Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy

A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

Background

The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia).

Results

The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph.

Conclusions

In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O₂ binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological tolerances of these gill-breathing animals. The observed differential expression of the two subunit types of mega-hemocyanin might allow individual respiratory acclimatization. We hypothesize that mega-hemocyanin is a key character supporting the adaptive radiation and invasive capacity of cerithioid snails.     

Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-Å cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-Å data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.     

Molluscan hemocyanin: structure,evolution, and physiology

Most molluscs have blue blood because their respiratory molecule is hemocyanin, a type-3 copper-binding protein that turns blue upon oxygen binding. Molluscan hemocyanins are huge cylindrical multimeric glycoproteins that are found freely dissolved in the hemolymph. With molecular masses ranging from 3.3 to 13.5 MDa, molluscan hemocyanins are among the largest known proteins. They form decamers or multi-decamers of 330- to 550-kDa subunits comprising more than seven paralogous functional units. Based on the organization of functional domains, they assemble to form decamers, di-decamers, and tri-decamers. Their structure has been investigated using a combination of single particle electron cryo-microsopy of the entire structure and high-resolution X-ray crystallography of the functional unit, although, the one exception is squid hemocyanin for which a crystal structure analysis of the entire molecule has been carried out. In this review, we explain the molecular characteristics of molluscan hemocyanin mainly from the structural viewpoint, in which the structure of the functional unit, architecture of the huge cylindrical multimer, relationship between the composition of the functional unit and entire tertiary structure, and possible functions of the carbohydrates are introduced. We also discuss the evolutionary implications and physiological significance of molluscan hemocyanin.     

Topology of the 10 subunits within the Decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.     

Five monomeric hemocyanin subunits from Portunus trituberculatus: Purification,spectroscopic characterization,and quantitative evaluation of phenol monooxygenase activity

Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [(μ-η²:η²-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5 M borate buffer (pH 9.0) and reacted with 4-methylphenol (p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3 M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo.     

Topology of the 10 subunits within the decamer of KLH,the hemocyanin of the marine gastropod Megathura crenulata

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.     

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1-h) reveals disulfide bridges

Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous "functional units" (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 ? cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a C(α) -trace at 4 ? resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 ?, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids.     

Characterization of a highly efficient heterodimeric xylosidase from Humicola insolens

A heterodimeric xylosidase (E.C. 3.2.1.37) with robust activity is secreted among the plant cell wall degrading enzymes produced by the saprophytic fungus Humicola insolens. The xylosidase has been purified to homogeneity by gel filtration and cation exchange chromatography, and demonstrated to be composed of two protein subunits of 68 and 17 kDa with a molecular mass in solution of approximately 85 kDa based on a combination of SDS-PAGE, size exclusion chromatography and analytical ultracentrifugation. Peptide sequence identities from the subunits indicate the 68 kDa subunit contains a catalytic protein domain and the 17 kDa subunit a carbohydrate binding module. The xylosidase has wide biotechnological potential with maximum activity exhibited at 70 °C and kinetic constants with p-nitrophenol xylopyranoside substrate that suggest it has the highest catalytic efficiency recorded to date (Vmax 22.17 μmoles/min/mg, Km 1.74 mM and Kcat 6787/s).     

The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.     

Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.     

Structural analysis and molecular modeling of the RvH2-e functional unit of Rapana venosa hemocyanin

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH–T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25 °C to 55 °C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0–8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20 °C and 35 °C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25 °C were calculated.     

Induced thermal stress on serotonin levels in the blue swimmer crab,Portunus pelagicus

The temperature of habitat water has a drastic influence on the behavioral, physiological and biochemical mechanisms of crustaceans. Hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. In crustaceans increased circulating crustacean hyperglycemic hormone (CHH) and hyperglycemia are reported to occur following exposure to several environmental stress. The biogenic amine, serotonin has been found to modulate the CHH levels and oxidation of serotonin into its metabolites is catalysed by monoamine oxidase. The blue swimmer crab, Portunus pelagicus is a dominant intertidal species utilized throughout the indo-pacific region and is a particularly important species of Palk bay. It has high nutritional value and delicious taste and hence their requirements of capture and cultivation of this species are constantly increasing. This species experiences varying and increasing temperature levels as it resides in an higher intertidal zone of Thondi coast. The present study examines the effect of thermal stress on the levels of serotonin and crustacean hyperglycemic hormone in the hemolymph of P. pelagicus and analyzes the effect of the monoamine oxidase inhibitor, pargyline on serotonin and CHH level after thermal stress. The results showed increased levels of glucose, CHH and serotonin on exposure to 26 °C in control animals. Pargyline injected crabs showed highly significant increase in the levels of CHH and serotonin on every 2 °C increase or decrease in temperature. A greater CHH level of 268.86±2.87 fmol/ml and a greater serotonin level of 177.69±10.10 ng/ml was observed at 24 °C. This could be due to the effect of in maintaining the level of serotonin in the hemolymph and preventing its oxidation, which in turn induces hyperglycemia by releasing CHH into hemolymph. Thus, the study demonstrates the effect of thermal stress on the hemolymph metabolites studied and the role of pargyline in elevating the levels of serotonin and CHH on thermal stress in the blue swimmer crab, P. pelagicus.     

Keyhole Limpet Hemocyanin Type 2 (KLH2): Detection and Immunolocalization of a Labile Functional Unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Corrélations entre l'évolution morphologique des membres inférieurs et la récupération fonctionnelle de sujets hémiplégiques

ObjectiveThe aim is to quantify the help provided by recent morphological indicators for hemiplegics patients, after a cerebral vascular accident, during functional rehabilitation. The correlations between functional recovery and the morphological evolution of the muscles and the segments of the lower limb are studied using a MRI system.Material and methodTen adult subjects suffering from vascular hemiplegia have been studied at the ELAN rehabilitation center in Wattrelos. Bobath rehabilitation technique was used to treat patients. Muscular and external volumes of the lower limbs segments, obtained through medical imagery, as well as functional tests (Functional Independence Measure (FIM), Bourges's score and indices of motricity of the pathological limb), were assessed when patients were admitted in and as they left from the rehabilitation centre. Spearman's rho coefficient was used in this study.ResultsThis non-parametric test was considered significant when P < 0.05. It demonstrated a relationship between the evolution of thigh's external and muscular volumes and changes in Bourges's score in seated position (r_s = 0.730 and r_s = 0.764, respectively). It also showed a relationship between pathologic knee motricity index and pathologic leg segment volume evolution (r_s = 0.675).Discussion–conclusionStatistical analysis demonstrated that pathologic thigh volume changes is beneficial to balance during seating as no volume evolution seems to influence balance during standing.     

Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.