/ . Biochem. 85, 1023-1028 (1979)
Purification and Some Properties of Rabbit Clr
Department of Microbiology, Faculty of Pharmaceutical Sciences, Higashi Nippon Gakuen University, Tobetsu, Ishikari-gun, Hokkaido 061-02 and 'Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060 Received for publication, November 7, 1978
Clr, an activated subcomponent of the first component of the complement system, was highly purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B followed by column chromatography on CM-Sephadex C-50. The Clr thus purified had a molecular weight of 105,000, consisting of two polypeptide chains connected by disulfide bonds; the molecular weights of the chains were 60,000 and 45,000. The Clr was found to reconstitute Cl complex when it reacted with rabbit Clq and Cls in the presence of Caa+, since Cls was able to bind to Clq bound on sensitized sheep erythrocytes only in the presence of Clr. On the other hand, an active Cls fragment derived by hydrolysis of the H chain without any loss of Cls activity [/. Biochem. 80, 1423-1427 (1976)] could not bind to Clq even in the presence of Clr. This result indicates that a part of the H chain of Cls not contributing to the structural integrity of an active site may be involved in the binding of Cls to Clr.
The first component of the complement system is a macromolecular complex consisting of Clq, Clr, and Cls which link together in the presence of Ca l+ (7). When Cl binds to immune complex via Clq, Clr activates autocatalytically to its protease form, Clr, and converts Cls to Cls. Much information on the molecular mechanism of activation of Clr has become available from many intensive studies on human Clr and Clr (2-7). Abbreviations: The complement nomenclature conforms to that described in Bull. Wld. Hlth. Org. 39, 935 (1968). Other abbreviations used are AAME, Af-»acetyl-L-arginine methylester; AGLME, acetylglycyl-Llysine methylester; ATEE, A'-a-acetyl-L-tyrosine ethylester; EDTA, ethylenediaminetetraacetate; SDS, sodium dodecyl sulfate; BGS, barbiturate-buffered saline supplemented with gelatin and CaCl t ; FE, formalized sheep erythrocytes; DFP, diisopropylfluorophosphate.
Vol. 85, No. 4, 1979
On the other hand, there are few reports on rabbit Clr and Clr, since rabbit serum is not a suitable source of complement for immune hemolysis (8). Recently, we found that the serum level of rabbit Cl was as high as the levels of human and guinea pig Cl and we succeeded in purifying rabbit Cls (9). In addition, our study revealed that, during purification, rabbit Cls with a molecular weight of 106,000 was partly converted to an active fragment with a molecular weight of 72,000 by proteolytic cleavage of the H chain by a contaminating protease, probably plasmin (9). In order to study rabbit Cl further, we have attempted to purify the Clr and to examine the binding ability of Cls or Cls fragment to Clr. In this paper, we report the purification of rabbit Clr and the site of Cls involved in the formation of Cl complex.
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Yoki MORI, Masahiko KOKETSU,* Nobuhito ABE, and Tiro KOYAMA*
1024
Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA
MATERIALS AND METHODS
Determination of Cls-Activating Activity of Clr—The Cls-activating activity of Clr was determined by the method of Naff and Ratnoff (4). Samples of Clr to be estimated were incubated with 0.1 unit (AGLME) of rabbit Cls in 1.0 ml of 0.1 M Tris-HCl buffer, pH8.1, for 30 min at 37°C, and the esterolytic activity of Cls thus activated was measured with AGLME at a final concentration of 5 mM in the same buffer. The Cls-activating activity was expressed in terms of units of Cls activated per 30 min at 37°C. Preparation of Rabbit Cls—Rabbit Cls was isolated by the method described by Sakai and Stroud (70) for the purification of human Cls with some modifications. Two hundred ml of freshly drawn, heparinized rabbit plasma was dialyzed against 4 mM phosphate buffer supplemented with 5 mM benzamidine, pH 7.5, for 16 h at 0°C. The precipitates formed were dissolved in 20 ml of 10 mM acetate buffer containing 0.45 M NaCl and 10 mM EDTA, pH 5.5. After centrifugation, the supernatant was diluted four-fold with 10 mM acetate buffer containing 10 mM EDTA, pH 5.5, and applied to a column of CM-Sephadex C-50 ( 2 x 5 cm) equilibrated with 10 mM acetate buffer containing 0 . 1 0 M NaCl and 10 mM EDTA, pH 5.5. The effluent was pooled, dialyzed against 10 mM phosphate buffer containing 50 mM NaCl and 10 mM EDTA, pH 7.5, and applied to a column of DEAE-cellulose (1.5x5 cm) equilibrated with the same buffer. Bound proteins were eluted with a linearly increasing NaCl concentration gradient. The Cls thus purified was used for experiments. Cls activity was determined as described by Sakai
The fractions containing Clq were pooled and diluted with 10 mM acetate buffer, pH 5.5, containing 10 mM EDTA to lower the NaCl concen/ . Biochcm.
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Determination of Esterolytic Activities of Clr and Cls—Esterolytic activities of Clr and Cls were determined with JV-a-acetyl-L-arginine methylester (AAME), acetylglycyl-L-lysine methylester (AGLME), and TV-a-acetyl-L-tyrosine ethylester (ATEE) (9). Samples of Clr and Cls to be estimated were incubated with each substrate at a final concentration of 5 mM in 1.0 ml of 0 . 1 M Tris-HCl buffer, pH8.1, for 30 min at 37°C, and amounts of the substrate hydrolyzed were measured using the hydroxamate reagent. One unit of the activity was defined as that hydrolyzing 1 /Jmol of each substrate per min.
and Stroud (70). One unit of Cls was defined as the activity capable of hydrolyzing 1 fimo\ of AGLME per min at 37°C when activated to Cls with Clr. Preparation of Rabbit Cls and Its Active Fragment—Purification of rabbit Cls and its active fragment, which have molecular weights of 100,000 and 72,000, respectively, was performed by DEAEcellulose column chromatography of Cl isolated by affinity chromatography on an IgG-Sepharose 6B column. The IgG-Sepharose 6B used was prepared by the method of Assimeh et al. (77). Details of the procedures were described in a previous paper (9). Preparation of Rabbit Clq—Rabbit Clq was purified from freshly drawn serum according to the method of Volanakis and Stroud with some modifications (72). Rabbit serum was dialyzed overnight against 4 mM phosphate buffer, pH 7.5. The precipitate was recovered by centrifugation and, after washing with the same buffer, was dissolved in 0.75 M NaCl containing 10 mM EDTA, pH 7.5. After removal of insoluble materials, the solution was dialyzed overnight against 10 mM phosphate buffer, pH 7.5, containing 10 mM EDTA. The recovered precipitate was washed with the same buffer and dissolved in 0.75 M NaCl containing 10 mM EDTA, pH 7.5. The solution was centrifuged and the supernatant was dialyzed overnight against 10 mM acetate buffer containing 10 mM EDTA, pH 5.5. The precipitate was dissolved in 0.75 M NaCl containing 10 mM EDTA and 10 mM acetate buffer, pH 5.5, and diluted four-fold with 10 mM acetate buffer, pH 5.5, containing 10 mM EDTA. The solution containing Clq was applied to a CM-Sephadex C-50 column equilibrated with 10 mM acetate buffer, pH 5.5, supplemented with 0.15 M NaCl and 10 mM EDTA. The column was washed with the same buffer and the adsorbed proteins were eluted with a linearly increasing NaCl concentration gradient. The activity of Clq was determined by measuring the hemagglutinating activity against sheep erythrocytes sensitized with rabbit IgG antibody. The amount of antibody used was adjusted to be sufficient to agglutinate the erythrocytes in the presence of Clq, but not in the absence of Clq.
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Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA The C l r gave a single band when it was analyzed by polyacrylamide disc gel electrophoresis (Fig. 3). Its molecular weight was estimated to be 105,000 from the mobility on SDS-polyacrylamide gel electrophoresis (14). On the other hand, SDS-polyacrylamide gel electrophoresis in the presence of 0.1 M 2-mercaptoethanol gave two polypeptide chains with molecular weights of
—H chain — L chain
Fig. 2. CM-Sephadex C-50 chromatography of rabbit Clr. The Cl fraction purified by affinity chromatography was applied to a column of CM-Sephadex C-50 (1x5 cm) equilibrated with lOmM acetate buffer containing 10 mM EDTA and 50 mM NaCl, pH 5.5. After washing the column with the same buffer, bound proteins were eluted with a linearly increasing NaCl concentration gradient in 10 mM acetate buffer containing 10 mM EDTA.
(1)
(2)
(3)
Fig. 3. Polyacrylamide gel electrophoresis of rabbit Clr. (1) Purified Clr in 5% polyacrylamide disc gel with acetate buffer, pH4.3; (2) Purified Clr in 5% polyacrylamide gel with 0.2% SDS and 0.2 M phosphate buffer, pH7.3; (3) 0 . 1 M 2-mercaptoethanol-treated Clr in 5% polyacrylamide gel with 0.2% SDS and 0.2 M phosphate buffer, pH 7.3.
TABLE I. Purification of rabbit Clr. Purification step Fractionation with polyethylene glycol
Total protein
Total activity* (units)
Specific activity (units//!«,,)
2,550
279/
0.109
0)
IgG-Sepharose 6B
16.0
96.0
6.0
CM-Sephadex C-50
3.4
51.0
15.1
Clr activity is expressed in terms of Cls-activating units with AGLME. / . Biochem.
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fraction, indicating complete separation of Clr from Cls (Fig. 2). The various steps in purification of rabbit C l r are shown in Table I. The purification was 150-fold with an overall yield of 20% relative to the activity of C l r in the precipitates with polyethylene glycol. Properties of Purified Clr—The purified C l r could hydrolyze AGLME and AAME but not ATEE, as in the case of human C l r (4). Its specific activities were 4.8 and 8.9 units/mg protein for AGLME and AAME, respectively. When 10 fig of the Clr was incubated with 10 fig of rabbit Cls for 30min at 37CC, the Clr completely activated the Cls to its protease form, Cls. Esterase activities of Clr were completely inactivated by highly purified rabbit Cl-inactivator (18) and diisopropylfluorophosphate (DFP) but not by soybean trypsin inhibitor.
1027
RABBIT Clr TABLE II. Binding of Cls or its fragment to sensitized FE in the presence of Clq and Clr. 1
2
4
;
0.05 0.25
0.07 0.22
+
Clq (36 fig) Clr (7.5 /ig) Cls (8.9 /ig) Cls fragment (13.6 /ig)
3
1 + 1
Reaction mixture
AGLME-hydrolyzing activity (units) Bound to FE Unbound to FE
0.22 0.05
0.07 0.20
Cls or its active fragment was incubated with Clq and Clr in the presence of Ca t+ and then with sensitized FE. After centrifugation, AGLME-hydrolyzing activities bound or unbound to FE were determined in a pH stat. Details of these procedures are described in the text. The AGLME-hydrolyzing activities of the Cls, its active fragment and Clr used were 0.27, 0.25, and 0.03 units, respectively. 60,000 and 45,000, indicating that rabbit Clr also consisted of two polypeptide chains, H and L chains, connected by disulfide bonds as in the case of human Clr (2).
Binding Properties of Clr with Cls and Clq— In order to examine whether the Clr could reconstitute Cl complex or not, it was incubated with purified Clq and Cls in the presence of Ca!+, and then FE sensitized with rabbit IgG antibody was added to the reaction mixture. As the AGLMEhydrolyzing activity of Cls used was high relative to that of Clr added, the binding of Cls to Clq bound on sensitized FE was estimated by measuring the esterase activities for AGLME. The Cls was found to bind to sensitized FE only in the presence of both Clr and Clq, indicating that Cls could form Cl complex with Clr and Clq (Table II). Furthermore, it was found that the binding of Cls to Clq-binding erythrocytes took place via Clr bound on the Clq, since the Cls added remained in the fluid phase when Clr was absent. On the other hand, the active fragment of Cls produced by proteolytic cleavage of the H chain during the procedures of purification (9) could not bind to Clq-treated FE even in the presence of Clr, since the AGLME-hydrolyzing activity remained completely in the fluid phase. This indicates that the ability of Cls to form Cl complex was not retained by the Cls fragment. A part of the H chain of Cls deleted by the proteolysis may be involved in the binding of Cls to Clr. Vol. 85, No. 4, 1979
DISCUSSION Human Clr has been shown to have a single polypeptide chain structure with a molecular weight of 83,000 and to be converted into Clr, having two polypeptide chains, when Cl binds to immune complex via Clq (2-7). Furthermore, it is known that unless protease inhibitors, such as DFP, are added to the serum initially and are added again at each stage of the purification, spontaneous activation of Clr occurs during the isolation. In agreement with this, rabbit Clr was also isolated as its active form alone since no precautions were taken to prevent activation, and affinity chromatography on an IgG-Sepharose 6B column was employed for the purification. The results presented in this paper demonstrate a marked structural similarity of rabbit Clr to human Clr; rabbit Clr is also composed of two chains which are similar to the H and L chains of human Clr (7). Furthermore, rabbit Clr is enzymatically similar to human Clr since it can hydrolyze AGLME and AAME, but not ATEE, and these esterolytic activities and also Clsactivating activity are easily inactivated by Cl inactivator isolated from rabbit serum, and DFP (7, 19). In the case of human Cls, Laurell and Martensson (20) reported that the Cls was able to bind to Clq in the presence of Clr, but not in the absence of Clr. Ziccardi and Cooper (3) also
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+
1028
REFERENCES 1. Lepow, I.H., Naff, G.B., Pensky, J., & Hinz, C.F. (1963) /. Exp. Med. 117, 983-1008 2. Takahashi, K., Nagasawa, S., & Koyama, J. (1975) FEBS Lett. 65, 20-23 3. Ziccardi, R.J. & Cooper, N.R. (1976) /. Immunol. 116, 496-503 4. Naff, G.B. & Ratnoff, O.D. (1968) /, Exp. Med. 128, 571-593 5. de Bracco, M.M. & Stroud, R.M. (1971) /. Clin. Invest. 50, 838-848 6. Valet, G. & Cooper, N.R. (1974) /. Immunol. 112, 1667-1673 7. Takahashi, K., Nagasawa, S., & Koyama, J. (1975) FEBS Lett. 55, 156-160 8. Linscott, W.D. (1968) lmmunochemistry 5, 311-314 9. Ishizaki, E., Mori, Y., & Koyama, J. (1976) / . Biochem. 80, 1423-1427 10. Sakai, K. & Stroud, R.M. (1973) /. Immunol. 110, 1010-1020 11. Assimeh, S.N., Bing, D.H., & Painter, R.H. (1974) /. Immunol. 113, 224-234 12. Volanakis, J.E. & Stroud, R.M. (1972) /. Immunol. Methods 2, 25-34 13. Reisfeld, R.A., Lewis, U.J., & Williams, D.E. (1962) Nature 195, 281-283 14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 15. Mori, Y., Kawai, N., & Koyama, J. (1973) / . Biochem. 73, 951-958 16. Gigli, 1., Porter, R.R., & Sim, R.B. (1976) Biochem. J. 157, 541-548 17. Reid, K.B.M., Lowe, D.M., & Porter, R.R. (1972) Biochem. J. 130, 749-763 18. Ishizaki, E., Mori, Y., & Koyama, J. (1977) / . Biochem. 82, 1155-1160 19. Ratnoff, O.D. (1969) / . Exp. Med. 129, 315-331 20. Laurell, A.B. & Martensson, U. (1974) Acta Path. Microbiol. Scand. B82, 585-589 21. Barkas, T., Scott, K., & Forthergill, J.E. (1973) Biochem. Soc. Trans. 1, 1219-1220 22. Nagasawa, S., Takahashi, K., & Koyama, J. (1974) FEBS Lett. 41, 280-282 23. Sim, R.B. & Porter, R.R. (1976) Biochem. Soc. Trans. 4, 127-129 24. Porter, R.R. (1977) Biochem. Soc. Trans. 5, 16601677
J. Biochem.
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observed that human Clr functioned not only as an activator for Cls, but also as a physical linkage between Clq and Cls in human Cl. The results obtained in the present study showed that rabbit Clr also serves as a physical linkage between Clq and Cls in a similar way. In a previous paper, we reported that an active Cls fragment with a molecular weight of 72,000 was isolated during the purification of rabbit Cls. Although this fragment was indistinguishable from Cls with regard to proteolytic activities tested, it differed from Cls in having an H chain of shorter length, which was almost equal to that of the L chain with a molecular weight of 37,000. The finding that the Cls fragment loses the ability to form Cl complex with Clq and Clr serves to identify the location of the site which is involved in the binding of Cls to Clr. This site of Cls may be located in the H chain, specially, in the portion which is deleted by action of a protease in rabbit serum (probably plasmin), and which does not contribute appreciably to the structural integrity of the active site. On the other hand, the active site of rabbit Cls may be in the L chain, as in the case of the human counterpart (21). The molecular mechanism of formation of the Cl complex seems to be complex. In the case of human Cl, Clr and Cls are thought to form a tight complex containing two molecules of each, giving a molecular weight of about 350,000, and possibly also an octomeric complex of double this size (22-24). Interaction of this complex with Clq is suggested by Porter to be weak relative to that of Clr to Cls (24). At present, we have no further information on the molecular mechanism of formation of rabbit Clr-Cls complex or on the location of the sites of Clr which are involved in the reconstitution of Cl complex. Further studies will be required to elucidate these problems.
Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA
Purification and Some Properties of Rabbit Clr
Department of Microbiology, Faculty of Pharmaceutical Sciences, Higashi Nippon Gakuen University, Tobetsu, Ishikari-gun, Hokkaido 061-02 and 'Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060 Received for publication, November 7, 1978
Clr, an activated subcomponent of the first component of the complement system, was highly purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B followed by column chromatography on CM-Sephadex C-50. The Clr thus purified had a molecular weight of 105,000, consisting of two polypeptide chains connected by disulfide bonds; the molecular weights of the chains were 60,000 and 45,000. The Clr was found to reconstitute Cl complex when it reacted with rabbit Clq and Cls in the presence of Caa+, since Cls was able to bind to Clq bound on sensitized sheep erythrocytes only in the presence of Clr. On the other hand, an active Cls fragment derived by hydrolysis of the H chain without any loss of Cls activity [/. Biochem. 80, 1423-1427 (1976)] could not bind to Clq even in the presence of Clr. This result indicates that a part of the H chain of Cls not contributing to the structural integrity of an active site may be involved in the binding of Cls to Clr.
The first component of the complement system is a macromolecular complex consisting of Clq, Clr, and Cls which link together in the presence of Ca l+ (7). When Cl binds to immune complex via Clq, Clr activates autocatalytically to its protease form, Clr, and converts Cls to Cls. Much information on the molecular mechanism of activation of Clr has become available from many intensive studies on human Clr and Clr (2-7). Abbreviations: The complement nomenclature conforms to that described in Bull. Wld. Hlth. Org. 39, 935 (1968). Other abbreviations used are AAME, Af-»acetyl-L-arginine methylester; AGLME, acetylglycyl-Llysine methylester; ATEE, A'-a-acetyl-L-tyrosine ethylester; EDTA, ethylenediaminetetraacetate; SDS, sodium dodecyl sulfate; BGS, barbiturate-buffered saline supplemented with gelatin and CaCl t ; FE, formalized sheep erythrocytes; DFP, diisopropylfluorophosphate.
Vol. 85, No. 4, 1979
On the other hand, there are few reports on rabbit Clr and Clr, since rabbit serum is not a suitable source of complement for immune hemolysis (8). Recently, we found that the serum level of rabbit Cl was as high as the levels of human and guinea pig Cl and we succeeded in purifying rabbit Cls (9). In addition, our study revealed that, during purification, rabbit Cls with a molecular weight of 106,000 was partly converted to an active fragment with a molecular weight of 72,000 by proteolytic cleavage of the H chain by a contaminating protease, probably plasmin (9). In order to study rabbit Cl further, we have attempted to purify the Clr and to examine the binding ability of Cls or Cls fragment to Clr. In this paper, we report the purification of rabbit Clr and the site of Cls involved in the formation of Cl complex.
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Yoki MORI, Masahiko KOKETSU,* Nobuhito ABE, and Tiro KOYAMA*
1024
Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA
MATERIALS AND METHODS
Determination of Cls-Activating Activity of Clr—The Cls-activating activity of Clr was determined by the method of Naff and Ratnoff (4). Samples of Clr to be estimated were incubated with 0.1 unit (AGLME) of rabbit Cls in 1.0 ml of 0.1 M Tris-HCl buffer, pH8.1, for 30 min at 37°C, and the esterolytic activity of Cls thus activated was measured with AGLME at a final concentration of 5 mM in the same buffer. The Cls-activating activity was expressed in terms of units of Cls activated per 30 min at 37°C. Preparation of Rabbit Cls—Rabbit Cls was isolated by the method described by Sakai and Stroud (70) for the purification of human Cls with some modifications. Two hundred ml of freshly drawn, heparinized rabbit plasma was dialyzed against 4 mM phosphate buffer supplemented with 5 mM benzamidine, pH 7.5, for 16 h at 0°C. The precipitates formed were dissolved in 20 ml of 10 mM acetate buffer containing 0.45 M NaCl and 10 mM EDTA, pH 5.5. After centrifugation, the supernatant was diluted four-fold with 10 mM acetate buffer containing 10 mM EDTA, pH 5.5, and applied to a column of CM-Sephadex C-50 ( 2 x 5 cm) equilibrated with 10 mM acetate buffer containing 0 . 1 0 M NaCl and 10 mM EDTA, pH 5.5. The effluent was pooled, dialyzed against 10 mM phosphate buffer containing 50 mM NaCl and 10 mM EDTA, pH 7.5, and applied to a column of DEAE-cellulose (1.5x5 cm) equilibrated with the same buffer. Bound proteins were eluted with a linearly increasing NaCl concentration gradient. The Cls thus purified was used for experiments. Cls activity was determined as described by Sakai
The fractions containing Clq were pooled and diluted with 10 mM acetate buffer, pH 5.5, containing 10 mM EDTA to lower the NaCl concen/ . Biochcm.
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Determination of Esterolytic Activities of Clr and Cls—Esterolytic activities of Clr and Cls were determined with JV-a-acetyl-L-arginine methylester (AAME), acetylglycyl-L-lysine methylester (AGLME), and TV-a-acetyl-L-tyrosine ethylester (ATEE) (9). Samples of Clr and Cls to be estimated were incubated with each substrate at a final concentration of 5 mM in 1.0 ml of 0 . 1 M Tris-HCl buffer, pH8.1, for 30 min at 37°C, and amounts of the substrate hydrolyzed were measured using the hydroxamate reagent. One unit of the activity was defined as that hydrolyzing 1 /Jmol of each substrate per min.
and Stroud (70). One unit of Cls was defined as the activity capable of hydrolyzing 1 fimo\ of AGLME per min at 37°C when activated to Cls with Clr. Preparation of Rabbit Cls and Its Active Fragment—Purification of rabbit Cls and its active fragment, which have molecular weights of 100,000 and 72,000, respectively, was performed by DEAEcellulose column chromatography of Cl isolated by affinity chromatography on an IgG-Sepharose 6B column. The IgG-Sepharose 6B used was prepared by the method of Assimeh et al. (77). Details of the procedures were described in a previous paper (9). Preparation of Rabbit Clq—Rabbit Clq was purified from freshly drawn serum according to the method of Volanakis and Stroud with some modifications (72). Rabbit serum was dialyzed overnight against 4 mM phosphate buffer, pH 7.5. The precipitate was recovered by centrifugation and, after washing with the same buffer, was dissolved in 0.75 M NaCl containing 10 mM EDTA, pH 7.5. After removal of insoluble materials, the solution was dialyzed overnight against 10 mM phosphate buffer, pH 7.5, containing 10 mM EDTA. The recovered precipitate was washed with the same buffer and dissolved in 0.75 M NaCl containing 10 mM EDTA, pH 7.5. The solution was centrifuged and the supernatant was dialyzed overnight against 10 mM acetate buffer containing 10 mM EDTA, pH 5.5. The precipitate was dissolved in 0.75 M NaCl containing 10 mM EDTA and 10 mM acetate buffer, pH 5.5, and diluted four-fold with 10 mM acetate buffer, pH 5.5, containing 10 mM EDTA. The solution containing Clq was applied to a CM-Sephadex C-50 column equilibrated with 10 mM acetate buffer, pH 5.5, supplemented with 0.15 M NaCl and 10 mM EDTA. The column was washed with the same buffer and the adsorbed proteins were eluted with a linearly increasing NaCl concentration gradient. The activity of Clq was determined by measuring the hemagglutinating activity against sheep erythrocytes sensitized with rabbit IgG antibody. The amount of antibody used was adjusted to be sufficient to agglutinate the erythrocytes in the presence of Clq, but not in the absence of Clq.
1026
Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA The C l r gave a single band when it was analyzed by polyacrylamide disc gel electrophoresis (Fig. 3). Its molecular weight was estimated to be 105,000 from the mobility on SDS-polyacrylamide gel electrophoresis (14). On the other hand, SDS-polyacrylamide gel electrophoresis in the presence of 0.1 M 2-mercaptoethanol gave two polypeptide chains with molecular weights of
—H chain — L chain
Fig. 2. CM-Sephadex C-50 chromatography of rabbit Clr. The Cl fraction purified by affinity chromatography was applied to a column of CM-Sephadex C-50 (1x5 cm) equilibrated with lOmM acetate buffer containing 10 mM EDTA and 50 mM NaCl, pH 5.5. After washing the column with the same buffer, bound proteins were eluted with a linearly increasing NaCl concentration gradient in 10 mM acetate buffer containing 10 mM EDTA.
(1)
(2)
(3)
Fig. 3. Polyacrylamide gel electrophoresis of rabbit Clr. (1) Purified Clr in 5% polyacrylamide disc gel with acetate buffer, pH4.3; (2) Purified Clr in 5% polyacrylamide gel with 0.2% SDS and 0.2 M phosphate buffer, pH7.3; (3) 0 . 1 M 2-mercaptoethanol-treated Clr in 5% polyacrylamide gel with 0.2% SDS and 0.2 M phosphate buffer, pH 7.3.
TABLE I. Purification of rabbit Clr. Purification step Fractionation with polyethylene glycol
Total protein
Total activity* (units)
Specific activity (units//!«,,)
2,550
279/
0.109
0)
IgG-Sepharose 6B
16.0
96.0
6.0
CM-Sephadex C-50
3.4
51.0
15.1
Clr activity is expressed in terms of Cls-activating units with AGLME. / . Biochem.
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fraction, indicating complete separation of Clr from Cls (Fig. 2). The various steps in purification of rabbit C l r are shown in Table I. The purification was 150-fold with an overall yield of 20% relative to the activity of C l r in the precipitates with polyethylene glycol. Properties of Purified Clr—The purified C l r could hydrolyze AGLME and AAME but not ATEE, as in the case of human C l r (4). Its specific activities were 4.8 and 8.9 units/mg protein for AGLME and AAME, respectively. When 10 fig of the Clr was incubated with 10 fig of rabbit Cls for 30min at 37CC, the Clr completely activated the Cls to its protease form, Cls. Esterase activities of Clr were completely inactivated by highly purified rabbit Cl-inactivator (18) and diisopropylfluorophosphate (DFP) but not by soybean trypsin inhibitor.
1027
RABBIT Clr TABLE II. Binding of Cls or its fragment to sensitized FE in the presence of Clq and Clr. 1
2
4
;
0.05 0.25
0.07 0.22
+
Clq (36 fig) Clr (7.5 /ig) Cls (8.9 /ig) Cls fragment (13.6 /ig)
3
1 + 1
Reaction mixture
AGLME-hydrolyzing activity (units) Bound to FE Unbound to FE
0.22 0.05
0.07 0.20
Cls or its active fragment was incubated with Clq and Clr in the presence of Ca t+ and then with sensitized FE. After centrifugation, AGLME-hydrolyzing activities bound or unbound to FE were determined in a pH stat. Details of these procedures are described in the text. The AGLME-hydrolyzing activities of the Cls, its active fragment and Clr used were 0.27, 0.25, and 0.03 units, respectively. 60,000 and 45,000, indicating that rabbit Clr also consisted of two polypeptide chains, H and L chains, connected by disulfide bonds as in the case of human Clr (2).
Binding Properties of Clr with Cls and Clq— In order to examine whether the Clr could reconstitute Cl complex or not, it was incubated with purified Clq and Cls in the presence of Ca!+, and then FE sensitized with rabbit IgG antibody was added to the reaction mixture. As the AGLMEhydrolyzing activity of Cls used was high relative to that of Clr added, the binding of Cls to Clq bound on sensitized FE was estimated by measuring the esterase activities for AGLME. The Cls was found to bind to sensitized FE only in the presence of both Clr and Clq, indicating that Cls could form Cl complex with Clr and Clq (Table II). Furthermore, it was found that the binding of Cls to Clq-binding erythrocytes took place via Clr bound on the Clq, since the Cls added remained in the fluid phase when Clr was absent. On the other hand, the active fragment of Cls produced by proteolytic cleavage of the H chain during the procedures of purification (9) could not bind to Clq-treated FE even in the presence of Clr, since the AGLME-hydrolyzing activity remained completely in the fluid phase. This indicates that the ability of Cls to form Cl complex was not retained by the Cls fragment. A part of the H chain of Cls deleted by the proteolysis may be involved in the binding of Cls to Clr. Vol. 85, No. 4, 1979
DISCUSSION Human Clr has been shown to have a single polypeptide chain structure with a molecular weight of 83,000 and to be converted into Clr, having two polypeptide chains, when Cl binds to immune complex via Clq (2-7). Furthermore, it is known that unless protease inhibitors, such as DFP, are added to the serum initially and are added again at each stage of the purification, spontaneous activation of Clr occurs during the isolation. In agreement with this, rabbit Clr was also isolated as its active form alone since no precautions were taken to prevent activation, and affinity chromatography on an IgG-Sepharose 6B column was employed for the purification. The results presented in this paper demonstrate a marked structural similarity of rabbit Clr to human Clr; rabbit Clr is also composed of two chains which are similar to the H and L chains of human Clr (7). Furthermore, rabbit Clr is enzymatically similar to human Clr since it can hydrolyze AGLME and AAME, but not ATEE, and these esterolytic activities and also Clsactivating activity are easily inactivated by Cl inactivator isolated from rabbit serum, and DFP (7, 19). In the case of human Cls, Laurell and Martensson (20) reported that the Cls was able to bind to Clq in the presence of Clr, but not in the absence of Clr. Ziccardi and Cooper (3) also
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REFERENCES 1. Lepow, I.H., Naff, G.B., Pensky, J., & Hinz, C.F. (1963) /. Exp. Med. 117, 983-1008 2. Takahashi, K., Nagasawa, S., & Koyama, J. (1975) FEBS Lett. 65, 20-23 3. Ziccardi, R.J. & Cooper, N.R. (1976) /. Immunol. 116, 496-503 4. Naff, G.B. & Ratnoff, O.D. (1968) /, Exp. Med. 128, 571-593 5. de Bracco, M.M. & Stroud, R.M. (1971) /. Clin. Invest. 50, 838-848 6. Valet, G. & Cooper, N.R. (1974) /. Immunol. 112, 1667-1673 7. Takahashi, K., Nagasawa, S., & Koyama, J. (1975) FEBS Lett. 55, 156-160 8. Linscott, W.D. (1968) lmmunochemistry 5, 311-314 9. Ishizaki, E., Mori, Y., & Koyama, J. (1976) / . Biochem. 80, 1423-1427 10. Sakai, K. & Stroud, R.M. (1973) /. Immunol. 110, 1010-1020 11. Assimeh, S.N., Bing, D.H., & Painter, R.H. (1974) /. Immunol. 113, 224-234 12. Volanakis, J.E. & Stroud, R.M. (1972) /. Immunol. Methods 2, 25-34 13. Reisfeld, R.A., Lewis, U.J., & Williams, D.E. (1962) Nature 195, 281-283 14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 15. Mori, Y., Kawai, N., & Koyama, J. (1973) / . Biochem. 73, 951-958 16. Gigli, 1., Porter, R.R., & Sim, R.B. (1976) Biochem. J. 157, 541-548 17. Reid, K.B.M., Lowe, D.M., & Porter, R.R. (1972) Biochem. J. 130, 749-763 18. Ishizaki, E., Mori, Y., & Koyama, J. (1977) / . Biochem. 82, 1155-1160 19. Ratnoff, O.D. (1969) / . Exp. Med. 129, 315-331 20. Laurell, A.B. & Martensson, U. (1974) Acta Path. Microbiol. Scand. B82, 585-589 21. Barkas, T., Scott, K., & Forthergill, J.E. (1973) Biochem. Soc. Trans. 1, 1219-1220 22. Nagasawa, S., Takahashi, K., & Koyama, J. (1974) FEBS Lett. 41, 280-282 23. Sim, R.B. & Porter, R.R. (1976) Biochem. Soc. Trans. 4, 127-129 24. Porter, R.R. (1977) Biochem. Soc. Trans. 5, 16601677
J. Biochem.
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observed that human Clr functioned not only as an activator for Cls, but also as a physical linkage between Clq and Cls in human Cl. The results obtained in the present study showed that rabbit Clr also serves as a physical linkage between Clq and Cls in a similar way. In a previous paper, we reported that an active Cls fragment with a molecular weight of 72,000 was isolated during the purification of rabbit Cls. Although this fragment was indistinguishable from Cls with regard to proteolytic activities tested, it differed from Cls in having an H chain of shorter length, which was almost equal to that of the L chain with a molecular weight of 37,000. The finding that the Cls fragment loses the ability to form Cl complex with Clq and Clr serves to identify the location of the site which is involved in the binding of Cls to Clr. This site of Cls may be located in the H chain, specially, in the portion which is deleted by action of a protease in rabbit serum (probably plasmin), and which does not contribute appreciably to the structural integrity of the active site. On the other hand, the active site of rabbit Cls may be in the L chain, as in the case of the human counterpart (21). The molecular mechanism of formation of the Cl complex seems to be complex. In the case of human Cl, Clr and Cls are thought to form a tight complex containing two molecules of each, giving a molecular weight of about 350,000, and possibly also an octomeric complex of double this size (22-24). Interaction of this complex with Clq is suggested by Porter to be weak relative to that of Clr to Cls (24). At present, we have no further information on the molecular mechanism of formation of rabbit Clr-Cls complex or on the location of the sites of Clr which are involved in the reconstitution of Cl complex. Further studies will be required to elucidate these problems.
Y. MORI, M. KOKETSU, N. ABE, and J. KOYAMA
