The subunits of Abarenicola affinis affinis (Ashworth) extracellular haemoglobin (erythrocruorin).

The subunits of the erythrocruorin of Abarenicola affinis affinis (Ashworth) were investigated by various physicochemical methods. Gel chromatography at pH 9.0 in Sephadex G-200 gave three main protein peaks with molecular weights of 240 000, 100 000 and 31 000, respectively. Polyacrylamide gel electrophoresis showed considerable heterogeneity in the subunits, although the main components gave similar molecular weight ranges to the above. Anomalous results were also obtained in SDS-polyacrylamide gel electrophoresis. In the analytical ultracentrifuge, the subunit from the first peak showed a sedimentation coefficient of 10 S and Mw 250 000, and the second yielded s20,w = 5 S. Functionally, the 10 S subunit showed cooperative oxygen binding, but the Hill coefficient was lower than that observed for the native molecule. A possible model for the subunit structure of Abarenicola affinis affinis erythrocruorin is discussed.

CD studies on the reversed heme orientation in monomeric Glycera dibranchiata hemoglobins.

Circular dichroism spectra of three monomeric components of Glycera dibranchiata hemoglobins are reported. Contrary to what is found for most hemoglobins and myoglobins, G. dibranchiata hemoglobins display largely negative dichroic spectra in the Soret region. Independent NMR measurements have shown that the same monomeric hemoglobin components contain the heme moiety predominantly (greater than 85%) oriented in a reversed way with respect to the orientation which occurs in most hemoglobins and myoglobins. On the basis of these independent NMR studies, and also of previous data on other invertebrate hemoproteins, a correlation appears evident between reversed heme orientation in hemoglobins and negative ellipticity in the Soret CD spectrum. This represents a simple tool to evaluate this aspect of heme asymmetric environment.

Molecular weight of Eudistylia vancouveri chlorocruorin and its subunits.

The chlorocruorin of the marine polychaete Eudistylia vancouveri has a molecular weight of 3.1-10(6) and a sedimentation coefficient (S020, w) of about 57 S at pH 8.0 in the presence of 0.01 M Mg2+. The quaternary structure of this pigment is unaffected by pH between 6.0 and 11.5 in the presence of 0.01 M Mg2+ whereas in 0l01 M EDTA, the pigment begins to dissociate above pH 9.0 into smaller submultiples. The chlorocruorin can be converted into subunits with molecular weights of about 14 000-15 000 and 30 000 as determined by sodium dodecyl sulfate-gel electrophoresis and 14 000-15 000 as measured by gel chromatography of the carboxy-methylated derivative in 8 M urea, 0.1 M 2-mercaptoethanol, or by sedimentation equilibrium in 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol. The pigment contains 0.212 +/- 0.008% iron corresponding to 1 g atom iron per 26 300 g chlorocruorin. The amino acid composition of this pigment is reported. The subunit structure of Eudistylia chlorocruorin and the polymeric annelid hemoglobins are similar in many respects.

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris.

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.

The physico-chemical and functional properties of Abarenicola affinis affinis (Ashworth) extracellular haemoglobin (erythrocruorin).

The purified erythrocruorin from the Arenicolid polychaete, Abarenicola affinis affinis (Ashworth) has an S020,w of 57.5 S, and a molecular weight of 3.29 . 10(6) as determined by sedimentation equilibrium at pH 7.0. In the electron microscope it displays a two-tiered hexagonal array characteristic of other annelid erythrocruorins and chlorocruorins, with dimensions of approximately 25.5 nm diameter and 17.0 nm height. The isoelectric point of the molecule is at pH 4.63, and its amino acid composition and absorption spectrum are similar to those of other annelid erythrocruorins. The haem and iron contents are 2.77% and 0.23%, corresponding to minimum molecular weights of 22 250 and 24 800, respectively. In pH-induced dissociation studies, the erythrocruorin is found to be stabilized by Ca2+ up to pH 8.6, while in the presence of EDTA, dissociation begins at pH 7.8. In discontinuous gel electrophoresis, dissociated erythrocruorin gives subunits with molecular weights 263 000, 117 500, 68 400, 35 900 and 28 800, while, in detergent electrophoresis in the presence of 2-mercaptoethanol, polypeptide chains with molecular weights 13 700, 15 800, 24 700, 27 800 and 33 600 are obtained. Abarenicola erythrocruorin is also shown to bind oxygen cooperatively with a Hill coefficient h = 4.0 and P50 = 6.0 mmHg at pH 7.0 and 25 degrees C. Over the pH range of 7.0 to 7.8 the molecule lacks an alkaline Bohr effect.

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid.

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.

Glycera dibranchiata hemoglobin. Structure and refinement at 1.5 A resolution.

The coelomic cells of the common marine bloodworm Glycera dibranchiata contain several hemoglobin monomers and polydisperse polymers. We present the refined structure of one of the Glycera monomers at 1.5 A resolution. The molecular model for protein and ordered solvent for the deoxy form of the Glycera monomer has been refined to a crystallographic R-factor of 12.7% against an X-ray diffraction dataset at 1.5 A resolution. The positions of 1095 protein atoms have been determined with a maximum root-mean-square (r.m.s.) error of 0.13 A, and the r.m.s. deviation from ideal bond lengths is 0.015 A and from ideal bond angles is 1.0 degree. The r.m.s. deviation of planar groups from their least-squares planes is 0.007 A, and the r.m.s. deviation for torsion angles is 1.2 degrees for peptide groups and 16.8 degrees for side-chains. A total of 153 water molecules has been located, and they have been refined to a final average occupancy of 0.80. Multiple conformations have been found for five side-chains, and a change has been suggested for the sequence at five residues. The heme group is present in the "reverse" orientation that differs only in the positions of the vinyl beta-carbons from the "normal" orientation. The doming of the heme towards the proximal side, and the bond distances and angles of the heme and proximal histidine are typical of most deoxy globin structures. The substitution of leucine for the distal histidine residue (E7) creates an unusually hydrophobic heme pocket.

Primary structure of myohemerythrin from the annelid Nereis diversicolor.

The metal-free form of Nereis diversicolor myohemerythrin was purified from whole animal extracts by trichloroacetic acid precipitation and ion exchange chromatography. The amino acid sequence of myohemerythrin has been determined. The protein is composed of 120 residues, possesses an unblocked N-terminus and is devoid of cysteine residues. It bears 62% sequence identity with Themiste zostericola myohemerythrin, the only other member of this subfamily sequenced to date. Within the family of hemerythrins, homology is particularly high in the segments involved in the binding of the two iron atoms and in the beta-turn-rich N-terminal segment.

Serotoninlike immunoreactivity in the central and peripheral nervous system of the scale worm Harmothoe imbricata (Polychaeta).

The distribution of serotoninergic neurons in the nervous system of the scale worm Harmothoe imbricata was visualized in the anterior half of the body by the peroxidase-antiperoxidase (PAP) immunohistochemical method with a specific antiserotonin antibody. Immunoreactive neuronal somata were localized in discrete ganglion cell masses of the dorsally situated cerebral ganglion and in segmental ganglia of the ventral nerve cord. They also make up the majority of neurons present in the parapodial ganglia. Large and small varicose fibers stained in the neuropile of all the above-mentioned ganglia but also in interganglionic connectives and segmental nerves. On the basis of soma size and location and of fiber distribution, the reactive neurons were identified as primarily interneuronal with a few motoneurons and presumptive afferent neurons. The presence of a motor component was substantiated by observations of several reactive varicose fibers spread over longitudinal muscle layers of the trunk. In addition, neurites of the subepidermal nerve plexus and enterochromaffinlike cells of the gut epithelium reacted with the serotonin antibody. It is concluded that serotoninergic pathways are ubiquitous elements in the organization of the central and peripheral nervous system of this polychaete. The significance of these findings in relation to other annelid groups and to the physiological role of serotonin is discussed.

Subunit structure of erythrocruorin from the polychaete Tylorrhynchus heterochaetus.

Sodium dodecyl sulfate (SDS)-gel electrophoresis of erythrocruorin from the polychaete Tylorrhynchus heterochaetus revealed the presence of four subunits with molecular weights of 12,000, 22,000, 23,500, and 54,000 in a molar ratio of 6 : 1 : 2 : 3, respectively. The largest subunit dissociates into polypeptide chains of 13,500 molecular weight in the presence of mercaptoethanol, whereas each of the other subunits consists of a single polypeptide chain. Hemochromogen determination gave a minimum molecular weight of 26,500 per mol of heme group. A model of the subunit structure of the erythrocruorin molecule is proposed, composed of 144 subunits (252 polypeptide chains) possessing 144 heme groups. The molecular weight of the erythrocruorin was calculated to be 3.636 x 10(6).

Authentic FMRFamide is present in the polychaete Nereis virens.

The tetrapeptide FMRFamide is but one member of a large family of invertebrate neuropeptides which includes another tetrapeptide, FLRFamide, and several longer peptides terminating in one or the other of these tetrapeptide sequences. These peptides have been isolated from both molluscs and arthropods, but so far not one has been isolated from an annelid. Since the annelid worms are believed to share a common ancestor with molluscs and arthropods, they should contain FMRFamide-like peptides. We found two immunoreactive peaks in Nereis virens, but microsequencing and fast atom bombardment mass spectrometry revealed that they represent only one native peptide, FMRFamide. (The other peak is its methionyl sulfoxide derivative.) Each worm contained only 100 to 600 fmols of peptide, which is at least 10-100 times less than the levels in molluscs. Our identification of a tetrapeptide, and only a tetrapeptide, in this worm suggests that the tetrapeptides are the more ancient members of the family, and were probably present in the common ancestors of the annelids, arthropods, and molluscs.

[Determination of apparent mean mass of proteins associated with heme in the hemoglobin molecule of Arenicola marina (L.), Annelida, Polychaeta]. / Détermination de la masse apparente moyenne des protéines associées à l'hème dans la molécule d'hémoglobine d'Arenicola marina (L.), Annélide Polychète.

Protein and iron concentrations and maximum combined oxygen concentration were measured in the blood of the lugworm Arenicola marina. The calculated mean molecular mass of the heme-associated proteins was higher than that reported for known invertebrate and vertebrate intracellular hemoglobins. The difference is probably due to the presence of polypeptide chains not linked to heme groups in the extracellular annelid hemoglobins.

Purification, characterization, and comparison of extracellular hemoglobins in coelomic fluid and blood vessel plasma of Travisia japonica (Polychaeta, Annelid).

1. Two kinds of extracellular gigantic hemoglobins in blood vessel plasma and in coelomic fluid of Travisia japonica (polychaeta, annelid) were purified. 2. Their properties including electron microscopic observation, gel filtration, amino acid analysis, SDS polyacrylamide gel electrophoresis, and immunological properties were studied and compared. No significant difference was found between them. 3. They had a mol. wt of ca. 3 X 10(6) and a shape consisting of 12 subunits with a mol. wt of ca. 2.4 X 10(5) arranged in two superimposed hexagonal rings with a diameter of 22-25 nm and a thickness of 15-16 nm, as for other annelid gigantic hemoglobins described in the literatures.

Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.

The coelomic haemoglobin of Glycera dibranchiata is known to be separable into monomeric and higher-Mr fractions. Although exhibiting homogeneity with respect to Mr, the extent of haemoglobin heterogeneity for the monomer fraction has never been adequately assayed. In the present paper we demonstrate that there exists in the monomer haemoglobin fraction reproducibly detectable heterogeneity regardless of the presence or absence of proteinase inhibitors during the isolations. These results show that, considered on the same time scale as previous preparations used for amino acid sequencing, crystallography and kinetics, the monomer haemoglobin fraction is highly heterogeneous. Application of ion-exchange chromatography and ion-filtration methods resulted in the isolation of four resolvable haem protein components from the Glycera monomer haemoglobin fraction. Three of these components were isolated in sufficient quantity to employ proton n.m.r. as a successful analytical tool for discriminating the individual haemoglobins. These results are not surprising. Several previous studies indicated less extensive heterogeneity in the monomer fraction. Moreover, the ability of the Glycera monomer haemoglobin to bind oxygen at even quite low partial pressures has been attributed to functional diversity originating in multiple haemoglobin components. The present work reveals the extent of the haemoglobin heterogeneity. The results show that it is more extensive than previously believed. Examination of this monomer fraction is particularly important, since crystallography indicates that one of the components of the monomer fraction lacks the E-7 (distal) histidine residue. As a consequence, the identification of such extensive heterogeneity is important to many previously published ligand-binding studies.

Amino acid sequence of polypeptide chain IIB of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus.

The giant extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded trimer of chains IIA, IIB, and IIC. The complete amino acid sequence of chain IIB was determined. This chain has 148 amino acid residues and a molecular weight of 17,236 including a heme group. Of the residues in chain IIB, 74 (50%) and 34 (30%) were found to be identical with those in the corresponding positions in Tylorrhynchus chains IIC and I, respectively (Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. 260, 3145-3154). Marked differences were found between the chains of Tylorrhynchus and Lumbricus in the COOH-terminal regions. Significant differences were predicted between the monomeric chain I and the "trimeric" chains (IIB and IIC) in the hydropathy profiles and alpha-helical contents.