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Three dimensional analysis of the membrane attack complex of complement recombined with phospholipid vesicles
ABSTRACTS
1395
BINDING OF CCBINDING PROTEIN BY AGGREGATED SERUM AMYLOID P COMPONENT M.B. Pepys*, F.C. de Beer, Marilyn L. Baltz, Sarah Ho/ford and A. Feinstein”. Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS and ‘ARC Institute of Animal Physiology, Babraham, Cambridge. C4-binding protein (C4bp) is an important regulatory protein of the classical complement pathway and it circulates, in part, in a complex with protein S, a vitamin K dependent protein of unknown function. We report here that isolated, pure serum amyloid P component (SAP), which had been aggregated either by direct conjugation to CNBr-Sepharose or by complexing with anti-SAP antibodies immobilized on Sepharose, selectively took up C4bp, protein S and the quite unrelated protein, fibronectin (Fn), from whole normal humand serum. The reaction was calcium-dependent and C4bp and Fn were bound independently of and in competition with each other. SAP in its native state in the serum was not complexed with either Fn or C4bp and there is no evidence that it affects complement activation or regulation in-vitro. The interaction with C4bp clearly depended on aggregation of SAP molecules and experiments with immobilized SAP-anti-SAP complexes suggested that close association of pairs of SAP molecules was required. The present findings extend the range of inter-relationships known to exist between complement and other protein systems and they may have biological relevance in that SAP, as well as being a normal plasma protein and a constituent of amyloid deposits, is also a normal tissue protein. It is present in the glomerular basement membrane and on the peripheral microfibrillar mantle of elastic fibres throughout the body and in these situations the molecular arrangement may enable it to express its reactivity for C4bp and Fn.
C5b-8 MEDIATED AND SPONTANEOUS POLYMERIZATION OF C9: FORMATION OF C9 POLYMERS WITH ULTRASTRUCTURAL RESEMBLANCE OF THE MEMBRANE ATTACK COMPLEX (MAC). E.R. Podack, J. Tschopp and H.J. Miiller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California 92037. Incubation of purified C9 at 0.4 mglml, physiological ionic strength and pH for 64 hr at 37” results in the spontaneous formation of soluble C9 polymers (poly C9). The ultrastructure of poly C9 resembles that of the MAC. Poly C9 appears as 16 nm long tubules with an internal diameter of 11 nm. One end of the poly C9 tubule consists of a 3 nm thick torus which has an inner and outer diameter of 11 nm and 21 nm, respectively. The other end of the tubule constitutes a-4 nm long hydrophobic domain as suggested by the manner in which poly C9 tubules aggregate. Poly C9 tubules are resistant to dissociation by SDS and migrate with an apparent molecular weight of lo6 upon SDS-PAGE suggesting a subunit composition of 12-16 protomers. Spontaneous C9 polymerization is highly temperature dependent. The following observations indicate that one of the functions of C5b-8 is to polymerize C9 within the forming MAC. a) The molar ratio of C9 to C8 in the reversible fluid phase C8-C9 complex is one. b) The interaction of C9 with C5b-8 at 0” is reversible and characterized by an association constant of 3x107M-‘. c) At 37” the association constant increases to-lO1’M-l. d) The molar ratio of C9 to C8 in the cell-bound C5b-9 complex varies with the cell surface density of C5b-8. C5b-8 monomers formed by binding of 3,000 C8 molecules to 25,000 C5b-7 complexes per rabbit erythrocyte bind 12 C9 molecules. In contrast when 25,000 C5b-8 are bound per cell 6 C9 molecules are bound per C8 molecule. Typical ultrastructural membrane lesions are observed at both C9/C8 ratios suggesting that the morphology of a lesion is primarily due to the C9 dodecamer regardless of whether it is bound to one or two C5b-8. (Supported by American Heart Association El no. 79-149 and USPHS Grants Al 17354, CA 27489 and HL 16411).
THREE DIMENSIONAL ANALYSIS OF THE MEMBRANE ATTACK COMPLEX OF COMPLEMENT RECOMBINED WITH PHOSPHOLIPID VESICLES. E.R. Podack and H.J. MUller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California. H. Horst and W. Hoppe. Max Planck Institute for Biochemistry, Martinsried, F.R. Germany. The three dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes have been analyzed by imaging of negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer aided three dimensional reconstruction. The information on electron micrographs obtained at 5” angular increments from +60” to -60” was digitized by densitometric scanning, Fourier transformed, corrected for imaging errors, cross correlated and synthesized to the three dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16 nm height, 2.0 nm wall thickness and a 3.0 nm torus at the free end having an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane binding site and only small pores of up to 2.0 nm diameter pentrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of-4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest that the MAC may mediate increased membrane permability by protein channel formation in addition to lipid reorientation. (Supported by Amer. Heart Assoc. El No. 79-149 and USPHS Grants Al 17354, CA27489 and HL 16411.)
1395
BINDING OF CCBINDING PROTEIN BY AGGREGATED SERUM AMYLOID P COMPONENT M.B. Pepys*, F.C. de Beer, Marilyn L. Baltz, Sarah Ho/ford and A. Feinstein”. Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS and ‘ARC Institute of Animal Physiology, Babraham, Cambridge. C4-binding protein (C4bp) is an important regulatory protein of the classical complement pathway and it circulates, in part, in a complex with protein S, a vitamin K dependent protein of unknown function. We report here that isolated, pure serum amyloid P component (SAP), which had been aggregated either by direct conjugation to CNBr-Sepharose or by complexing with anti-SAP antibodies immobilized on Sepharose, selectively took up C4bp, protein S and the quite unrelated protein, fibronectin (Fn), from whole normal humand serum. The reaction was calcium-dependent and C4bp and Fn were bound independently of and in competition with each other. SAP in its native state in the serum was not complexed with either Fn or C4bp and there is no evidence that it affects complement activation or regulation in-vitro. The interaction with C4bp clearly depended on aggregation of SAP molecules and experiments with immobilized SAP-anti-SAP complexes suggested that close association of pairs of SAP molecules was required. The present findings extend the range of inter-relationships known to exist between complement and other protein systems and they may have biological relevance in that SAP, as well as being a normal plasma protein and a constituent of amyloid deposits, is also a normal tissue protein. It is present in the glomerular basement membrane and on the peripheral microfibrillar mantle of elastic fibres throughout the body and in these situations the molecular arrangement may enable it to express its reactivity for C4bp and Fn.
C5b-8 MEDIATED AND SPONTANEOUS POLYMERIZATION OF C9: FORMATION OF C9 POLYMERS WITH ULTRASTRUCTURAL RESEMBLANCE OF THE MEMBRANE ATTACK COMPLEX (MAC). E.R. Podack, J. Tschopp and H.J. Miiller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California 92037. Incubation of purified C9 at 0.4 mglml, physiological ionic strength and pH for 64 hr at 37” results in the spontaneous formation of soluble C9 polymers (poly C9). The ultrastructure of poly C9 resembles that of the MAC. Poly C9 appears as 16 nm long tubules with an internal diameter of 11 nm. One end of the poly C9 tubule consists of a 3 nm thick torus which has an inner and outer diameter of 11 nm and 21 nm, respectively. The other end of the tubule constitutes a-4 nm long hydrophobic domain as suggested by the manner in which poly C9 tubules aggregate. Poly C9 tubules are resistant to dissociation by SDS and migrate with an apparent molecular weight of lo6 upon SDS-PAGE suggesting a subunit composition of 12-16 protomers. Spontaneous C9 polymerization is highly temperature dependent. The following observations indicate that one of the functions of C5b-8 is to polymerize C9 within the forming MAC. a) The molar ratio of C9 to C8 in the reversible fluid phase C8-C9 complex is one. b) The interaction of C9 with C5b-8 at 0” is reversible and characterized by an association constant of 3x107M-‘. c) At 37” the association constant increases to-lO1’M-l. d) The molar ratio of C9 to C8 in the cell-bound C5b-9 complex varies with the cell surface density of C5b-8. C5b-8 monomers formed by binding of 3,000 C8 molecules to 25,000 C5b-7 complexes per rabbit erythrocyte bind 12 C9 molecules. In contrast when 25,000 C5b-8 are bound per cell 6 C9 molecules are bound per C8 molecule. Typical ultrastructural membrane lesions are observed at both C9/C8 ratios suggesting that the morphology of a lesion is primarily due to the C9 dodecamer regardless of whether it is bound to one or two C5b-8. (Supported by American Heart Association El no. 79-149 and USPHS Grants Al 17354, CA 27489 and HL 16411).
THREE DIMENSIONAL ANALYSIS OF THE MEMBRANE ATTACK COMPLEX OF COMPLEMENT RECOMBINED WITH PHOSPHOLIPID VESICLES. E.R. Podack and H.J. MUller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California. H. Horst and W. Hoppe. Max Planck Institute for Biochemistry, Martinsried, F.R. Germany. The three dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes have been analyzed by imaging of negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer aided three dimensional reconstruction. The information on electron micrographs obtained at 5” angular increments from +60” to -60” was digitized by densitometric scanning, Fourier transformed, corrected for imaging errors, cross correlated and synthesized to the three dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16 nm height, 2.0 nm wall thickness and a 3.0 nm torus at the free end having an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane binding site and only small pores of up to 2.0 nm diameter pentrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of-4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest that the MAC may mediate increased membrane permability by protein channel formation in addition to lipid reorientation. (Supported by Amer. Heart Assoc. El No. 79-149 and USPHS Grants Al 17354, CA27489 and HL 16411.)