Isolation and characterization of methylated sugars from the tube of the hydrothermal vent tubiculous annelid worm Alvinella pompejana.

The tube of Alvinella pompejana contains in its carbohydrate fraction, 3 methylated monosaccharides: 2-mono-O-methyl-L-fucose, 3-mono-O-methyl-L-fucose and 2,4-di-O-methyl-L-fucose. The present work appears to be the first report of the occurrence of 2-mono-O-methyl-L-fucose and 3-mono-O-methyl-L-fucose in the animal kingdom. Moreover, it is the first time that 2,4-di-O-methyl-L-fucose is found in nature.

Iron and heme contents of the extracellular hemoglobins and chlorocruorins of annelids.

1. A survey of the literature on the extracellular hemoglobins and chlorocruorins of over 30 species of annelids, covering the last 30 years, shows that the range of iron content is 0.211-0.265 wt.% (mean = 0.228 +/- 0.013, N = 28) and the range of the heme content is 1.83-3.64 wt.% (mean = 2.60 +/- 0.38, N = 29). 2. There is relatively little scatter in the values of the experimental iron contents and only one of the 28 values is clearly outside the standard deviation range. 3. The values of heme contents are much more scattered, with seven values, clearly outside the standard deviation limits. 4. The aberrant cases are discussed and it is noted that the mean heme content of 2.60 wt.% corresponds to an iron content of 0.236 wt.% in excellent agreement with the mean iron content of 0.228 wt.%. 5. This result suggests strongly that experimental values of iron and heme contents outside the ranges of 0.211-0.243 and 2.3-2.7 wt.%, respectively, corresponding to a minimum molecular mass outside the range 23,000-26,000, should be regarded with caution.

Heterogeneity of the subunits of Lumbricus terrestris extracellular hemoglobin dissociated at alkaline pH.

1. The gel filtration profiles of dissociated extracellular hemoglobin of Lumbricus terrestris obtained in 0.1 M borate buffers at pH greater than 9, using columns of Sephadex G100, Sephacryl S200 and Ultragel AcA 44, consist of at least two peaks A and B. 2. SDS-PAGE of several fractions across the complete elution profile demonstrates that only the fractions under the right hand portion of peak B are homogenous and consist of the monomer (M) subunit (Mr = 17,000). 3. The fractions under the first peak contain the remaining subunits, a disulfide-bonded trimer (T) subunit (Mr = 50,000) and of two subunits (D1 and D2) of ca 30,000. 4. Densitometry of the SDS-PAGE patterns suggests that the proportions of these subunits vary across the two peaks, implying that peak A does not consist of a complex of fixed stoichiometry of the T and D1 and D2 subunits. 5. Furthermore, the D1 and D2 subunits overlap the M subunit in the trough between peaks A and B and are present in the left hand portion of peak B, probably because of the self-association of the M subunit. 6. In addition, SDS-PAGE experiments with a single fraction of peak A, where the load and the duration of staining were varied, suggests that the relative proportions of the subunits are independent of these two variables.

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex.

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.

Severe granulomatous arthritis due to spinous injury by a "sea mouse" annelid worm.

A case of destructive arthritis and soft tissue granulomatous inflammation occurred in a 25 year old man who had injured his right index finger while snorkelling in the Mediterranean. It was initially thought that he had fallen on a sea-urchin. He removed some spines at the time of injury but the finger became stiff, swollen, and painful, and after eight months with no symptomatic improvement amputation through the proximal phalanx was performed. Examination showed an exuberant granulomatous and foreign body type inflammation in the dermis and subcutaneous tissues and affecting the bone, with erosion of the cartilaginous surfaces of the proximal interphalangeal joint. Spines present in soft tissue sections contained no calcium but did contain chitin as shown by a von Wisseling reaction for chitosan. It is concluded that the chitinous spines almost certainly came from a sea-mouse (Phylum Annelida, family Aphroditidae). Sea mice are inconspicuous creatures which live on the sea floor and which may cause some injuries thought to be attributable to sea-urchins.

Primary structure of a constituent polypeptide chain (AIII) of the giant haemoglobin from the deep-sea tube worm Lamellibrachia. A possible H2S-binding site.

The deep-sea tube worm Lamellibrachia, belonging to the Phylum Vestimentifera, contains two giant extracellular haemoglobins, a 3000 kDa haemoglobin and a 440 kDa haemoglobin. The former consists of four haem-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the haem-containing chains [Suzuki, Takagi & Ohta (1988) Biochem. J. 255, 541-545]. The tube-worm haemoglobins are believed to have a function of transporting sulphide (H2S) to internal bacterial symbionts, as well as of facilitating O2 transport [Arp & Childress (1983) Science 219, 295-297]. We have determined the complete amino acid sequence of Lamellibrachia chain AIII by automated or manual Edman sequencing. The chain is composed of 144 amino acid residues, has three cysteine residues at positions 3, 74 and 133, and has a molecular mass of 16,620 Da, including a haem group. The sequence showed significant homology (30-50% identity) with those of haem-containing chains of annelid giant haemoglobins. Two of the three cysteine residues are located at the positions where an intrachain disulphide bridge is formed in all annelid chains, but the remaining one (Cys-74) was located at a unique position, compared with annelid chains. Since the chain AIII was shown to have a reactive thiol group in the intact 3000 kDa molecule by preliminary experiments, the cysteine residue at position 74 appears to be one of the most probable candidates for the sulphide-binding sites. A phylogenetic tree was constructed from nine chains of annelid giant haemoglobins and one chain of vestimentiferan tube-worm haemoglobin now determined. The tree clearly showed that Lamellibrachia chain AIII belongs to the family of strain A of annelid giant haemoglobins, and that the two classes of Annelida, polychaete and oligochaete, and the vestimentiferan tube worm diverged at almost the same time. H.p.l.c. patterns of peptides (Figs. 4-7), amino acid compositions of peptides (Table 2) and amino acid sequences of intact protein and peptides (Table 3) have been deposited as Supplementary Publication SUP 50154 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin.

Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.

Primary structure of a linker subunit of the tube worm 3000-kDa hemoglobin.

The deep-sea tube worm Lamellibrachia contains two giant extracellular hemoglobins, a 3000-kDa hemoglobin and a 440-kDa hemoglobin. The former consists of four heme-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the heme-containing chains. The 440-kDa hemoglobin consists of only four heme-containing chains (Suzuki, T., Takagi, T., and Ohta, S. (1988) Biochem. J. 255, 541-545). The complete amino acid sequence of a linker subunit (chain AV) has been determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase and endoproteinase Asp-N. The chain is composed of 224 amino acid residues, and the molecular mass for the protein moiety was calculated to be 24,894 Da. An Asn-X-Thr sequence which is possible as a glycosylation site was suggested at positions 108-110. A computer-assisted homology search showed that the sequence shows no notable homology with any other globins and proteins. However a careful alignment of the linker sequence with a heme-containing chain sequence suggested that there is a slight, but significant homology between the two sequences. The alignment also suggested that the linker resulted from gene duplication of a heme-containing chain with a three exon-two intron structure, and that the first exon of domain 1 and the last exon of domain 2 had been lost during evolution. In our alignment, domain 1 has the heme-binding proximal histidine, but domain 2 does not. This is the first linker subunit to be sequenced completely.

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis.

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).

N-terminal amino acid sequence of the deep-sea tube worm haemoglobin remarkably resembles that of annelid haemoglobin.

The deep-sea giant tube worm Lamellibrachia, belonging to the phylum Vestimentifera, contains two extracellular haemoglobins, an Mr 3,000,000 haemoglobin and an Mr 440,000 haemoglobin. The former has a hexagonal bilayer structure and consists of six polypeptide chains (AI-VI); a study of its haem content shows that not all of the chains contain haem. The Mr 440,000 haemoglobin consists of four haem-containing chains (BI-IV). We isolated most of the chains by reverse-phase chromatography and determined the amino acid sequences of the 21-45 N-terminal residues. Eight chains (AI-IV and BI-IV) showed significant homology with haem-containing chains of annelid giant haemoglobin. The highest homology was found between Lamellibrachia chain AI and Tylorrhynchus chain I; surprisingly, 18 out of the 20 N-terminal residues are identical. On the other hand, chain AV, with an unusual Mr of 32,000, showed a rather different sequence and is likely to be a non-haem chain which might act as a linker protein in the assembly of the haem-containing chains. From these results, we conclude that the tube worm Mr 3,000,000 haemoglobin is highly homologous with annelid haemoglobin.

The active site of hemerythrin as determined by X-ray absorption fine structure.

Extensive X-ray absorption fine structure measurements and analysis have been made on azidomet- and methemerythrin and on the native forms of oxy- and deoxyhemerythrin. Due to the availability of models that have been synthesized to mimic the active site of hemerythrin, it was possible to make a thorough assessment of the various errors in the structural parameters determined by the analysis. It is found that the largest source of error is the lack of complete transferability of amplitude and phase between the standards and hemerythrin. This is of particular importance in distinguishing the contributions of the second-shell low-Z atoms and, thus, has a substantial influence on the determination of the iron-iron distance. The internal consistencies of the various checks and a new formulation of error analysis for the structural parameters give us confidence in the structure determined for the active site. The main result is that as O2 is released from oxyhemerythrin, the mu-oxo bridge between the two iron atoms in the active site with an Fe-O distance of 1.8 A converts to a mu-hydroxo bridge in deoxyhemerythrin, expanding the Fe-O distance to 2.0 A. The Fe-Fe distance expands proportionally from 3.24 A in oxyhemerythrin to 3.57 A in deoxyhemerythrin so as to keep the Fe-O-Fe bridging angle approximately constant. These conclusions provide experimental support for the structures of oxy- and deoxyhemerythrin proposed previously on the basis of spectroscopic and preliminary X-ray crystallographic data.

Molecular symmetry of Lumbricus erythrocruorin.

X-ray diffraction data to a minimum Bragg spacing of 5.5 A have been collected from crystals of Lumbricus terrestris erthrocruorin, a 3.9 x 10(6)-dalton respiratory protein. Self-rotation function calculations from these data reveal D6 symmetry to a resolution of at least 6 A. These calculations show that erythrocruorin molecules pack in their crystals with molecular diads coincident with crystallographic diads along the a axis. Packing constraints limit the position of the molecular center to within 40 A of x = 1/4a.

The calcium, copper and zinc content of some annelid extracellular haemoglobins.

The extracellular haemoglobins of Lumbricus and Tylorrhynchus contain 50 and 61 tightly bound calcium atoms per molecule, respectively. In addition, they contain one to four tightly bound copper and zinc atoms. Although the role of the latter is unknown, that of calcium is probably structural, assisting in the maintenance of the native hexagonal bilayer structure.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata.

Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7 position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here we demonstrate that, for all of the Glycera monomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparations. Similar behavior is also demonstrated for horse heart myoglobin. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displays two bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handling prior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin.(ABSTRACT TRUNCATED AT 250 WORDS)

Two globin strains in the giant annelid extracellular haemoglobins.

The constituent polypeptide chains I, II, III and IV of the giant extracellular haemoglobin of the oligochaete Lumbricus terrestris were isolated by mono Q ion-exchange chromatography and C8 reverse-phase chromatography. The N-terminal amino acid sequences of Lumbricus chains I, III and IV were determined and aligned with those of Lumbricus chain II and the four chains of the extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus. Three invariant amino acid residues, Cys-7, Val-15 and Trp-19, were found to occur in the N-terminal segments (17-22 residues) of the eight chains of Lumbricus and Tylorrhynchus haemoglobins. In addition, it was found that the eight sequences could be separated into two groups: 'A', consisting of Lumbricus chains I and II and Tylorrhynchus chains I and IIA, having invariant Lys-14 and Lys-16, and 'B', consisting of Lumbricus chains III and IV and Tylorrhynchus IIB and IIC, having invariant Cys-6, Ser-8 and Asp-11. This result suggests that there are two strains of globin chain in the annelid extracellular haemoglobins.

Structure and oxygen equilibrium of the three coelomic cell hemoglobins of the echiuran worm Thalassema mellita (Conn).

1. The three coelomic cell hemoglobins from Thalassema mellita have been isolated to purity; the two major components have dimeric structure while the third minor component has monomeric structure. 2. Acid-urea Triton gel electrophoresis of the isolated hemoglobins identified three polypeptides among the three hemoglobins, one of the dimeric hemoglobins is a heterodimer (pI = 4.9) with one polypeptide sharing identity with the monomeric hemoglobin (pI = 6.3), while the other dimer is a homodimer (pI = 4.5) consisting of the third polypeptide. 3. SDS gel electrophoresis suggests that the two dimeric hemoglobins have interpolypeptide disulfide bonds. 4. Coelomic cell suspensions and lysed coelomic cells have PO2 at half saturation (P50) of 2.5-3.0 mmHg and cooperativity values (n) of 1.5-1.93. 5. All three isolated hemoglobins have higher oxygen affinities and lower cooperativity values (P50 = 1-2 mmHg, n = 1-1.3) than lysed coelomic cells suggesting some heterotrophic and homotrophic interactions.

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris.

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.