Limulus Polyphemus Hemocyanin Research Articles

Allosteric control in Limulus polyphemus hemocyanin: functional relevance of interactions between hexamers.

Hemocyanin of the horseshoe crab Limulus polyphemus is composed of 48 oxygen-binding subunits, which are arranged in eight hexameric building blocks. Allosteric interactions in this oligomeric protein have been examined by measurement of high-precision oxygen-equilibrium curves, using an automated Imai cell. Several models were compared in numerical analysis of the data. A number of conclusions can be drawn with confidence. (1) Oxygen binding by Limulus hemocyanin cannot satisfactorily be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118] for allosteric transitions with either the hexamer or dodecamer as the allosteric unit. (2) Of the models tested, the data sets can be best described by an extended MWC model that allows for an equilibrium, within the 48-subunit ensemble, between cooperative hexamers and cooperative dodecamers. The model invokes T and R states for both hexamers (T6 and R6) and dodecamers (T12 and R12). Allosteric effectors modulate oxygen affinity and cooperativity by affecting the R to T equilibria within hexamers and dodecamers and by shifting the equilibria between hexamers and dodecamers. (3) The fitted model parameters show that under most conditions the intersubunit contacts within T-state hexamers are more constrained than those within T-state dodecamers. (4) The oxygen affinities of the hexameric and dodecameric R states are the same, but under all conditions examined the conformation of the fully oxygenated molecule is that of the dodecameric R state. (5) Between pH 7.4 and pH 8.5 the dodecameric T state has a higher affinity for oxygen than the hexameric T state, allowing for "T-state cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)

A Synthetic Peptide-directed Antibody as a Probe of the Phosphorylation Site of Pyruvate Dehydrogenase

A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies. A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes. The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex. The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies.

Dimerization of native Limulus polyphemus hemocyanin in the presence of calcium ions and sucrose

In the presence of both sucrose and millimolar concentrations of calcium, native Limulus polyphemus hemocyanin, having a sedimentation coefficient of ~ 60S, forms a dimer with a sedimentation coefficient of ~ 90S. Dimer formation was observed by sucrose gradient ultracentrifugation and gel filtration. It occurs at pH 7.0 but not at pH 8.5 or 6.0, and it is dependent on the calcium concentration. Treatment with EDTA to remove calcium reverses the dimerization process. The effect of sucrose may be due to its known preferential exclusion from the vicinity of proteins [15]. Such preferential exclusion would favor the formation of dimers if the excluded volume of the protein were to decrease on dimerization. Formation of dimer is accompanied by the binding of calcium to hemocyanin, as determined by analysis of gradient fractions by atomic absorption spectroscopy. In addition, stoichiometric amounts of europium (III), known to occupy the calcium binding sites of hemocyanin, also promotes dimer formation. Dimer formation is not induced by 100 mM sodium chloride. The observation of a 90S form of Limulus hemocyanin indicates that there is nothing inherent in the structure of the 60S molecule that prevents further aggregation.

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin.

The reaction of Limulus polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained one binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was about 10 times as high as that for the deoxy form. Conversely, the dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration, whereas the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in two steps. The first bimolecular step is characterized by an increase in the apparent pKa of the bound dye, while the second unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was found to be ahead of the increase in the average degree of O2 saturation. It was concluded that the structural changes in hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence.

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

Immune complexes can trigger specific, T cell-dependent, autoanti-IgG antibody production in mice.

Immunization of mice with a combination of passively administered syngeneic IgG (anti-p-azophenylarsonate [anti-Ars]) antibody and a soluble, multivalent form of the antibody's corresponding antigen (Limulus polyphemus hemocyanin conjugated with Ars [Lph-Ars]) resulted in specific autoanti-IgG Fc (rheumatoid factor) production. The response was rapid and only anti-IgG of the IgM isotype is found. Because immunization with either the IgG antibody or the antigen alone did not result in rheumatoid antibody production, immune complexes appear to be the active form of the immunogens. Antibody/antigen ratios that resulted in maximal anti-IgG antibody responses were the same as those required for peak in vitro immunoprecipitation, i.e., equivalence. Previous exposure of the mice to the exogenously supplied antigen was not required for the response. The response to immune complexes is specific because mice immunized with IgG2a-containing complexes produced autoanti-IgG2a, while mice immunized with IgG1-containing complexes produced anti-IgG1 with little reactivity to other IgG isotypes. IgG2a blocked in its complement-fixing capacity was more effective in eliciting the anti-IgG2a response than native IgG2a, suggesting a possible role for the complement system in modulating the anti-IgG2a response. Induction of rheumatoid factor production by immune complexes could be induced in xid mice but not in nu/nu mice, indicating T lymphocyte dependence of the response. In contrast, the B lymphocyte activator lipopolysaccharide was able to elicit vigorous rheumatoid factor production in both nu/nu and normal mice, demonstrating that nu/nu mice contain B cells capable of making the response. Rheumatoid antibody produced in the immune complex- or LPS-induced responses is Fc specific and has relatively low affinity for IgG that is not bound to antigen.

Self-association and oxygen-binding characteristics of the isolated subunits of Limulus polyphemus hemocyanin

The hemocyanin of the horseshoe crab Limulus polyphemus is characteristic of arthropod hemocyanins in that it is a high-molecular-weight oligomer composed of functionally and structurally distinct subunits. The protein forms a 48-subunit complex, the largest form of arthropod hemocyanin, whose oxygen-binding characteristics are modulated by subunit interaction within the oligomer. It has previously been shown that a number of electrophoretic isozymes, which are identical immunochemically, are present in dissociated Limulus hemocyanin. In this study it is demonstrated that the electrophoretic differences in the antigenically identical subunits are not reflected in their oxygen-binding and self-assembly properties or in the roles they play in reassembly and function of the 48-subunit native molecule. The chloride-dependent modulation of the oxygen-binding properties of those Limulus subunits which do not self-assemble, as documented here, illustrates that this allosteric effect may be operable at the tertiary level. For each of the purified subunits the effects of pH and calcium ions on oxygen-binding characteristics and self-assembly reactions are reported, and the roles of specific subunits in reassembly of distinct aggregation states are further documented.

The role of multivariate image analysis in solving the architecture of the limulus polyphemus hemocyanin molecule

Multivariate analysis facilitates the study of the structure of complex biomolecular assemblies of which Limulus polyphemus hemocyanin is an example. Both the analysis of classes of molecule views and the investigation of continuous structure-related variations have provided new information on the architecture of hemocyanin.

Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: a model system for analysis of structure-function relationships in the absence of subunit heterogeneity

Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of immunolabels by electron microscopy and image averaging

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

Architecture of Limulus polyphemus hemocyanin.

The architecture of the 48-meric hemocyanin of the horseshoe crab Limulus polyphemus has been determined from electron micrographs of whole (48-mer) molecules and half- (24-mer) molecules. The assembly of hexamers of kidney-shaped subunits can produce two dodecameric enantiomorphs, designated as right and left. The assembly of 24-mers can again result in two enantiomorphs. By taking into account the rocking effect described by Van Heel and Frank [Van Heel, M., & Frank, J. (1981) Ultramicroscopy 6, 187-194], we deduced that the 24-meric half-molecule is made up of two copies of the left dodecameric enantiomorph. In addition, the two constituent dodecamers of the half-molecule are shifted with respect to a symmetric head-to-tail arrangement, which makes it possible to distinguish two different faces of the 24-mer, termed flip and flop. A model of the whole molecule was built from two copies of the 24-meric half-molecule. This model presents the four distinct views observed in the electron microscope (pentagon, ring, cross, and bowtie). In addition, the model shows the pentagonal view to exist in two varieties: symmetric and asymmetric. An analysis of electron micrographs presenting the pentagonal view by image processing using the statistical technique of correspondence analysis confirmed the existence of two types of pentagonal view, representing projections of a molecule built from two copies of the left 24-meric enantiomorph. In addition, the best fit between the averaged molecule images and the possible models was observed with a flop-flop inter 24-mer contact. The final model is shown in a series of stereo views produced by computer graphical techniques.

Comparison of 4 × 6-meric hemocyanins from three different arthropods using computer alignment and correspondence analysis

We have compared 4 × 6-meric hemocyanin molecules from Androctonus australis and Eurypelma californicum with the similar 4 × 6-meric half-molecules of Limulus polyphemus hemocyanin. This comparison was performed using multivariate statistical analysis applied to computer-aligned electron microscopical images of the molecules: a method that was recently proposed by van Heel & Frank (1980,1981). Our study shows that the molecules are very similar indeed, and that the evidence for a tetrameric arrangement of the hexamers found in 4 × 6-meric Limulus molecules (van Heel & Frank, 1980,1981) also holds for Androctonus and Eurypelma hemocyanin. The model proposed for this molecule by Lamy et al. (1981) is therefore based on correct assumptions. In addition, our study shows that correspondence analysis, as a method of multivariate statistical analysis, is capable of detecting subtle differences between similar structures as well as differences in preparative conditions, effects that were hitherto not accessible to quantitative analysis.

Effect of uv laser radiation on copper-proteins

The results of irradiation studies of human ceruloplasmin, mushroom tyrosinase and Limulus polyphemus hemocyanin with a nitrogen laser (Λ = 337.1 nm) are presented. In all the proteins we have considered the absorption band at 330 nm, characteristic of dimer Type 3 copper centres, decreases, while an increase of the absorbance at 280 nm is observed. Moreover a new band at about 400 nm appears in irradiated ceruloplasmin. Finally sharp oscillations of the absorbance at 610 and 794 nm are recorded in ceruloplasmin, when the irradiation is switched off.

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10 6-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen ( J. Comp. Physiol. Ser. B 130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie ( Arch. Int. Physiol. Biochem. 87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. ( Arch. Biochem. Biophys. 196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.

Catabolism of hemocyanin in the American lobster, Homarus americanus

1. 1. Cross-reactivity studies by double immunodiffusion suggest no immunological similarity between Homarus americanus hemocyanin and Panulirus argus hemocyanin. 2. 2. Slow hemolymph clearance rates ( t 1 2 of 15–30 days) for 125I- Homarus americanus and Panulirus argus hemocyanins were determined after injection in both species, suggesting similar structure of the two hemocyanins relative to clearance recognition sites. 3. 3. No single tissue emerged as the site of clearance and degradation of endogenous 125I-labelled hemocyanin in Homarus americanus, possibly due to its slow clearance rate. 4. 4. 125I- Limulus polyphemus hemocyanin was rapidly cleared from the hemolymph of Homarus americanus ( t 1 2 = 45 min ) with the epidermis indicated as the initial site of uptake and degradation.

Dissociation and Reassembly of Limulus polyphemus Hemocyanin

Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.

Subunit heterogeneity in arthropod hemocyanins: I. Chelicerata

1. The hemocyanins of 2 spiders (Eurypelma californicum, Cupiennius salei), 2 scorpions (Pandinus pallidus, Androctonus australis), a whipscorption (Mastigoproctus brasilianus) and 2 whipspiders (Tarantula palmata, Trichodamon froesi) were analyzed for subunit heterogeneity by high resolution polyacrylamide electrophoresis (PAGE). For comparison,Limulus polyphemus hemocyanin was subjected to the same analytical scheme. 2. All of the species, except forLimulus (predominantly 60 S-hemocyanin) andCupiennius (16 S- and 24 S-hemocanin) possess only hemocyanin sedimenting in the 33 S to 37 S range. A second, major blood protein was observed in each species, safeMastigoproctus. This second, non-respiratory protein sediments with ca. 16 S, but with about 24 S in the case of the scorption species. 3. Upon incubation with sodium dodecylsulfate (SDS) and β-mercaptoethanol and electrophoresis in polyacrylamide gradients, between 2 and 5 hemocyanin bands are obtained, with average molecular weights between 70,000 and 75,000. The non-respiratory proteins yield two chains in each case, with molecular weights between 95,000 and 130,000. 4. Each hemocyanin could be dissociated at pH 9.6 to yield “native” subunits. By gel filtration, these were separated into monomers (4.5S) and dimers (6 S). No dimers were observed after dissociation ofPandinus and ofLimulus hemocyanin. Only in the case ofCupiennius the dimer is formed by a disulfide bridge. — PAGE of the dissociation mixture shows complex patterns which display 6–7 bands. 5. By preparative isolation of “native” subunits and subsequent analysis in SDS-PAGE, the two patterns of hemocyanin bands could be related to each other, and the total number of different polypeptide chains established with some certainty. It ranges from 5 inCupiennius 16 S-hemocyanin to 8 inAndroctonus hemocyanin and possibly 12 inLimulus hemocyanin. A loose correlation between oligomer size and number of different chains is suggested.

Subunit association and heterogeneity of Limulus polyphemus hemocyanin.

The molecular weights of the 6S, 24S, 36S, and 60S components of Limulus polyphemus hemocyanin were determined by high speed sedimentation equilibrium to be 69 400, 856 000, 1 690 000, and 3 160 000. The behavior of this hemocyanin appears to be similar to that of other arthropod hemocyanins where the first aggregation step is the formation of a hexamer of the 6S monomer. Here the larger aggregated states (24S, 36S, and 60S) are successive dimers of an unobserved hexamer (16S). The 24S-36S-60S association was found to be heterogeneous, suggesting that 24S components of different composition may be present.

Crystals of a functional 70,000 molecular weight subunit of hemocyanin from Limulus polyphemus

Crystals have been obtained of a subunit of Limulus polyphemus hemocyanin. The blue crystals have the symmetry of the space group R32 with hexagonal lattice parameters a = 115 a ̊ , c = 285 A ̊ . There is one 70,000 molecular weight subunit per asymmetric unit. Each subunit contains two non-heme copper atoms and can reversibly bind one oxygen molecule.

Oxygen binding by Limulus polyphemus hemocyanin: allosteric modulation by chloride ions.

Oxygen binding by Limulus polyphemus hemocyanin: allosteric modulation by chloride ions.