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Ultrastructural studies of the human complement components C3 and factor B and of cobra venom factor (CVF)
ABSTRACTS
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ULTRASTRUCTURAL STUDIES OF THE HUMAN COMPLEMENT COMPONENTS C3 AND FACTOR B AND OF COBRA VENOM FACTOR (CVF). CA. Smith*, M.K. Pangburn, C.-W. Vogel and H.J. MiillerEberhard. Research institute of Scripps Clinic, La Jolla, California 92037. Elucidation of the morphology of alternative pathway components was begun with high resolution transmission electron microscopy studies of C3, C3b, C3c, B, Bb, CVF and CVF, Bb. Negatively stained human C3 and C3b appear very similar in structure, both exhibiting a distorted droplet morphology. By several distinct aspects the macromolecule appears to consist of a large and small domain separated by a shallow groove perpendicular to the long axis of the molecule. The large domain, which contains approximately 75% of the mass of the molecule, is oval shaped and often displays substructure, while the small domain has a tail-like appearance and is usually visualized as displaced from the long axis. Monoclonal antibody specific for the C3c fragment attached to the large domain of C3. C3c images with an oval morphology similar to the large domain of C3, lacking a pronounced tail region. CVF bears a striking resemblance to C3c and to the large domain of C3 and C3b. C3b dimers were produced from C3b monomers by chemically cross-linking the free sulfhydrl groups in the d-domains (metastable binding sites). The dimer appeared as a complex of two rod-like monomers separated by slight stain penetration with little suggestion of a tail-like domain. These results suggest that C3c, not C3 or C3b, is the vertebrate analog of CVF and that the tail structure of C3b contains the metastable binding site. Factor B, Bb and the stable C3 convertase CVF, Bb were also examined. Factor B appears quite globular, Bb about 70% as large and somewhat elongated. CVF and Bb could be distinguished in mircrographs from CVF, Bb which allowed construction of a tentative molecular model of this C3 convertase, and, by analogy, of the naturally occurring C3 convertase, C3b, Bb.
ISOLATION AND CHARACTERIZATION OF A BIOLOGICALLY ACTIVE C2 DERIVED OLIGOPEPTIDE. M. Lopez-Trascasa, M. Moisy, E. Pirotzky, Y. Blouquit, C. Blanc and A.T. Sobe/*, lnserm U139, U 200, U91, Hopital Henri-Mondor, 94010, Creteil, France. C2 was purified to homogeneity by successive ion exchange and affinity (C4bSepharose) chromatographies. Upon SDS-PAGE, under reducing conditions, native hemolytically active C2 had a single polypeptic chain (105,000 daltons) which was cleaved by purified Cls into the known C2a and C2b (72,000 and 33,000 daltons respectively). Further limited proteolysis with trypsin (1% w/w, 1 min.) released a peptide with an estimated m.w. of 1,400 daltons. This fragment increased vascular permeability of guinea pig and rat skin, provoked contraction of rat uterus and dose-dependent degranulation of rat peritoneal mast cells. The molar concentrations capable of inducing a biological response ranged between 2x10-‘and Ix~O-~M. Guinea pig ileum was unresponsive under similar conditions. The vascular response was quantitated for pharmacological modulation. It was not prevented by prior treatment with Hl-type antihistaminics but was considerably altered in the rat skin by inhibitors of serotonin (response: 5% of controls), of kinins (17% of controls) and of mast cells degranulation (0 to 10% of controls). In contrast, diethyldithiocarbamate, a kininase inhibitor, markedly enhanced the skin vascular effect. Carboxypeptidase B did not affect the reactions. When C2a and C2b were separated upon PAGE and independently recovered, the biologic activity was found to be derived from C2b. This was confirmed with fragments obtained by separation on a C4bSepharose column. Aminoacid analysis of purified preparations of the oligopeptide revealed 8 different amino acids (without arginine), representing 12 residues. The data suggested that this C2 derived pept de, structurally and functionally different from the classical anaphylatoxins, may be identical to the “kinin-like” factor previously described in and partly purified from the plasma of patients with hereditary angioneurotic edema. (Supported by grants of Iserm, Dgrst and Paris XII university.)
STUDIES OF THE INTERACTION OF C8 AND C9 WITHIN THE MEMBRANE-BOUND CYTOLYTIC COMPLEX OF COMPLEMENT. J.B. Monahan, B.E. Welbaum and J.M. Sodetz’, Department of Chemistry and School of Medicine, University of South Carolina, Columbia, South Carolina. The proximity of C8 to C9 in the C5b-9 complex of human complement was examined using an intrinsic photosensitive cross-linking reagent located in either the (a-~) or (,9)subunit of C8. Incorporation of a photosensitive functional group into each subunit was accomplished by reacting purified (0-r) or (p) with N-succinimidyl-6(4’-azido2’nitrophenylamino) hexanoate. Both subunits were found to incorporate 5-7 moles of photolabile 6(4’-azido-2’nitrophenylamino) hexanoate (ANH) per mole of subunit. When tested in recombination experiments, both ANH-(a-‘~) and ANH-(8) exhibited 80-90% ability to reassociate with unmodified(p) and e-y), respectively. Human C8 containing a single modified subunit was prepared in this manner and, regardless of the subunit modified, the resulting ANHC8 possessed hymolytic activity comparable to that of normal C8. To examine the proximity of C8 subunits to C9 in C5b-9, EACl-7 cells were incubated separately with each modified form of ANHC8, washed and exposed to saturating amounts of purified 1251-C9.Controls were prepared similarly but contained ‘?-ANHC8 and unlabeled C9. After lysis, membranes were irradiated, solubilized in sodium dodecylsulfate and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of the radiolabeled component to a higher molecular weight. While controls showed that both forms of Y-ANH-C8 cross-link to some membrane and/or C5b-9 constituents, results from corresponding experiments using ANH-C8 and Y-C9 indicated that cross-linking between C8 subunits and C9 was negligible. These results suggest that most or ail of the C9 found in C5b-9 may not be in close proximity to C8. (Supported by American Cancer Society Grant IM-282.) !,.IM.M.19 I I-11
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ULTRASTRUCTURAL STUDIES OF THE HUMAN COMPLEMENT COMPONENTS C3 AND FACTOR B AND OF COBRA VENOM FACTOR (CVF). CA. Smith*, M.K. Pangburn, C.-W. Vogel and H.J. MiillerEberhard. Research institute of Scripps Clinic, La Jolla, California 92037. Elucidation of the morphology of alternative pathway components was begun with high resolution transmission electron microscopy studies of C3, C3b, C3c, B, Bb, CVF and CVF, Bb. Negatively stained human C3 and C3b appear very similar in structure, both exhibiting a distorted droplet morphology. By several distinct aspects the macromolecule appears to consist of a large and small domain separated by a shallow groove perpendicular to the long axis of the molecule. The large domain, which contains approximately 75% of the mass of the molecule, is oval shaped and often displays substructure, while the small domain has a tail-like appearance and is usually visualized as displaced from the long axis. Monoclonal antibody specific for the C3c fragment attached to the large domain of C3. C3c images with an oval morphology similar to the large domain of C3, lacking a pronounced tail region. CVF bears a striking resemblance to C3c and to the large domain of C3 and C3b. C3b dimers were produced from C3b monomers by chemically cross-linking the free sulfhydrl groups in the d-domains (metastable binding sites). The dimer appeared as a complex of two rod-like monomers separated by slight stain penetration with little suggestion of a tail-like domain. These results suggest that C3c, not C3 or C3b, is the vertebrate analog of CVF and that the tail structure of C3b contains the metastable binding site. Factor B, Bb and the stable C3 convertase CVF, Bb were also examined. Factor B appears quite globular, Bb about 70% as large and somewhat elongated. CVF and Bb could be distinguished in mircrographs from CVF, Bb which allowed construction of a tentative molecular model of this C3 convertase, and, by analogy, of the naturally occurring C3 convertase, C3b, Bb.
ISOLATION AND CHARACTERIZATION OF A BIOLOGICALLY ACTIVE C2 DERIVED OLIGOPEPTIDE. M. Lopez-Trascasa, M. Moisy, E. Pirotzky, Y. Blouquit, C. Blanc and A.T. Sobe/*, lnserm U139, U 200, U91, Hopital Henri-Mondor, 94010, Creteil, France. C2 was purified to homogeneity by successive ion exchange and affinity (C4bSepharose) chromatographies. Upon SDS-PAGE, under reducing conditions, native hemolytically active C2 had a single polypeptic chain (105,000 daltons) which was cleaved by purified Cls into the known C2a and C2b (72,000 and 33,000 daltons respectively). Further limited proteolysis with trypsin (1% w/w, 1 min.) released a peptide with an estimated m.w. of 1,400 daltons. This fragment increased vascular permeability of guinea pig and rat skin, provoked contraction of rat uterus and dose-dependent degranulation of rat peritoneal mast cells. The molar concentrations capable of inducing a biological response ranged between 2x10-‘and Ix~O-~M. Guinea pig ileum was unresponsive under similar conditions. The vascular response was quantitated for pharmacological modulation. It was not prevented by prior treatment with Hl-type antihistaminics but was considerably altered in the rat skin by inhibitors of serotonin (response: 5% of controls), of kinins (17% of controls) and of mast cells degranulation (0 to 10% of controls). In contrast, diethyldithiocarbamate, a kininase inhibitor, markedly enhanced the skin vascular effect. Carboxypeptidase B did not affect the reactions. When C2a and C2b were separated upon PAGE and independently recovered, the biologic activity was found to be derived from C2b. This was confirmed with fragments obtained by separation on a C4bSepharose column. Aminoacid analysis of purified preparations of the oligopeptide revealed 8 different amino acids (without arginine), representing 12 residues. The data suggested that this C2 derived pept de, structurally and functionally different from the classical anaphylatoxins, may be identical to the “kinin-like” factor previously described in and partly purified from the plasma of patients with hereditary angioneurotic edema. (Supported by grants of Iserm, Dgrst and Paris XII university.)
STUDIES OF THE INTERACTION OF C8 AND C9 WITHIN THE MEMBRANE-BOUND CYTOLYTIC COMPLEX OF COMPLEMENT. J.B. Monahan, B.E. Welbaum and J.M. Sodetz’, Department of Chemistry and School of Medicine, University of South Carolina, Columbia, South Carolina. The proximity of C8 to C9 in the C5b-9 complex of human complement was examined using an intrinsic photosensitive cross-linking reagent located in either the (a-~) or (,9)subunit of C8. Incorporation of a photosensitive functional group into each subunit was accomplished by reacting purified (0-r) or (p) with N-succinimidyl-6(4’-azido2’nitrophenylamino) hexanoate. Both subunits were found to incorporate 5-7 moles of photolabile 6(4’-azido-2’nitrophenylamino) hexanoate (ANH) per mole of subunit. When tested in recombination experiments, both ANH-(a-‘~) and ANH-(8) exhibited 80-90% ability to reassociate with unmodified(p) and e-y), respectively. Human C8 containing a single modified subunit was prepared in this manner and, regardless of the subunit modified, the resulting ANHC8 possessed hymolytic activity comparable to that of normal C8. To examine the proximity of C8 subunits to C9 in C5b-9, EACl-7 cells were incubated separately with each modified form of ANHC8, washed and exposed to saturating amounts of purified 1251-C9.Controls were prepared similarly but contained ‘?-ANHC8 and unlabeled C9. After lysis, membranes were irradiated, solubilized in sodium dodecylsulfate and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of the radiolabeled component to a higher molecular weight. While controls showed that both forms of Y-ANH-C8 cross-link to some membrane and/or C5b-9 constituents, results from corresponding experiments using ANH-C8 and Y-C9 indicated that cross-linking between C8 subunits and C9 was negligible. These results suggest that most or ail of the C9 found in C5b-9 may not be in close proximity to C8. (Supported by American Cancer Society Grant IM-282.) !,.IM.M.19 I I-11