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E R Podack

Publications and source records attributed to E R Podack.

At least 145 records · Page 8Linked to original sources
PubMed1981

Membrane attack complex of complement. Evidence for its dimeric structure based on hybrid formation.

Molecular hybridization experiments provided new evidence for the dimeric nature of the membrane attack complex (MAC) of complement. Monomeric C5b-6, which constitutes the first intermediate complex in MAC formation, was prepared in two differentially labeled forms: biotin-125I-C5b-6 and 131I-C5b-6. Using a mixture of the differentially labeled C5b-6, the MAC was assembled on phospholipid vesicles upon addition of C7, C8, and C9. The assembled MAC containing biotin-125I and 131I was extracted from the vesicles with deoxycholate, purified, and exposed to avidin-Sepharose. Biotin-mediated binding of the MAC to avidin-Sepharose not only effected binding of 125I, but also of 131I, indicating that both radiolabels resided in the same molecular entity. When equimolar amounts of differentially labeled C5b-6 were available for MAC formation, 50% of MAC formed contained one molecule of each form. Theoretical analysis of the experimental data clearly favored the dimer structure over the structure of a higher oligomer. In contrast, fluid phase SC5b-9 was clearly monomeric on the basis of the same analysis. The electron microscopic appearance of the biotinated MAC hybrid closely resembled that of the characteristic membrane lesions of complement lysed cells. An avidin-ferritin conjugate attached itself to the ring-shaped portion of the biotinated MAC and not to its perpendicular structures, suggesting that C5b-6 is an integral part of the ring structure of the MAC.

Biotin↗
PubMed1981

Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.

Membrane destruction by complement is effected by the membrane attack complex (MAC) which is the dimer of a fusion product of the complement proteins C5b, C6, C7, C8, and C9. Phospholipid bilayer vesicles were used as target membranes for the MAC and its intermediate complexes. The subunits of these membrane-bound complexes were explored as to their relative exposure to the hydrocarbon phase of the lipid bilayer and to water surrounding the lipid vesicles. Protein exposed to the aqueous phase was labeled with 125I; protein exposed to the hydrocarbon phase was labeled by using tritiated azido phospholipids and irradiation. Analysis of the membrane-bound MAC showed that subunits C5b, C8 beta, and C9 were exposed to the aqueous phase. The subunits C8 alpha-gamma and C9 were primarily in contact with the hydrocarbon phase. C6 and C7 were little exposed to either phase, suggesting that these proteins are inaccessible within the MAC. Analysis of the intermediate complexes showed that C5b was the subunit most exposed to water in membrane-bound C5b-7, and C5b and C8 beta were the water-exposed subunits in C5b-8. Subunit exposure to the hydrocarbon phase of the lipid bilayer changed during MAC assembly. Whereas all three subunits of C5b-7 carried the phospholipid photolabel; most of the label was bound to the C8 subunit in C5b-8 and to C9 in the MAC. It is proposed that contact with the hydrocarbon core of membranes is established by C5b-7 through each of its subunits, by C5b-8 through C8, and by the MAC through C8 and, particularly, C9.

Affinity Labels↗
PubMed1981

The role of C9 in complement-mediated killing of Neisseria.

During the routine examination of a healthy 31-yr-old woman, we found an incomplete deficiency of the 9th component of complement (C9). By hemolytic assay her serum C9 activity was 10 to 15% of normal. Limited family studies suggested that she inherited the deficiency as an autosomal codominant trait. She had no history of unusual or severe infections. When tested for bactericidal activity against serum-sensitive Neisseria gonorrhoeae and N. meningitidis, her serum reacted comparably to normal serum. Normal serum depleted immunochemically of C9 and sera from congenitally C9-deficient patients were also bactericidal against serum-sensitive Neisseria but required 120 min to kill the same numbers of gonococci that intact serum killed within 30 min. In the electron microscope, N. gonorrhoeae incubated with C9-depleted serum were fragmented but lacked the typical C lesions. Therefore, serum lacking C9 can kill serum-sensitive Neisseria, unlike sera deficient in the other terminal C components.

Adult↗
PubMed1981

Suppression of the anti-erythrocyte immune response in mice by the C5b--9 complex of complement.

The C5b--9 complex of complement associated with sheep erythrocyte membranes suppresses the immune response of mice to sheep erythrocytes as measured with the Jerne plaque technique. This type of immune suppression is independent of early complement components and antibody, and is mediated by both human and guinea-pig complement components. The degree of immune suppression correlates with the number of C5b--9 complexes per cell used for immunization: 21,000 C5b--9 complexes per erythrocyte lead to a 97% inhibition of the immune response in comparison to untreated erythrocytes. Inhibition requires the full assembly of the C5b--9 complex including C8 and C9. Virtually no inhibition was observed by the C5b--7 complex. C5b--9 and IgG-mediated immune suppression do not function additively. From dose-response experiments it is concluded that separate and mutually independent sites mediate suppression by C5b--9 and IgG, respectively.

Animals↗
PubMed1981

The membrane attack mechanism of complement: photolabeling reveals insertion of terminal proteins into target membrane.

We have utilized a membrane-restricted, photoactivable glycolipid probe to investigate the protein-lipid interactions involved in complement (C) mediated lysis of a target membrane. The purified C proteins C5b-6, C7, C8, and C9 were added to artificial membrane vesicles containing the 14C-labeled photoreactive probe anchored in the outer monolayer of the membrane, and 6-carboxyfluorescein trapped in the lumen as an indicator for effective lysis. Irradiation of the membrane samples at different stages of functional complex assembly resulted in labeling of each of the 5 terminal C proteins, indicating that all 5 proteins become inserted into the hydrophobic milieu of the membrane during some stage of complex assembly. However, at the final stage of complex assembly, only C9 appeared to be labeled. Because we can demonstrate that the photoreactive probe has no strong affinity for C9 over the other terminal components (C5b-C8), the extensive change in labeling specificity during assembly is evidence for substantial changes in protein-lipid and possibly protein-protein interactions during formation of the C lesion.

Cell Membrane↗
PubMed1980

Membrane attack complex of complement: a structural analysis of its assembly.

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement through structural analysis. Four intermediate complexes and the MAC were examined by electron microscopy and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 x 60 x 60 A. At protein concentrattions greater than 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting a tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of four half-rings, each measuring 200 x 50 A, which are connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-A-long and 30-A-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of approximately 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces the formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles with DOC has a sedimentation rate of 33S, which is characteristic of the C5b-9 dimer. It is concluded that dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200- x 50-A half-ring which is attached to the DOL-vesicle by a 100- x 80-A appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. It is proposed that protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are events that facilitate dimerization of C5b-9 and thus MAC formation.

Binding Sites↗
PubMed1980

The interaction of C5 with C3b in free solution: a sufficient condition for cleavage by a fluid phase C3/C5 convertase.

We have measured the interactions of C3b with C5 in free solution under conditions that favor detecting weak binding interactions (high C3b and low C5 concentrations and low ionic strength). When a mixture of 125I-C5 (2 X 10(-8) M) and unlabeled C3b (3.8 x 10(-5) M) was ultracentrifuged in a sucrose gradient, virtually all of the C5 sedimented to the position of a 13 to 14S complex. In contrast, a sedimentation rate for C5 of 9S was obtained in the absence of C3b. The ability to bind C5 was observed to be a property of C3b since native C3 was unable to bind C5. It was also found that beta 1H by itself could inhibit the binding of C5 to cell-bound C3b. From inhibition studies, we estimate that the association constant for the C3b-C5 interaction is on the order of 2 x 10(6) M-1 in a low ionic strength buffer (mu = 0.06) and 5-fold weaker at physiologic ionic strength. C5 bound to C3b in free solution was cleaved by nephritic factor-stabilized fluid phase C3bB. C5 activation did not occur on omitting C3b. We conclude that the ability to bind C5 is a property of C3b molecules whether surface bound or in free solution and that when C5 is bound in either fashion, it can be cleaved by a fluid phase C3/C5 convertase.

Animals↗
PubMed1979

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

Blood Bactericidal Activity↗
PubMed1979

C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

Animals↗
PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗
PubMed1979

Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

Binding Sites↗
PubMed1979

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.

Cholesterol↗
PubMed1979

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.

Chromatography, Affinity↗
PubMed1979

Structural similarities between C6 and C7 of human complement.

A new method for the isolation of C6 and C7 by affinity chromatography of human serum with anti-C6 and anti-C7 coupled to Sepharose is described. C6 and C7 prepared by this method are hemolytically fully active, homogeneous proteins obtained in 25% yield. A comparison of the properties of isolated C6 and C7 gave the following results: The amino acid composition of the two proteins is very similar. The m.w. calculated from the amino acid content is 124,800 for C6 and 120,800 for C7. Both components are single chain glycoproteins migrating upon electrophoresis at pH 8.6 as beta 2-globulins, Both proteins are polymorphic as detected by isoelectrofocusing in polyacrylamide gels and range in their isoelectric points from pH 6.15 to 6.7. The UV spectra reveal only minor differences; the extinction coefficients are: EC6 = 1.71 cm2 X mg-1 and EC7 = 1.92 cm2 X mg-1. CD-spectra show 8% alpha-helix and 10% beta-structure for C6 and 10% alpha-helix and 14% beta-structure for C7. The structural similarities of C6 and C7 suggest their evolution from a common ancestral gene.

Chemical Phenomena↗