Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.     

Antibody-independent C1 activation by E. coli

Antibody-independent interactions of C1 with several E. coli strains were examined. Purified C1 was directly activated by the semi-rough mutant E. coli J-5, its parental wild-type strain, E. coli 0111:B4, and two clinical isolates, E. coli (P) and E. coli (A), in the absence of C1 inhibitor. E. coli J-5 activated C1 about 10-fold more rapidly and bound approximately threefold more C1 than the other strains. E. coli J-5, but not the other strains, also bound C1s2, provided that the subcomponent was offered to the bacteria in the presence of C1q and calcium; such binding was thus independent of the presence or absence of C1r2. After C1 activation in the absence of C1 inhibitor, activated C1s spontaneously dissociated from E. coli 0111:B4, (P), and (A), but remained associated with E. coli J-5. The regulatory protein C1 inhibitor prevented C1 activation by the weaker activators, E. coli strains 0111:B4, (P), and (A), but had no effect on C1 activation by E. coli J-5. Although C1 inhibitor thus failed to modulate C1 activation by E. coli J-5, it did block the enzymatic activity of activated C1 bound to this strain. Analyses of the molecular processes involved revealed differences with other systems. In the presence of C1 inhibitor, the C1s subunit of C1 activated by E. coli J-5 underwent further cleavage with the release into the supernatant of C1s fragments and complexes of C1 inhibitor with light chain fragments. Such fragments were not disulfide-linked to the remainder of the C1s molecule. The bulk of the heavy chain remained adherent to the surface of E. coli J-5. This finding documents the presence of a binding site for activated C1s on the surface of E. coli J-5 and localizes this site to the heavy chain. These studies thus indicate that several E. coli strains are direct C1 activators. Furthermore, E. coli J-5 provides another example of a direct C1 activator having binding sites not only for C1q but also for dimeric C1s. The studies also show that there are multiple properties of particles which determine the ability to activate C1, the rate of activation, the possibility of regulation of the activation process by C1 inhibitor, and the fate of activated C1.     

Antibody-independent activation of C1, the first component of complement, by cardiolipin

Lipid vesicles containing phospholipids known to be present in substantial amounts in mitochondrial membranes were tested for their capacity to activate C1. Among them, only cardiolipin (CL) was highly efficient in C1 activation; no such effect was observed with phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. CL was shown to bind specifically C1q, because only unlabeled C1q competed with 125I-C1q for binding to CL. The requirement for C1q was confirmed by the finding that only fully reconstituted macromolecular C1, containing C1q, was activated by CL. The specificity of CL-induced activation of C1 was also demonstrated by introducing adriamycin, an agent known to interact with CL. Whereas adriamycin did not decrease C1 activation induced by immune complexes, it abrogated C1 activation by CL. The latter was shown to be a strong nonimmune activator of C1, because C1-INH did not inhibit CL-induced activation. When the concentration of CL in vesicles was decreased in the presence of phosphatidylcholine, C1 activation was detected only above a critical level of 35 mol% CL, compatible with a minimal density or clustering of CL molecules in the plane of the membrane. Moreover, C1 activation by CL was modulated by the addition of cholesterol. The threshold of CL required for C1 activation was lowered by the incorporation of more than 35 mol% cholesterol into the vesicles. These results show that CL incorporated into liposomes can be a potent nonimmune activator of C1. The negatively charged phosphate groups in CL are likely candidates for Clq-binding.     

Activation of C1r by proteolytic cleavage.

C1r was unable to cleave and activate proenzyme C1s unless first incubated at 37 degrees C in the absence of calcium before the addition of C1s. The acquisition of ability to activate C1s was associated with, and paralleled by, cleavage of each of the two noncovalently bonded 95,000 dalton chains of the molecule into disulfide linked subunits of 60,000 and 35,000 daltons, respectively. Thus, C1r is converted from an inactive form into an enzyme, C1r, able to cleave and activate C1s by proteolytic cleavage in marked analogy to the activation of several other complement enzymes. Trypsin was also found to cleave C1r but at a different site, and its action did not lead to C1r activation. C1r activation was inhibited by calcium, polyanethol sulfonate, C1 inactivator, and DFP but not by a battery of other protease inhibitors. C1 inactivator inhibited C1r by forming a complex with C1r via sites located on the light chain of the molecule. In other studies, cleavage of C1r was not accelerated by the addition of C1r ot C1s. C1r and C1r were found to have the same m.w., sedimentation coefficient, and diffusion coefficients. They differed, however, in charge with C1r migrating as a Beta-globulin and C1r as a gammaglobulin on electrophoresis in agarose. The amino acid composition of C1r and of each of the two polypeptide chains of Clr was determined. Both chains contained carbohydrate. Proteolytic cleavage of the C1r molecule was found to occur on addition of aggregated IgG to a mixture of C1q, C1r, and C1s in the presence of calcium. Neither C1q, C1s nor aggregated IgG alone, not C1r nor C1s induced C1r cleavage. Liquoid, an inhibitor of C1 activation, inhibited C1r cleavage. Thus, proteolytic cleavage of C1r appears to be a biologically meaningful event occurring during the activation of C1.     

(Na+ + K+)-adenosine triphosphatase of mammalian brain. Catalytic and regulatory K+ sites distinguishable by selectivity for Li+.

Li+, K+, and Rb+ are compared as activators of the hydrolysis of p-nitrophenylphosphate by beef brain (Na+ + K+)-ATPase. Previous experiments have established two classes of K+ binding sites that are involved in this reaction: "catalytic sites" have the higher affinity, their occupation is essential for catalytic activity, and they appear to correspond to the extracellular binding sites for active K+ transport; regulatory sites appear to have an allosteric function to "unmask" the catalytic sites. A separate set of Na+-binding regulatory sites bring about a similar unmasking of catalytic sites under phosphorylating conditions. Rb+ can activate p-nitrophenylphosphate hydrolysis both in the presence and absence of Na+ and, thus, can interact effectively with both K+ regulatory and catalytic sites. Li+ does not activate p-nitrophenylphosphate hydrolysis at 25 degrees C in the absence of other monovalent ligands. Li+ does activate when the catalytic sites are exposed by Na+ + ATP. Thus, K+ regulatory and catalytic sites differ in their cation selectivity. At temperatures less than 25 degrees C Li+ is able to activate the phosphatase reaction in the absence of other monovalent ligands: maximum activity occurs at 10-12 degrees C. A plot of the ratio, Li+ activation/K+ activation, as a function of temperature shows that the allosteric transition that unmasks catalytic sites occurs spontaneously with decreasing temperatures.     

Structures of activated fructose-1,6-bisphosphatase from Escherichia coli. Coordinate regulation of bacterial metabolism and the conservation of the R-state

The enteric bacterium Escherichia coli requires fructose-1,6-bisphosphatase (FBPase) for growth on gluconeogenic carbon sources. Constitutive expression of FBPase and fructose-6-phosphate-1-kinase coupled with the absence of futile cycling implies an undetermined mechanism of coordinate regulation involving both enzymes. Tricarboxylic acids and phosphorylated three-carbon carboxylic acids, all intermediates of glycolysis and the tricarboxylic acid cycle, are shown here to activate E. coli FBPase. The two most potent activators, phosphoenolpyruvate and citrate, bind to the sulfate anion site, revealed previously in the first crystal structure of the E. coli enzyme. Tetramers ligated with either phosphoenolpyruvate or citrate, in contrast to the sulfate-bound structure, are in the canonical R-state of porcine FBPase but nevertheless retain sterically blocked AMP pockets. At physiologically relevant concentrations, phosphoenolpyruvate and citrate stabilize an active tetramer over a less active enzyme form of mass comparable with that of a dimer. The above implies the conservation of the R-state through evolution. FBPases of heterotrophic organisms of distantly related phylogenetic groups retain residues of the allosteric activator site and in those instances where data are available exhibit activation by phosphoenolpyruvate. Findings here unify disparate observations regarding bacterial FBPases, implicating a mechanism of feed-forward activation in bacterial central metabolism.     

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.     

The structural basis for neutrophil inactivation of C1 inhibitor.

Limited proteolysis of C1 inhibitor (C1-INH) by neutrophil elastase, Pseudomonas elastase and snake venoms resulted in initial cleavage within the molecule's N-terminus followed by further cleavage within the molecule's C-terminally placed reactive centre. N-Terminal proteolysis occurred at peptide bonds 14-15, 36-37 and 40-41. This had no effect on either the inhibitory activity or the heat-stability of C1-INH. Proteolysis within the reactive centre occurred at peptide bonds 439-440, 440-441, 441-442 and 442-443. Cleavage at any one of these sites inactivated C1-INH and conferred enhanced heat-stability upon a previously heat-labile molecule. Released neutrophil proteinases also cleaved and inactivated C1-INH, suggesting that they may physiologically regulate C1-INH during inflammatory episodes.     

Study of the effect of high molecular weight kininogen upon the fluid-phase inactivation of kallikrein by C1 inhibitor

Plasma kallikrein and factor XIa circulate bound to high molecular weight kininogen, and such binding has been reported to protect these enzymes from inactivation by their respective inhibitors. However, this observation is controversial, and the effect of high molecular weight kininogen upon the interaction between kallikrein and C1 inhibitor (C1-INH) has been questioned. We have re-evaluated this reaction and studied the rate of inhibition of kallikrein by C1-INH in the presence and absence of high molecular weight kininogen. The second-order rate constant of inhibition of kallikrein by C1-INH was unaffected by saturating concentrations of high molecular weight kininogen. Our results suggest that although high molecular weight kininogen clearly augments the rate of formation of kallikrein and other enzymes of the contact activation pathway, it has no effect on the rate of enzyme inhibition by C1-INH.     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

Activation of human C1: analysis with Western blotting reveals slow self-activation

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.     

StcE,a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.     

Characterization of the StcE protease activity of Escherichia coli O157:H7

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.     

Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli

The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L-quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor.     

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance

Background

Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states.

Scope of research

We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout.

Major conclusions

Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation.

General significance

This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies.     

Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli.

Using as substrates a series of chimeric proteins containing various fragments of the hepatitis C virus precursor polyprotein between Escherichia coli maltose binding protein and dihydrofolate reductase, we analyzed the substrate requirements of hepatitis C viral serine proteinase (Cpro-2) for intermolecular polypeptide cleavage in E. coli. Cpro-2-dependent substrate cleavage was observed in E. coli cells simultaneously transformed with expression plasmids for the Cpro-2 molecule and substrate protein. The cleavage sites were estimated by determining the amino (N)-terminal amino acid sequences of dihydrofolate reductase-fused processed products purified partially by affinity chromatography from the lysates, indicating that cleavage occurred at sites identical to those observed in eukaryotic cells. Mutation analysis using the chimeric substrate indicated that the presence of cysteine and small uncharged residues at positions P1 and P1', respectively, of the putative cleavage site is necessary for cleavage and that acidic residues in the region upstream of the cleavage site are required for efficient cleavage.     

Escherichia coli E-39 ADPglucose synthetase has different activation kinetics from the wild-type allosteric enzyme

Kinetic and binding studies have shown that Lys39 of Escherichia coli ADPglucose synthetase is involved in binding of the allosteric activator. In order to study structure-function relationships at the activator binding site, this lysine residue was substituted by glutamic acid (Lys39----Glu) by site-directed mutagenesis. The resultant mutant enzyme (E-39) showed activation kinetics different from those of the wild-type enzyme. The level of activation of the E-39 enzyme by the major activators of E. coli ADPglucose synthetase, 2-phosphoglycerate, pyridoxal phosphate, and fructose-1,6-phosphatase was only approximately 2-fold compared to activation of 15- to 28-fold respectively, for the wild-type enzyme. NADPH, an activator of the wild-type enzyme, was unable to activate the mutant enzyme. In addition, the concentrations of the above activators necessary to obtain 50% of the maximal stimulation of enzyme activity (A0.5) were 5-, 9-, and 23-fold higher, respectively, than those for the wild-type enzyme. The E-39 enzyme also had a lower apparent affinity (S0.5) for the substrates ATP and MgCl2 than the wild-type enzyme and the values obtained in the presence or absence of activator were similar. The concentration of inhibitor giving 50% of enzyme activity (I0.5) was also similar for the E-39 enzyme in the presence or absence of activator. These results indicate that the E-39 mutant enzyme is not effectively activated by the major activators of the E. coli ADPglucose synthetase wild-type enzyme, and that this amino acid substitution also prevents the allosteric effect that the activator has on the wild-type enzyme kinetics, either increasing its apparent affinity for the substrates or modulating the enzyme's sensitivity to inhibition.     

Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes

We have studied the turnover of the third component of C (C3) and capture of the major cleavage fragment of C3 produced during C activation (C3b) that occurs when soluble antibody/DNA immune complexes (IC) active C. We used the Amersham RIA kit for the minor cleavage fragment of C3 produced during C activation (C3a), and a new assay utilizing mAb to C3b to measure the fraction of active C3 in a C source after the IC activate C. These mAb, along with a mAb to human IgG, allowed us to measure IC stoichiometries. The efficiency of C3 turnover by the IC is quite high, and under conditions of Ab excess, the maximum number of IgG bound per dsDNA corresponds to 1 IgG/20 to 30 base pairs. The maximum number of C3b found in the IC corresponds to less than 1 C3b/IgG, and the vast majority of the captured C3b is bound to the IgG, and not to the DNA. We identified several IC that consumed large amounts of C3, and captured large amounts of C3b, but did not bind to human E via C3b receptors (C receptor type 1). This finding suggests that the ability of IC to bind to human E depends upon the number and distribution of captured C3b molecules and the conformation and size of the DNA Ag, which reflects the need for multivalent binding between several properly arrayed C3b and a "cluster" of C receptor type 1 on the human E membrane. IC that activate C3 but do not bind to E would presumably "escape" the E IC clearance mechanism, but could deposit in susceptible organs and tissues and play a role in the pathogenesis of SLE because of their potential to generate the inflammatory products of C activation.     

In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site

The pyruvate oxidase of Escherichia coli is a peripheral membrane flavoprotein that is dramatically activated by lipids. The enzyme strongly binds to phospholipid vesicles in vitro. In vivo, in addition to enzyme activation, binding is thought to be important to provide access of the enzyme to ubiquinone dissolved in the lipid bilayer. It was unclear if both or either of these attributes is needed for enzyme function in vivo. To differentiate between activation and lipid binding, we have constructed, using recombinant DNA techniques, a mutant gene that produces a truncated protein. The truncated protein lacks the last 24 amino acids of the C-terminus of the oxidase (due to introduction of a translation termination codon) and thus is closely analogous to the activated species produced in vitro by limited chymotrypsin cleavage [Recny, M.A., Grabau, C., Cronan, J.E., Jr., & Hager, L.P. (1985) J. Biol. Chem. 260, 14287-14291]. The truncated protein (like the protease-derived species) is fully active in vitro in the absence of lipid, and its activity is not further increased by addition of lipid activators. Moreover, the truncated enzyme fails to bind Triton X-114, a detergent that binds to and activates the wild-type oxidase. Strains producing the truncated protein were devoid of oxidase activity in vivo. This result indicates that binding to membrane lipids is specifically required for function of the oxidase in vivo; activation alone does not suffice.     

Formation and Ultrastructure of Extra Membranes in Escherichia coli

A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).