Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway

In this study the physiological role of properdin and the differential subunit composition of the solid phase enzymes of the pathway have been explored. Cell-bound C3 and C5 convertase differ in their C3b requirement. Apparently one molecule of C3b is sufficient to allow formation of C3 convertase (C3b,B), whereas two or more are required for generation of C5 convertase (C3bn,B). This conclusion was drawn from results indicating the critical role of the spacial distribution of C3b molecules on the cell surface in enzyme formation. While the C3/C5 convertase is fully capable of acting on C5 and thereby initiating the assembly of the cytolytic membrane attack complex, it is exceedingly labile and vulnerable to destruction by the C3b inactivator. It is the apparent role of properdin to confer a degree of stability upon the labile enzyme and to protect its C3 convertase function against enzymatic destruction. To achieve these effects, precursor properdin (pre-P) is recruited in a binding-activation reaction by the labile C3/C5 convertase. Multiple C3b molecules appear to be needed for the formation of properdin-activating principle. Three modes of regulation have been described, which involve spontaneous dissociation enzymatic degradation by C3b inactivator and disassembly by beta1H. The functional differences of pre-P and activated properdin (P) were delineated, pre-P displaying a weak affinity for C3b and P the capacity of strong interaction, P generating a soluble C3 convertase in serum and pre-P being unable to do so. Because of the profound differences between native pre-P and the laboratory product P, the question was raised as to whether soluble P represents an unphysiological form of the protein. On the basis of this and other studies, the conclusion was reached that in vitro properdin recruitment constitutes the terminal event of the properdin pathway, and that properdin augments the function of C3/C5 convertase without changing its substrate specificity.     

Cardiolipin synthase from mammalian mitochondria

Factor I deficiency causes a permanent, uncontrolled activation of the alternative pathway resulting in an increased turnover of C3 and consumption of factor B, factor H and properdin. Factor I deficiency is clinically associated with recurrent bacterial infections already in early infancy, mainly affecting the upper and lower respiratory tract, or presenting as meningitis or septicemia. We here report on a Brazilian family (n = 9) with known consanguinity, where in 3/7 children, suffering from chronic otitis, meningitis, and respiratory infections, a complete factor I deficiency was recognized. One of the patients died after fulminant sepsis. Hemolytic activity of the alternative pathway was not detectable in the patients' sera due to decreased plasma concentrations of C3, factor B and properdin. As a consequence of factor I deficiency, C3b could not be metabolized with the result that no C3-derived split products (C3dg/C3d) were detectable in the patients' sera. In vitro reconstitution with purified factor I restored the regulatory function in the patients' sera with the subsequent cleavage of C3b to C3c and C3dg. Factor H levels were decreased in all patients' sera and found to be tightly complexed with C3b resulting in a modified electrophoretic mobility. Upon factor I reconstitution, factor H was released from C3b regaining its beta 1 electrophoretic mobility. Complement-mediated biological functions like opsonization of bacteria, chemotactic activity and phagocytosis in these patients were impaired. The parents (cousins, 2nd degree) and 3/4 siblings had significantly reduced factor I plasma levels without further alteration in their complement profile. 3 of these obviously heterozygously deficient family members suffered from recurrent bacterial infections of different frequency and severity.     

The chromosomal order of genes controlling the major histocompatibility complex, properdin factor B, and deficiency of the second component of complement

The relationship of the genes coding for HLA to those coding for properdin Factor B allotypes and for deficiency of the second component of complement (C2) was studied in families of patients with connective tissue disorders. Patients were selected because they were heterozygous or homozygous for C2 deficiency. 12 families with 15 matings informative for C2 deficiency were found. Of 57 informative meioses, two crossovers were noted between the C2 deficiency gene and the HLA-B gene, with a recombinant fraction of 0.035. A lod score of 13 was calculated for linkage between C2 deficiency and HLA-B at a maximum likelihood value of the recombinant fraction of 0.04. 18 families with 21 informative matings for both properdin Factor B allotype and HLA-B were found. Of 72 informative meioses, three recombinants were found, giving a recombinant fraction of 0.042. A lod score of 16 between HLA-B and Factor B allotypes was calculated at a maximum likelihood value of the recombinant fraction of 0.04. A crossover was shown to have occurred between genes for Factor B and HLA-D, in which HLA-D segregared with HLA-A and B. These studies suggest that the genes for Factor B and C2 deficiency are located outside those for HLA, that the order of genese is HLA-A, -B, -D, Factor B allotype, C2 deficiency, that the genes coding for C2 deficiency and Factor B allotypes are approximately 3--5 centimorgans from the HLA-A and HLA-B loci, and that the apparent lack of recombinants between the Factor B gene and C2 deficiency gene suggests that these two genes lie in close proximity to one another.     

Dependence of mutation induction on fast-neutron energy in a human epithelial teratocarcinoma cell line (P3)

Cultivation of Candida albicans NIH B-792 (serotype B) at high temperature (37 degrees C) for 48 h in yeast extract-containing Sabouraud liquid medium (YSLM) provided the following findings in comparison with the findings obtained after incubation at 27 degrees C. Growth of the blastoconidia of this strain was decreased, with a dry weight of 9%, and the cells were deficient in cytokinesis. The cells did not undergo agglutination with serum factor 5 from a commercially available serum factor kit (Candida Check). Mannan (B-37-M) obtained from the cells cultured at 37 degrees C had partially lost its reactivity against serum factor 4 and lost most of its reactivity against serum factor 5 in an enzyme-linked immunosorbent assay (ELISA) in contrast to that (B-27-M) at 27 degrees C. Both cells and mannan prepared by cultivation first at 37 degrees C and then at 27 degrees C entirely recovered their reactivities with serum factors 4 and 5. 1H-nuclear magnetic resonance analysis also revealed that B-37-M had lost a beta-1,2-linked mannopyranose unit and retained a phosphate group. Similar changes were observed in the three other serotype B strains used in the study. The beta-1,2-linked mannooligosaccharides longer than mannotetraose were not included among the products released from B-37-M by mild acid treatment. The results of the inhibition ELISA with a series of beta-1,2-linked mannooligosaccharides from biose to octaose (M2 to M8, respectively) showed that the reactivity against serum factor 4 was inhibited most strongly by the oligosaccharides M4 to M8 and that the reactivity against serum factor 5 was inhibited completely by relatively longer oligosaccharides, M5 to M8, indicating their participation as the antigenic factor 5 epitopes.     

Evidence for the presence of components of the alternative (properdin) pathway of complement activation in respiratory secretions

Activation of the alternative complement pathway by respiratory secretory IgA was demonstrated by incubating purified, aggregated preparations of serum and secretory IgA with neat human serum. No depletion of the early components (C1-4) was observed, but 63 and 70% of C3-9, respectively, were consumed. The C3-9-consuming capacity of heat-aggregated nasal secretions from an IgA-deficient volunteer was compared with heat-aggregated nasal secretions from a normal volunteer known to have secretory IgA. The deficient secretions consumed C3-9, whereas the IgA deficient secretions did not. Reconstitution of the nasal secretions from the IgA-deficient volunteer with purified secretory IgA produced alternative pathway activation. Factor B of the alternative complement pathway was found to be present in 16 of 18 bronchoalveolar lavage samples (BALF) from normal volunteers. Simultaneous measurement of lavage and serum albumin and Factor B concentrations rendered it unlikely that Factor B was merely a transudative product from serum in half the samples but rather suggested that it may be a component of lower respiratory tract secretions. The presence of an intact alternative complement pathway in BALF was indicated by showing that cobra venom factor and endotoxin cleaved functionally pure human C3 when mixed with BALF, but had no effect on C3 in the absence of BALF.     

Membranoproliferative glomerulonephritis with disruption of the glomerular basement membrane

Seven patients with a form of membranoproliferative glomerulonephritis distinct in its glomerular ultrastructure from other forms are described. The use of silver impregnated electron micrographs revealed contiguous subepithelial and subendothelial deposits associated with basement membrane disruption, replication and layering of lamina densalike material. By light and fluorescence microscopy the appearance was distinctive but not diagnostic. Immunohistology consistently showed abundant C3 and properdin in a granular pattern while immunoglobulins and Clq were variably present. Low serum C3 concentrations were observed at some time in each patient, often accompanied by low levels of properdin, whereas the concentrations of Clq and C4 were normal. The patients were indistinguishable in their clinical course from those with other types of MPGN.     

The rabbit properdin system: I. Identification of a new factor and purification of rabbit properdin

Pure preparations of rabbit properdin were obtained from rabbit serum by ion-exchange chromatography. These preparations functioned as properdin when they were measured with the zymosan assay or with a serum reagent selectively depleted of properdin by a specific immunoabsorbent. Properdin in these preparations was in its activated state. A new serum factor was required to measure properdin activity when purified preparations of rabbit properdin were tested with the zymosan assay. This factor was designated as ZBP, or zymosan-binding protein. ZBP appeared to be distinct from known components of the alternative complement pathway and the classical complement system, and it did not appear to be an immunoglobulin.     

Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold

The effect of exposure to low temperatures (5 degrees C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 degrees C compared to that of animals kept at 22 degrees C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 degrees C for the same length of time. Mean serum complement activity after 1 month at 5 degrees C was significantly reduced in comparison to animals held at 22 degrees C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 degrees C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 degrees C) in frogs returned to 22 degrees C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 degrees C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 degrees C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 degrees C failed to cause death or observable symptoms of disease in frogs that were returned to 22 degrees C.     

Formation in the presence of C3 nephritic factor (C3NeF) of an alternative pathway C3 convertase containing uncleaved B

C3 nephritic factor (C3NeF) interacts with native C3 and B in the absence of D to generate a C3 convertase containing an uncleaved form of B. Dose response studies with C3NeF and B, respectively, revealed incremental C3 inactivation without loss of B. These findings are in agreement with the previous isolation from such reaction mixtures of a 10S complex containing haemolytically inactive C3 and active B and manifesting C3 convertase activity. Functional contamination of C3 with C3b was negated by demonstrating that pretreatment of C3 with C3bINA had no effect on its subsequent interaction with B and C3NeF to generate C3 convertase activity, while pretreatment of C3b eliminated its effective interaction with B and C3NeF. Relatively higher concentrations of C3bINA present during interaction of C3, B and C3NeF suppressed C3 inactivation, indicating its dependence on amplification by utilization of the initial C3b generated. Trace quantities of D were not found by functional analyses of C3, B and C3NeF and pretreatment of these proteins with a concentration of DFP sufficient to suppress D activity had no effect on their effective interaction. The introduction of D to mixtures of C3NeF, B, and C3 resulted in B clevage and more efficient expression of C3 convertase function as defined by a reduced requirement for C3NeF.     

Complement-fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection

Hamster to rat cardiac xenografts undergo delayed rejection as compared with the hyperacute rejection of discordant xenografts. Elicited xenoreactive Abs (EXA) are thought to initiate hamster to rat cardiac xenograft rejection. In this study, we demonstrate that following transplantation of a hamster heart, rats generated high levels of EXA. Adoptive transfer into naive recipients of purified IgM, IgG2b, or IgG2c, but not IgG1 or IgG2a EXA, induced xenograft rejection in a complement-dependent manner. Ability of EXA to cause rejection correlated with complement activation, platelet aggregation, and P-selectin expression in the xenograft endothelium. Cyclosporin A (CyA) administration, after transplantation, totally suppressed IgG1, IgG2a, IgG2b, and IgG2c EXA, and inhibited IgM EXA production, but failed to overcome rejection. Administration of cobra venom factor (CVF), 1 day before and at the time of transplantation, resulted in complement inhibition during 3 days after transplantation, which failed to overcome rejection. Combination of CyA and CVF, which we have previously shown to overcome rejection, resulted in suppression of IgG EXA production and in the return of IgM XNA to preimmunization serum levels, 3 to 7 days after xenotransplantation, while complement remained inhibited. Thus, under CyA/CVF treatment, complement activation by hamster cells was suppressed following xenotransplantation, and presumably for this reason xenograft rejection did not occur. In conclusion, our data demonstrate that EXA play a pivotal role in the pathogenesis of xenograft rejection and that CyA and CVF suppress xenograft rejection by preventing exposure of xenograft endothelial cells to complement activation by EXA.     

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.     

Effect of chilling on the survival of Bacteroides fragilis

Factors affecting the susceptibility of Bacteroides fragilis subsp. fragilis to low temperature were examined. Predetermined numbers of cells were spread on agar media or suspended in enriched Trypticase soy broth and exposed to low temperature under both aerobic and anaerobic conditions. Exposure of 18-h growth of a freshly isolated B. fragilis strain to 4 degrees C aerobically or anaerobically resulted in a loss of at least 50% viability after 12 h. B. fragilis cells in early growth (6 h) were more tolerant to exposure at 4 degrees C than older cells (18 h). When the freshly isolated strain was repeatedly subcultured in the laboratory it was uniformly more cold tolerant than fresh clinical isolates. The incorporation of 1.0 M sucrose and 5 mM magnesium chloride into liquid media partially alleviated the lethal effects of cold temperature on B. fragilis subsp. fragilis.     

Complement activation of electrogenic ion transport in isolated rat colon

The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.     

Enhancement of cyclophosphamide cytotoxicity in vivo by the benzamide analogue pyrazinamide

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.     

Cutting at the right place--the importance of selective limited proteolysis in the activation of proproteinase E

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin-like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X-ray crystallography for the intact native form, with a 11-residue N-terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387-4394]. Also for a N-terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla-Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763-1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N-terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.     

Classical complement pathway activation on nucleated cells. Role of factor H in the control of deposited C3b

The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major physiologic function of the complement regulatory protein factor H. In this study, we provide evidence that factor H is also a restriction factor of classical pathway activation on the surface of nucleated cells. We found that C3b was rapidly converted to inactivated C3b (iC3b) on human SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglioside surface Ag GD3. The SK-MEL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleaving protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MCP (CD46) molecules/cell. FACS analysis and direct quantitation using [125I]factor H revealed high level binding of factor H to the melanoma cells (5.6 x 10(6) molecules/cell) during classical pathway activation. The binding of factor H could be inhibited under conditions that inactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classical pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclonal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increased amount of C3b on these cells correlated with a 2.65-fold greater rate of cell death. In contrast, the increase in cell death effected by neutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Collectively, these data provide experimental evidence that factor H, through its cofactor activity for C3b degradation, is involved in the restriction of the classical pathway of complement on the surface of nucleated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.     

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

A conserved element in the serine protease domain of complement factor B

Factor B and C2 are serine proteases that carry the catalytic sites of the complement C3 and C5 convertases. Their protease domains are activated by conformational changes that occur during convertase assembly and are deactivated upon convertase dissociation. Factor B and C2 share an 8-amino acid conserved sequence near their serine protease termini that is not seen in other serine proteases. To determine its importance, 24 factor B mutants were generated, each with a single amino acid substitution in this region. Whereas most mutants were functionally neutral, all five different substitutions of aspartic acid 715 and one phenylalanine 716 substitution severely reduced hemolytic activity. Several aspartic acid 715 mutants permitted the steps of convertase assembly including C3b-dependent factor D-mediated cleavage and activation of the high affinity C3b-binding site, but the resulting complexes did not cleave C3. Given that factor B and C2 share the same biological substrates and that part of the trypsin-like substrate specificity region is not apparent in either protein, we propose that the conserved region plays a critical role in the conformational regulation of the catalytic site and could offer a highly specific target for the therapeutic inhibition of complement.     

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica)

This paper presents the results of viscosity determinations on aqueous solutions of bovine serum albumin (BSA) at a wide range of concentrations and at temperatures ranging from 5 degrees C to 45 degrees C. On the basis of these measurements a general formula connecting the relative viscosity eta r with temperature T and concentration c of the dissolved proteins was established: [formula: see text] The quantities alpha, beta, B, D and delta E are described in the text below. A simple substantiation of the formula was also given. This relation gives immediately the Mooney approximation and allows the prediction of the values of the parameter S and a self-crowding factor K in this approximation. By applying an asymptotic form of the formula such rheological quantities as the intrinsic viscosity and Huggins coefficient were calculated.