Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina.

A multi-angle laser-light scattering and electrospray-ionisation mass-spectrometry analysis.

Eur. J. Biochem., 1997, 243, 85-92.
Zal F., Green B.N., Lallier F.H., Vinogradov S.N. and Toulmond A.
To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser light scattering (MALLS) and to electrospray ionisation mass spectrometry (ESI-MS). It was also subjected to SDS-PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 ± 24 kDa and a gyration radius of 11.3 ± 1.7 nm. Maximum entropy analysis of the multiply-charged electrospray spectra of the native dehaemed reduced and carbamidomethylated Hb forms provided its complete polypeptide chain and subunit composition. We found in the reduced condition eight globin chains of molecular masses 15952.5 Da (a1) 15974.8 Da (a2) 15920.9 Da (b1) 16020.1 Da (b2) 16036.2 Da (b3) 16664.8 Da (c) 16983.2 Da (d1) 17033.1 Da (d2) and two linker chains L1 25174.1 Da and L2 26829.7 Da. In the native Hb chains b c d occur as five disulphide-bonded trimer subunits T with masses of 49560.4 Da (T1) 49613.9 Da (T2) 49658.6 Da (T3) 49706.8 Da (T4) 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide-bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide-bonded heterodimer L1-L2 (D2) of 51981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of 5 cysteine residues. Semi-quantitative analysis of ESI-MS data allowed us to propose the following model for the one-twelfth protomer: [(3a1)(3a2)2T] (T corresponding to either T3 T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains each twelfth being in contact with 3.5 linker subunits providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI-MS relative intensities and the model proposed above the globin:linker ratio gave 0.71:0.29 and 0.73:0.27 respectively. The estimation of haem content by pyridine haemochromogen and by HiCN methods also support the globin chain number provided by ESI-MS.
Request for reprintCopy the reference above and mail it to me with your address

Inorganic carbon acquisition by the hydrothermal vent tubeworm Riftia pachyptila depends upon high external Pco2 and on proton elimination by the worm.

J. Exp. Biol., 1997, 200, 883-896.
Goffredi S.K., Childress J.J., Desaulniers N.T., Lee R.W., Lallier F.H. and Hammond D.
Riftia pachyptila is the most conspicuous organism living at deep sea hydrothermal vents along the East Pacific Rise. To support its large size and high growth rates, this invertebrate relies exclusively upon internal chemosynthetic bacterial symbionts. The animal must supply inorganic carbon at high rates to the bacteria, which are far removed from the external medium. We found substantial differences in body fluid total inorganic carbon (CO2) both within and between vent sites when comparing freshly captured worms from a variety of places. However, the primary influence on body fluid CO2 was the chemical characteristics of the site from which the worms were collected. Studies on tubeworms, both freshly captured and maintained in captivity, demonstrate that the acquisition of inorganic carbon is apparently limited by the availability of CO2, as opposed to bicarbonate, and thus appears to be accomplished via diffusion of CO2 into the plume, rather than by mediated transport of bicarbonate. The greatly elevated Pco2 measured at the vent sites (up to 12.6 kPa around the tubeworms), which is a result of low environmental pH (as low as 5.6 around the tubeworms), and elevated CO2 (as high as 7.1 mmol l-1 around the tubes) speeds this diffusion. Moreover, despite large and variable amounts of internal CO2, these worms maintain their extracellular fluid pH stable, and alkaline, in comparison with the environment. The maintenance of this alkaline pH acts to concentrate inorganic carbon into extracellular fluids. Exposure to N-ethylmaleimide, a non-specific H+-ATPase inhibitor, appeared to stop this process, resulting in a decline in extracellular pH and CO2. We hypothesize that the worms maintain their extracellular pH by active proton-equivalent ion transport via high concentrations of H+-ATPases. Thus, Riftia pachyptila is able to support its symbionts' large demand for inorganic carbon owing to the elevated Pco2 in the vent environment and because of its ability to control its extracellular pH in the presence of large inward CO2 fluxes.
Request for reprintCopy the reference above and mail it to me with your address

Hemocyanin oxygen-binding properties of a deep-sea hydrothermal vent shrimp: evidence for a novel cofactor.

J. Exp. Zool., 1997, 277, 357-364.
Lallier F.H. and Truchot J.P.
Rimicaris exoculata is a caridean shrimp from the family Alvinocarididae which forms the dominant species around deep-sea hydrothermal vents from the Mid-Atlantic Ridge (MAR). Seeking respiratory adaptations to the hydrothermal environment, we have analysed the oxygen-binding properties of Rimicaris hemocyanin (Hc) in relation with temperature, pH and lactate variations. Rimicaris native Hc is mostly composed of hexamers. It showed a rather high oxygen affinity (P50 ca. 3 Torr at pH 7.5, 15°C), a large Bohr effect ((DELTA)logP50/(DELTA)pH = -1.87±0.25, n=6), a moderate lactate effect ((DELTA)logP50 /(DELTA)log[lac] = -0.12) and almost no temperature effect ((DELTA)H = -1.23 kJ.mol-1 15-35°C). Most surprisingly, dialysis of native hemolymph elicited a large increase of Hc-O2 affinity, an effect opposite to all previous observations made on crustacean Hcs. Moreover, this increase in affinity could be reversed by adding an ultrafiltrate of native hemolymph to a dialysed sample, thus unveiling the existence of a dialysable, yet unknown cofactor which decreases Hc-oxygen affinity.
Request for reprintCopy the reference above and mail it to me with your address

Sulfide acquisition by the hydrothermal vent tubeworm Riftia pachyptila is via diffusion of HS- rather than H2S.

J. exp. Biol., 1997, 200, 2609-2616
S. K. Goffredi, J. J. Childress, N. T. Desaulniers and F. H. Lallier
Deep-sea hydrothermal vents are home to a variety of invertebrate species, many of which host chemosynthetic bacteria in unusual symbiotic arrangements. The vent tubeworm Riftia pachyptila (Vestimentifera) relies upon internal chemolithoautotrophic bacterial symbionts to support its large size and high growth rates. Because of this, R. pachyptila must supply sulfide to the bacteria, which are far removed from the external medium. Internal (SIGMA)H2S ([H2S+HS-+S2-]) can reach very high levels in R. pachyptila (2-12mmoll-1 in the vascular blood), most of which is bound to extracellular hemoglobins. The animal can potentially take up sulfide from the environment via H2S diffusion or via mediated uptake of HS-, or both. It was expected that H2S diffusion would be the primary sulfide acquisition mechanism, paralleling the previously demonstrated preferential uptake of CO2. Our data show, however, that the uptake of HS- is the primary mechanism used by R. pachyptila to obtain sulfide and that H2S diffusion into the worm apparently proceeds at a much slower rate than expected. This unusual mechanism may have evolved because HS- is less toxic than H2S and because HS- uptake decouples sulfide and inorganic carbon acquisition. The latter occurs via the diffusion of CO2 at very high rates due to the maintenance of an alkaline extracellular fluid pH. (SIGMA)H2S accumulation is limited, however, to sulfide that can be bound by the hemoglobins, protecting the animal from sulfide toxicity and the symbionts from sulfide inhibition of carbon fixation.
Request for reprintCopy the reference above and mail it to me with your address

Investigation by electrospray ionization mass spectrometry of the extracellular hemoglobin from the polychaete annelid Alvinella pompejana: An unusual hexagonal bilayer hemoglobin

Biochemistry, 1997, 36, 11777-11786
F. Zal, B. N. Green, F. H. Lallier and A. Toulmond
Alvinella pompejana inhabits deep-sea, hydrothermal vent sites along the East-Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. This worm is the most thermophilic metazoan known to date. In Alvinella, as in other alvinellids, oxygen transport is mainly achieved by an extracellular Hb dissolved in the vascular blood. This Hb has a molecular mass of 3833 +/- 14 kDa as revealed by multiangle laser light scattering (MALLS). Native and derivative Hb (reduced, carbamidomethylated, and deglycosylated) were analyzed by electrospray ionization mass spectrometry (ESI-MS). The data were processed by the maximum entropy deconvolution system (MaxEnt). We identified three groups of peaks for Alvinella Hb, at cn. 16, 23-26, and 50 kDa corresponding to (i) four monomeric globin chains, al (16 633.4), a2 (16 532.4), a3 (16 419.6), and a4 (16 348.9); (ii) four linker chains, L1-L4 (22 887.1, 24 230.5, 26 233.6, and 25 974.4); and (iii) one disulfide-bonded trimer T (51 431.9) composed of globin chains b (16 477.5), c (16 916.1), and d(18 048.8). These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. In addition, using the ESI-MS data we propose two alternative models for the quaternary structure of Alvinella's Hb.
Request for reprintCopy the reference above and mail it to me with your address

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: Implication on the knowledge of the sulfide binding-site

Proteins, 1997, 29, 562-574
F. Zal, T. Suzuki, Y. Kawasaki, J. J. Childress, F. H. Lallier and A. Toulmond
Request for reprintCopy the reference above and mail it to me with your address

Hydrothermal-vent alvinellid polychaete dispersal in the eastern Pacific .1. Influence of vent site distribution, bottom currents, and biological patterns

Limnol Oceanogr, 1997, 42, 67-80
P. Chevaldonne, D. Jollivet, A. Vangriesheim and D. Desbruyeres
Deep-sea hydrothermal-vent habitats are typically linear, discontinuous, and short-lived. Some of the vent fauna such as the endemic polychaete family Alvinellidae are thought to lack a planktotrophic larval stage and therefore not to broadcast-release their offspring. The genetic evidence points to exchanges on a scale that seems to contradict this type of reproductive pattern. However, the rift valley may topographically rectify the bottom currents, thereby facilitating the dispersal of propagules between active vent sites separated in some cases by 10s of kilometers or more along the ridge axis. A propagule flux model based on a matrix of intersite distances, long-term current-meter data, and information on the biology and ecology of Alvinellidae was developed to test this hypothesis. Calculations of the number of migrants exchanged between two populations per generation (N-m) allowed comparisons with estimates obtained from genetic studies. N, displays a logarithmic decrease with increasing dispersal duration and reaches the critical value of 1 after 8 d when the propagule Aux model was run in standard conditions. At most, propagule traveling time cannot reasonably exceed 15-30 d, according to the model, whereas reported distances between sites would require longer lasting dispersal abilities. Two nonexclusive explanations are proposed. First, some aspects of the biology of Alvinellidae have been overlooked and long-distance dispersal does occur. Second, such dispersal never occurs in Alvinellidae, but the spatial-temporal dynamics of vent sites over geological timescales allows short-range dispersal processes to maintain gene flow.
Request for reprintCopy the reference above and mail it to me with your address

Ribosomal (rDNA) variation in a deep-sea hydrothermal vent polychaete, Alvinella pompejana Desbruyères & Laubier, from 13°N on the East Pacific Rise

J. mar. Biol. Ass. UK, 1997, 78, 1-17
D. Jollivet, L. R. J. Dixon, D. Desbruyères and D. R. Dixon
The rDNA repeat-unit of the vent polychaete Alvinella pompejana Desbruyères and Laubier was investigated using restriction analysis. Mapping revealed evidence of rDNA polymorphism within and between individuals which was due to individual restriction site variation and sequence rearrangements involving spacer regions. The size of the repeat unit was 10.5 kb with virtually no evidence of length variation. Sequence inversions indicated the presence of two spatially-distinct sub-families of repeats, probably on different chromosome pairs. Animals from contrasting vent habitats with respect to age and chemical emissions (young versus old chimneys and white versus black smokers) from within the 13°N/EPR (East Pacific Rise) vent sector were analysed for evidence of population differentiation. Based on individual restriction site variation, average FST estimates across neighbouring populations were about 0.05 and differed significantly from zero. This level of genetic differentiation is comparable to values reported previously for allozymes. Spatial and temporal allelic frequency variances estimated from pairwise combinations (i.e. s2T and s2S) strongly suggested that differences in allelic frequency were the result of repeated extinction/recolonization events associated with the vent instability. Estimates of the effective population size derived from standardized temporal allelic frequency variances Fks were very low compared to actual population size indicating great temporal fluctuations in the former. Theoretically, such an effective population size is not sufficient to maintain the observed level of polymorphism within the 13°N/EPR vent sector. Results are therefore consistent with a "propagule" colonization-type model in which extinction/recolonization rates are high. In Alvinella, planktonic larval dispersal appears sufficient to overcome any genetic differentiation resulting from drift, but these findings also indicate that propagules may only be capable of dispersing a few 10s of kilometres per generation.
Request for reprintCopy the reference above and mail it to me with your address
Equipe SBR Les gens Les thèmes Les publis Les liens