled or unlabeled form had the properties of native AP in serum. Radiolabeled AP was added separately to isolated AP, serum or EDTA serum and the mixtures subjected to SDG analyses. AP exhibited the same single symmetrical peak and 9.5 S sedimentation rate whether detected by Ouchterlony analyses or by radioactivity. None was found in the 16 S or heavier regions ofthe SDG whether sedimented in the presence of calcium or of EDTA.

Interaction of AP with agarose and IgG andaggregation by calcium

A mixture of highly purified unlabeled and radiolabeled AP was passed through Sepharose, Sepharose-IgG and Sepharose-IgG-CIq columns in the presence of 10 mM calcium. The columns were subsequently washed with buffer containing calcium and then eluted with buffer containing 10 mM EDTA. The presence of AP in the eluates was determined by Ouchterlony analysis and by radioactivity measurement. AP was present only in the EDTA eluates of the Sepharose, Sepharose-IgG and Sepharose-IgG-CIq columns and none was found in the calcium washes of these columns. Radioactivity recovery in the EDTA eluates was 74, 68 and 86% of that applied to the Sepharose, Sepharose-IgG and Sepharose-IgG-C lq columns, respectively.

SDG analyses showed that AP had some ability to bind to IgG. In each of 7 experiments (Table 1) in which 3 different preparations of ‘251-AP (1 pg) were incubated with monomeric or aggregated IgG (100 pg) in the presence of 0.01 M calcium, there was an increment in the proportion of the AP found on the

Fig. 1. SDS-PAGE analysis of Isolated AP m the presence (right) and absence (left) of DTT. Fifty micrograms of AP were electrophoresed, after which the gels were stained with

Coomassie blue. The anode is at the bottom.

bottom of the centrifuge tube. There was no apparent difference in the results depending on whether monomeric or aggregated IgG was used. Binding to IgG was completely reversed by EDTA (Table 1).

As also indicated in Table 1, a variable proportion of the AP sedimented alone in the presence of 0.3 mM calcium was found on the bottom of the centrifuge tubes. In another centrifuge tube of experiment 7 (Table 1) in which the calcium concentration was increased to 10 mM, the proportion of AP pelleted increased from 16 to 30%. EDTA reversed the calcium-dependent aggregation process (Table 1).

Lack ~Jrequirement jbr AP.for complement activity

AP was depleted from human serum by passage through an anti-AP immune adsorbent in the presence of EDTA. Absence of the protein was verified by Ouchterlony analysis. After dialysis and recon-

824 XEIL R COOPER and ROBERT .I. %IC‘<‘iZRDI

stitution with calcium and magnesium. W,, of the

CH50 activity was recovered, which was not further increased by restitution of physiological levels ot P-component (20 jcp’ml).

AP is not a constituent of c’1

As we have recently observed. macromolecular Cl can be directly demonstrated in human serum as a single fused line of precipitation developed with antisera to Clq, Clr and Cls (Ziccardi & Cooper, 1978), a pattern which indicates that all three antigens arc on the same molecule. This technique allowed us to directly demonstrate that AP was not 21 constituent of macromolecular Cl in serum, since. as shown in Fig. 2, the precipitin line produced by AP crosses that due to Cl.

In the tirst of three approaches employed to determine whether AP interacted with the Cl sub- components or with Cl. radiolabeled AP was

Cat+

incubated with Cl sub-components and then subjected to SDG ultracentrifugation. In a typical experiment, 1 pg of ‘*‘I-AP was incubated with 3 fig each ofeither Clq. Clr or Cls alone. or in all possible combinations for 30 min at 37°C in buffer containing 1 mM calcium before SDG analysis. The labeled AP sedimented as ;I single symmetrical 9.5 S peak in the presence or absence of the Cl sub-components as shown in Fig. 3.

In a second approach, unlabeled AP was Incubated with serum or with Cl reconstituted from purified unlabeled C 1 q, C 1 r and C I s, after which the mixtures were subjected to SDG analyses and the fractions analyzed for Cl hemolytic activity. Ten micrograms of AP were generally added either to a mixture composed of8/cgofClq,9/cgofCIrand lO~~gofCIsorto50~l of serum. Cl hemolytic activity has invariably found only in a single symmetrical peak sedimenting at 16 S regardless of the presence or absence of AP. Cl hemolytic activity was never detected in the lower portion of such gradients.

In ;I third approach. I /ig 01‘ “‘LAP was incubated

EDTA

Fig. 2. Immunodiffwon analysis of Cl, Cl sub-components and Clt (AP) In normal human serum. Normal human serum was allowed to diffuse in agarose containing calcium or IO mM EDTA for 48 hr at 4<C. The precipitin line produced by AP crosses that due to Cl in the pattern on the left, indicating that AP

is not a constituent of Cl

Clt and Complement 825

5’0 1do Percent of Gradient

Fig. 3. Sucrose density gradient ultracentrifugation of 12cI- Clt (AP) in the presence and absence of Cl. A sedimentation rate of 9.5 S was obtained when AP was sedimented alone (top panel) or in the presence of Cl reconstituted from isolated Clq,Clr andCls(lowerpanel). Patternsidentical to that in the lower panel were also obtained when LZSI-AP was sedimented in the presence of isolated Clq, Clr or Cls

individually or in any combination.

for 30 min at 30°C with 3 x lo8 EA (IgM) or EA(IgG) in the presence of either 7 pg of Clq or with Cl reconstituted from 7 pgeach ofClq, Clr and Cls in a low ionic strength buffer containing 3 mM calcium. The cells were then centrifuged through a cushion of sucrose before being analyzed for radioactivity. Negligible binding of iZSI-P-component (2-5%) was observed in all combinations. Similar studies were carried out with 1 pg of **SI-Cls which was incubated with EA (IgM) and either 5 pg of C 1 q and 7 pg of C 1 r or with 5 pg of Clq and 6 pg of AP. In the Clq-Clr-iz51-Cls mixtures, 16”/, of the radiolabeled

Table 2. Hemolytic activity of Clr and AP

Reagent added Amount Percent to EAC4, Clq, Cls kg) Treatment Lysis

None 0

AP No. I 5 None 59 AP No. 1 0.5 None 54 AP No. I 0.05 None 23

AP No. 3 5 None 100 AP No. 3 0.5 None 99 AP No. 3 0.05 None 90

AP No. 3 5 DFP 5

AP No. 8 5 None 0 AP No. 8 0.5 None 0 AP No. 8 0.05 None 0

Clr 5 None 100 Clr 0.5 None 100 Clr 0.05 None 100

Clr 5 DFP 0

=4 E 20

=3 15 Z E2 10 El

-1 5

10 20 30 10 20 30 Fraction Number

Fig. 4. SDS-PAGE analysis of Cl activation. Cl reconstituted from Clq, Clr and ‘z”l-Cls, or a mixture of Clr and lzsI-Cls were incubated with aggregated IgG or with Clt (AP), after which the reaction mixtures were subjected to SDS-PAGE. Cls in the reaction mixtures containing Cl (upper left panel) is primarily in the 87,000 dalton proenzyme form. After addition of aggregated IgG (lower left panel), the rz51-Cls in the reconstituted Cl was cleaved into a 59,000 dalton fragment (and an unlabeled 28,000 dalton fragment), thus showing that the reconstituted Cl is activatable. Clt, however, was unable to activate Cl (lower right panel) as the ‘zsI-Cis remained in the 87,000 dalton proenzyme form. AP also did not activate a Clr-“cI-

Cls mixture (upper right panel).

Cls became bound. On substituting AP for Clr, 67, of the labeled C 1 s became bound, a value which AP does not significantly exceed the background level obtained with EA Clq and izSI-Cls (5”/,). AP also did not potentiate the binding of iZSI-Clr to EA (IgM) as there was 50% and 47’2 binding of iz51-C lr, respectively, in reaction mixtures containing EA, C I q, *251-ClrandClsandEA,Clq,LZ51-Clr,APandCls. The above studies together indicate that AP does not bind to the Cl sub-components or to C 1.

Lack of modulation of Cl jtinction by AP

Assimeh et al. have reported that AP has Clr-like hemolytic activity in a test system containing EAC4, 60 ng of Clq and 10 pg of Cls (Assimeh & Painter, 1975a; Assimeh & Painter, 19756). Nine preparations were examined in a similar test system except that 50 ng each of Clq and Cls were used. Although several of the AP preparations had hemolytic activity in this type of test, they varied considerably in this regard (Table 2). The most highly purified preparations (i.e. preparation 8 as shown in Fig. 1) were devoid of such hemolytic activity, even in high doses (Table 2). The hemolytic activity manifested by the other preparations was destroyed by incubation for 10 min at 37”C, a procedure which activates Clr (Ziccardi & Cooper, 1976a) followed by treatment with DFP as shown for preparation 3 in Table 2.

In another approach, two sera genetically deficient in Clr and containing no detectable Clr were found to contain 26 and 29 pg/ml of Clq, 12 and 10 pg/ml of C 1 s and 32 and 35 pg/ml of AP, respectively (these sera were kindly furnished by Drs. N. K. Day and F. B. Taylor). The AP values are almost twice normal for that protein (1 l-25 pg/ml). The sera did not produce

826 UE.lL R (‘OOPER and ROBERT J. LIC‘CARDI

lysis in Cl hemolytic assays but this activity was reconstituted with physiologic C 1 I_ concentrations. It ix thus likely that pure AP is devoid of Clr-like hemolytic activity and that the activitv sometimes observed IX due to contamination with ;I scrine csterase, most likely C I I-.

In other studies, AP did not enhance the Cl hemolyticactivityofClq-Clr-Clsmixtures.Thus the addition of 0.6 ng of AP to a mixture of 2.2 ng of Clq, 0.5 ng of Clr and I. I ng of Cls did not increase hemolytic Lictivity (3.4 x IO’ E.M. vs 4.3 x IO’ E.M.).

Varying amounts of AP were incubated with proen7yme Cl reconstituted from Clq, Clr and “‘I- Cls in a Cl activation assay (Cooper & Ziccardi. 1977). As shown in Fig. 4. AP did not activjate Cl.

Amyloid P-component (AP) isolated from strum by two different methods had the sub-unit structure reported earlier for this protein. In both unlabeled and radiolabeled forms, AP was indistinguishable from native AP in serum in sedimentation rate (9.5 S) and charge (a, ). AP interacted with agarose via a caicium- dependent bond as previously documented (Pepys et al., 1977~; Pepys rt ol., 1977h; Painter. 1977). In addition, AP interacted with IgG to ;I variable extent in the presence of calcium. Calcium also induced aggregation of AP which could be reversed by chelation. Serum immunodepleted of AP retained complement activity confirming studies showing that the protein vvas not essential for hcmolytic activity (Ziccardi & Cooper. 1977: Gigli e/ ul., 1976: Painter, 1977). Ouchterlony studies with anti-Clq. Clr, Cls and AP directly showed that AP was not associated with, or a constituent of Cl, a conclusion suggested by earlier indirect approaches. indicating that Cl and AP behaved independently (Ziccardi & Cooper. 1977). AP did not interact in sucrose density gradients with isolated Clq, Clr or Cls. individually, or in any combination. Similarly, AP did not enhance the binding of izSI-Clr to EAClq in the presence (or absence) of C I s or mediate the binding of ’ z ‘1-C I s to EA or EAClq and iz51-AP did not itself bind to any cellu1ai intermediate. Although several AP preparations had variable Clr-like hcmolytic activity, this was due to variable contamination with a 37 C heat activatable serine esterose, probably Clr. AP did not substitute for C I r in providing any C I function to set-a genetically deficient in Clr. It :IISO did not activate

precursor native Cl or enhance the activity of

Clq-Clr-Cls mixtures.

Multiple pieces of data led to the earlier conclusion (Assimeh &Painter, 1975a; Assimeh & Painter, 1975b) that AP was a fourth Cl sub-component. These included the calcium dependent binding of AP to Sepharose IgG columns along with the Cl sub- components. This was a fortuitous occurrence as AP is now known to bind to Sepharose via :I calcium bond independently of the presence of IgG or Clq, Clr and Cls, as shown here and by others (Pepys et al., 1977a; Pepys et al., 19776: Painter, 1977). However, the previous observation that AP binds only to Sepharose IgG-Clq and not to Sepharose IgG (Assimeh &

Painter. lY75~: Assimeh & Painter, IY751~). which could not be confirmed here. rcmkiins uncxplainrd. Another piece of evidence leading to the concept of ;I

fourth Cl sub-component was the co-sedimentation of APalong with Cly. Clr and Cls in heavier regions of density gradients carried out with serum in the prcsencc of calcium and sedimentation in the hghtct regions of the gradient. together with Clq. Clr and Cls when EDTA was added. This adventitious association of AP with regions of the gradient containing Clq. Clr and Cls is probably due to aggregation of AP in the presence ofcalcium as shown here. The earlier finding that AP increased the sedimentation rate of Clq-Clr-Cls mixtures presumably retlects the inadvertent addition of C I r, along with AP, to the Clq-Cls mixture as recently postulated (Painter, 1977). as studies carried out here failed to shovv any aftinity of AP for the Cl sub- components either individually ot- m any combination. The present study further indicates that the Clr-like hemolytic activity reported earlier (Assimeh & Painter, 1975~1; Assimeh & Painter. 1975h: Taylor et ul.. 1977) is the result of contamination of some AP preparations with ;I 37 C heat activatable serine esterase. probably Cl I-. Cl r contamination also explains the apparent ability of AP preparations to facilitate Cls attachment to EAC lq,4 and increase the sedimentation rate of Clq-Cls mixtures (Painter, lY77).

The present experiments along with earlier studies thus furnish alternativ,e explanations for the earlier data which suggested that AP was a fourth Cl sub- component. Cumulatively, these studies indicate that AP is neither 2 functional nor non-functional constituent of Cl and further show that AP possesses no ability to activate Cl or to physically interact with, or modulate the activity of Cl or any of its sub-units.

4 c~ktlon /1%/,,1’“K”1/.\ WC vvtsh to thank Susan Host fat outstanding technical assistance and Carol Davis for skilled secretarial help.

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Assimeh S. N.. Bing D. H. & Painter R. H. (1974). A simple method for the isolation of the auhcomponents of the tirst component ofcomplement by aftintty chromatography. J. In1mu,r. 113, 225.

Assimeh S. N. & Painter R. H. (1975~) The identification ofa previously unrecognized subcomponent of the first component of complement. .I. Iftzmun. I IS, 482.

Assimeh S. N. & Painter R. H. (197%) The macromolecular structure of the first component of complement. J. Immun. 115, 488.

Bladen H. A., Nylen M. U. Br Glenner G. G. (1966) The ultrastructure of human amylotd as revealed by the negative statning technique. J. L~lrrusrruc. Res. 14, 449.

Calcott M. & Miller-Eberhard (1972) Clq protein of human complement. Biochemi.,/r~~ 1 I, 3443.

Cooper N. R. &Ziccardi R. J. (1977) Reconstitution ofC1 m native, proenzyme form and its use in a quantitative Cl activation test. J. Immun. 119, 1664.

Gigli I.. Porter R. R. & Sim R. R. (1976) The unactivated form of the first component of human complement. Cl. Riochew. J. 157, 541.

C I t and Complement 827

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Mayer M. M. (1961) Complement and complement fixation. In E.uperimcwtrrl Immunochemistry (Edited by Kobat E. A. & Mayer M. M.) p. 133. C. C. Thomas, Springfield.

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Pinteric L., Assimeh S. N.. Kells D. 1. C. & Painter R. H. (1976) The ultra-structure of Cit. a subcomponent of the first component of complement: An E.M. and ultracentrifuge study. J. Im&m. 117, 79.

Skinner M., Benson M. D. & Cohen A. S. (1976) Amyloid P-component: Sequence analysis and comparison to the

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