Protofibrin clots induced by calcium and zinc

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin-activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration-dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)- or Zn(II)-induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation-sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.     

Albumin indirectly modulates fibrin and protofibrin ultrastructure

Albumin modulation of fibrin and protofibrin coagulation parameters was studied. Cation-depleted, fatty acid free, human and bovine albumins decrease fibrin clot turbidity in a concentration-dependent manner. Albumin also inhibits the formation of protofibrin gels, induced by addition of 25 microM Zn(II) to protofibrils, though it does not bind to (proto)fibrin. In order to verify that competition for cations underlies the influence of albumin, fibrinogen was dialyzed against cation-depleted albumin. Elemental analysis indicates a redistribution of Zn(II) from the fibrinogen to the albumin compartment, and the resultant fibrin clots are less turbid. Apparently, cation-depleted albumin acts as a competitor for divalent cations. The ability of albumin to compete for available Zn(II) was also expressed in gels formed by pH-jump experiments, in which fibrin monomer, maintained soluble at pH 4.9, is induced to change phase by addition of NaOH to pH 7.4. While turbidimetric evidence indicates that individual fibrin fibers simply become thinner with albumin, scanning electron micrographs (SEM) reveal a more complex effect on ultrastructure. Though albumin does not bind to the gels, fibrin gels produced with albumin show major changes in fiber ultrastructure, particularly evident in gels formed in the presence of Zn(II). These structural modifications are discussed within the context of the "excluded volume" effect, in which "crowding" by albumin alters (proto)fibrin reactivity and ultrastructure.     

Zinc binding to fibrinogen and fibrin

Zinc binding to fibrinogen and fibrin was studied by two techniques. Scatchard analysis of ultrafiltration eluates reveals that fibrinogen has multiple Zn(II)-binding sites, KD (fibrinogen) = 18 microM; n = 6. The zinc content of the "collapsed" fibrin gel supernatant was also determined by atomic absorption spectroscopy and analyzed by a Scatchard plot (KD (fibrin) = 8 microM, n = 6). In other experiments, Zn(II) did not displace 45Ca(II) from fibrin. It appears that the binding of zinc to fibrinogen or fibrin is distinct from that of calcium, and that the zinc-binding characteristics of fibrinogen and fibrin are not significantly affected by the transformation of one into the other.     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.

The structural basis of the wide variability of the physical properties of fibrin clots and the process of assembly of the clot were investigated by electron microscopy of fibers formed under various ionic conditions. In addition, highly specific proteolytic enzymes from different snake venoms were used to remove selectively only the A (batroxobin) or the B (venzyme) fibrinopeptides from fibrinogen, in contrast to thrombin, which removes both pairs. Fibers produced by cleavage of only the B fibrinopeptides displayed a characteristic band pattern indistinguishable from that of fibers formed upon removal of either the A fibrinopeptides alone or of both pairs. Computer modeling studies suggest that there is a unique molecular packing that gives rise to this fibrin band pattern. These findings imply that the release of either fibrinopeptide triggers similar modes of aggregation; the intermolecular binding sites can be localized to particular molecular domains. The diameters of fibers formed with each condition of enzyme, pH, salt concentration, and temperature were measured from electron micrographs. All fibers, except for those produced at both high ionic strength and pH, had about the same average diameter of 85 +/- 13 nm. The degree of lateral aggregation of the fibers themselves varied greatly, however; fibers aggregated more readily with cleavage of both pairs of fibrinopeptides and at lower pH and salt concentrations. The formation of such thick fiber bundles increases the stability of the clot and its resistance to proteolytic dissolution.     

Zinc-induced self-assembly of goat brain tubulin: some novel aspects

Unlike normal microtubule assembly, the $\text{in vitro}$ assembly of DEAE-purified goat brain tubulin in presence of Zn(II) is not inhibited by suprastoichiometric concentrations of antimicrotubular drugs like colchicine and podophyllotoxin. However, assembly in the presence of Zn(II) is inhibited by vinblastine. Vinblastine sensitivity of the assembly process depends on the Mg(II) concentration in the assembly medium. Like normal microtubules, Zn(II)-induced polymers are sensitive to cold. The polymers assembled in presence of Zn(II) are readily disassembled on treatment with Zn(II)-chelators like EDTA or o-phenanthroline, indicating that the binding of Zn(II) to tubulin is essential for maintaining the polymeric structure.     

Decorin modulates fibrin assembly and structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and H??k, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.     

Experimental tests of a geometrical abstraction of fibrin polymerization

By combining measurements of the enzymatic release of fibrinopeptide A (FPA) with measurements of intensity and linewidth of Rayleigh scattering from fibrin polymer solutions prior to gelation, we have systematically tested a variety of predictions that can be made on the basis of a simple geometrical abstraction of fibrin polymerization. The experimental investigations include FPA content of fibrin polymers, aggregation of fibrin with fibrinogen, enzyme kinetics, shift of the chemical equilibrium by adding Gly-Pro-Arg-Pro or fibrinogen to the polymer solution, evolution of the polymerization, and influence of fibrinopeptide B release. Among the considered geometrical abstractions there is only one that survives the experimental tests and at the same time is compatible with the electron micrographs by other authors. The main conclusions that can be drawn are (1) the location of binding sites A must be taken from the structure of the fibrinogen molecule proposed by Hoeprich and Doolittle [Biochemistry (1983) 22 , 2049–2055], (2) The fibrinogen monomer is basically centrosymmetric, (3) the state of polymerization is reversible and corresponds to a chemical equilibrium, and (4) Michaelis–Menten enzyme kinetics can be applied.     

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.     

Intracellular Ca2+ and Zn2+ levels regulate the alternative cell density-dependent secretion of S100B in human glioblastoma cells

In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca(2+)- (Zn(2+)- and Cu(2+))-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca(2+) levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), the Ca(2+)-chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn(2+) levels. Treatment of cells with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn(2+)-binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca(2+) levels, and even more so, a decrease in Zn(2+) concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway.     

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.     

Divalent cations and cytochalasin B-sensitive processes in activation,synthesis, and release of mixed leukocyte reaction suppressor factor

We have examined the possible rotes of divalent cations and of cytochalasin B-sensitive processes in the production of mixed leukocyte reaction suppressor factor (MLR-TsF). Ca but not Mg was strictly required for MLR-TsF production. A requirement for Mg could be demonstrated only under conditions of Ca depletion, suggesting an intimate linkage in divalent cation requirements. Ca was required for synthesis as well as release of the suppressor factor. Cytochalasin B inhibited MLR-TsF production only when present during the first 4 hr of culture; such inhibition was reversible with incubations of up to 16 hr. This indicated that cytochalasin B inhibited early events of activation, rather than synthesis or release of MLR-TsF. It was found that both Ca and Mg were required either before or during the cytochalasin-sensitive steps in the activation sequence. The relationship of cytochalasin B-sensitive events in early activation to the antigenic stimulus per se was established by limited exposure of suppressor cells to allogeneic stimulators fixed to poly-l-lysine (PLL)-coated plates. Maximal stimulation was observed with a 4-hr exposure, and this antigen-dependent commitment was cytochalasin B sensitive.     

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.     

Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.     

Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bβ12–46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the αC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calciumbinding sites of fibrin(ogen) and the functional role of Ca²⁺ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.     

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

Remarkable affinity and selectivity for Cs+ and uranyl (UO22+) binding to the manganese site of the apo-water oxidation complex of photosystem II.

The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP). Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding of large alkali cations (Cs+ > Rb+ > K+ > Na+ > Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (>/=5000) for any known chelator. Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ > Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me2+aq --> [MeOH]+aq + H+aq. Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH]+ is the active species that forms upon binding of Mn2+aq to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH]+, a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)2Mn]3+ or apo-WOC-[Mn(mu-OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)2]+). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay prior to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH]+.     

Effect of divalent cations on structure-function relationships of the antitumor protein alpha-sarcin

alpha-Sarcin binds one Zn(II) cation per protein molecule, with a Kd value of 0.9 mM, determined by equilibrium dialysis experiments. Ca(II), Mg(II), and Mn(II) do not bind to alpha-sarcin. Cd(II) and Co(II) also behave as Zn(II). The binding produces local modifications on the protein conformation affecting the microenvironment of tryptophan residues. The three cations modify the fluorescence emission of the protein. The near-u.v. circular dichroism spectrum of the protein is also altered. The binding of Zn(II) and related cations does not modify the secondary structure of the protein. The ribonucleolytic activity of alpha-sarcin is inhibited upon Zn(II) binding, but no alteration of the ability of the protein to aggregate phospholipid vesicles has been observed.     

Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.