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J Tschopp

Publications and source records attributed to J Tschopp.

At least 253 records · Page 14Linked to original sources
PubMed1984

Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement.

Poly(C9), the tubular 27 S complex forming the transmembrane channel of the membrane attack complex of complement, was purified to homogeneity by gel filtration and sucrose density gradient ultracentrifugation. The molecular weight of poly(C9) was determined by two independent methods in addition to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. First, sedimentation equilibrium analysis using 0.2% SDS or 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 X 10(6) and a weight average molecular weight of all poly(C9) complexes of 1,050,000 +/- 40,000 (S.D.). SDS and deoxycholate binding to poly(C9) was measured in an air-driven ultracentrifuge and was determined to be 0.53 +/- 0.065 (S.D.) g of SDS and 0.26 +/- 0.015 (S.D.) g of deoxycholate/g of poly(C9), respectively. Second, the mass of 27 S poly(C9) devoid of bound detergent was determined by electron scattering of unstained specimens in the scanning transmission electron microscope. The molecular weight obtained by this method was 1,078,000 +/- 194,000. The inner diameter of poly(C9) tubules imaged in top view projections by negative staining electron microscopy varied between 9 and 12 mm. The accumulated data suggest a true heterogeneity of the molecular weight of poly(C9) due to polymers with varying protomer numbers. Using a mean value of 73,500 for the molecular weight of monomeric C9, the protomer number of poly(C9) tubules appear to vary between 12 and 18 C9 subunits. Approximately 50-75% of the tubules have 14 to 16 subunits as deduced from the mass distribution determined by electron scattering and from ring size measurements. It is suggested that poly(C9) tubules with various protomer numbers may arise due to limited flexibility in the C9-C9 interaction.

Complement C9↗
PubMed1984

Membrane attack by complement.

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.

Animals↗
PubMed1984

Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.

Amino Acid Transport Systems↗
PubMed1983

Two distinct subfractions in isolated Saccharomyces cerevisiae plasma membranes.

The plasma membrane from Saccharomyces cerevisiae X2180-1A and a secretion-blocked mutant, secl (P. Novick and R. Schekman, Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979) has been purified. Cell walls were digested by treatment with lyticase followed by concanavalin A coating of spheroplasts. alpha-Methylmannoside treatment after lysis, sonication at high salt concentration, and fractionation on a Renografin gradient resulted in two highly purified membrane fractions sedimenting at densities of 1.15 and 1.17 g/cm3. Yields determined by recovery of vanadate-sensitive ATPase activity were 11 to 18%, and those determined by recovery of the spheroplast surface label 125I were 17 to 29%. Iodinated cells have most of their label in sedimentable, nonspheroplast material. However, both membrane populations contain some 125I surface label and show ATPase activity with pH optima only at 5.5. The apparent Vmax of the plasma membrane ATPase equals 360 to 560 nmol of ATP hydrolyzed per min per mg of protein, with a Km for ATP of 0.7 mM. ATPase specific activity is not decreased in mutant plasma membrane. Analysis of 125I-labeled plasma membrane proteins by two-dimensional gel electrophoresis revealed seven major proteins on the plasma membrane surface.

Adenosine Triphosphatases↗
PubMed1982

Formation of transmembrane tubules by spontaneous polymerization of the hydrophilic complement protein C9.

The ninth component of complement C9 can undergo circular polymerization in the fluid phase and on lipid membranes. The concomitant hydrophilic-amphiphilic transition is the result of a conformational reorganization of C9 and allows insertion of poly C9 into membranes in the form of a transmembrane protein channel. The ultrastructure of poly C9 resembles that of membrane lesions caused by complement.

Cell Membrane↗
PubMed1982

Kinetics of activation of the first component of complement (C1) by IgG oligomers.

The rate of activation of the first component of complement, Cl, by IgG oligomers was investigated. The kinetics of Clr activation exhibited a pronounced lag phase at low IgG concentration and were followed by a rapid conversion of the proenzyme Cls to Cls. Activation of Cl resulted in a conformational change of this complex. Bases on these results, a model of Cl activation by IgG oligomers is proposed: The efficiency of Cl activation by IgG oligomers is in parallel to their binding affinity to Clq. Clr undergoes a slow rearrangement to a conformation which is not stable and is autoactivated, then Clr rapidly converts Cls to Cls.

Antigen-Antibody Complex↗
PubMed1982

Dissociation of C1 and concentration dependence of its activation kinetics.

The activation of the zymogen C1s to the enzyme C1s in the human C1 complex [C1q(c1rC1s)2] was studied as a function of the concentrations of (C1rC1s)2 and C1q which were saturated with oligomers of rabbit IgG. A large concentration dependence of the sigmoidal kinetics was observed in the 2-180 nM concentration range. This was explained by association-dissociation equilibria between the antibody-saturated C1q and various forms of the (C1rC1s)2 complex (unactivated to activated). The establishment of these equilibria (binding constant 2 x 10(7) M-1) was assumed to be fast as compared to the rates of the activation steps (rate constants 10(-3) and 10(-2) sec-1 at 30 degrees C). The fast re-equilibration of the C1 complex explains the finding that small amounts of antibody-saturated C1q catalysed the activation of large amounts of C1s. The interpretation of the kinetic results was supported by a direct demonstration of the dissociation of C1 into C1q and (C1rC1s)2 by analytical and density gradient centrifugation. No difference was found between the rates of activation and the dissociation properties of reconstituted C1 and C1 isolated from serum.

Centrifugation, Density Gradient↗
PubMed1982

Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement.

The ninth component of complement (C9) has a marked propensity to polymerize. C9 polymers [poly(C9)] formed spontaneously in Veronal-buffered saline upon incubation of purified C9 for 64 hr at 37 degrees C or within 2 hr at 46--56 degrees C. Poly(C9) formed at 37 degrees C was visualized by electron microscopy as a tubular structure with an internal diameter of 110 A and a length of 160 A. Its ultrastructure suggested a dodecameric composition and resembled that of the membrane attack complex of complement. The wider end of the tubular structure was formed by an approximately 30-A-thick torus with inner and outer diameters of 110 A and 220 A, respectively. Because the dimensions of C9 within poly(C9) were 160 x 55 A (maximal) and 20 A (minimal) and because monomeric C9 has dimensions of approximately 80 x 55 A, it is proposed that monomeric C9 unfolds during polymerization into tubules. Polymerization also occurred upon treatment of C9 for 1 hr at 37 degrees C with 0.6 M guanidine . HCl, 0.1 M octyl glucoside, or 1.5% sodium deoxycholate. Guanidine . HCl-induced C9 polymers consisted of elongated highly curved strands 55--80 A wide, suggesting that these polymers were formed by globular C9 that had not unfolded.

Cell Membrane Permeability↗
PubMed1982

Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Complement C9↗
PubMed1981

Occurrence of an incomplete C8 molecule in homozygous C8 deficiency in man.

Sera from unrelated individuals with recurrent Neisserial infections lacked C8 hemolytic activity, but contained a protein that is antigenically related to C8. Immunochemical analysis revealed complete identity of the C8-related protein of all three sera and a marked antigenic deficiency compared with normal C8. The C8-related protein was isolated from serum by adsorption to immobilized anti-C8 IgG, elution with 3 M guanidine, and subsequent gel filtration. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the abnormal protein resembled the alpha-gamma subunit of normal C8 with respect to mobility and its ability to be cleaved upon reduction into the alpha and gamma chains. The beta chain present in normal C8 was absent. Sedimentation equilibrium analysis indicated a molecular weight of 86,000 for the abnormal C8 protein, which is identical to that of the alpha-gamma subunit of normal C8. Amino acid analysis revealed no significant difference between the abnormal C8 and normal alpha-gamma. Unlike normal C8, the abnormal protein did not bind to EAC1-7 or to SC5b-7; however, upon addition to the deficient serum of beta chain isolated from normal C8, hemolytic activity was restored and formation of SC5b-9 occurred. We concluded that the dysfunctional C8 protein in the three individuals' serum is identical to the alpha-gamma subunit of normal C8 and that this form of C8 deficiency is distinct from the C8 deficiencies previously reported in which the entire three-chain protein is lacking.

Animals↗
PubMed1980

Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues.

An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed. It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously. Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues. Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not. N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex. It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate. As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex. The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit. Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding. The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites. Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate.

Binding Sites↗
PubMed1980

Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit.

The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques. Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin. Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction. These findings are consistent with nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism. A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants. In particular, the same value of L0 = [T0]/[R0] = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands. The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar to the high-affinity R state and the nicked apo beta 2 subunit is similar to the low-affinity T state of the apo beta 2 subunit. The slow isonerization involved in the cooperative binding of the ligands to the intact apo beta 2 subunit is discussed in terms of local and concerted conformational changes involving the two autonomously folding domains of the beta protomer.

Apoenzymes↗
PubMed1980

Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies.

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at -70 degrees C.

Animals↗
PubMed1980

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.

Animals↗