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PubMed2003

Effect of heavy metals on the uptake of [3H]-L-histidine by the polychaete Nereis succinea.

Integumentary uptake of [3H]-L-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid L-leucine in 60% artificial seawater (ASW). The time course of 10 microM [3H]-L-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 microM zinc and when calcium in the incubation medium was reduced from 6 mM to 5 microM the stimulatory effect of zinc on amino acid accumulation was reduced and uptake under the latter conditions was approximately half that of the control. Zinc stimulation of [3H]-L-histidine influx was a hyperbolic function of zinc concentration over the range 0 to 50 microM metal and displayed an apparent activation or affinity constant of 385+/-127 nM Zn(2+). The hyperbolic stimulatory effect of 1 microM Zn(2+) on the time course of 10 microM [3H]-L-histidine uptake was abolished in the presence of 25 microM L-leucine, suggesting that this amino acid shared the same transport system as [3H]-L-histidine and acted as a potential competitive inhibitor. Influx of [3H]-L-histidine was a hyperbolic function of external amino acid concentration and displayed an apparent affinity constant (Km) of 23.71+/-5.02 microM and an apparent aximal velocity (J(max)) of 4701+/-449 pmol/g dry wt.x15 min. Addition of 0.5 microM zinc resulted in a four-fold increase in J(max) and a doubling of K(m), suggesting the effect of the metal was mostly on the rate of amino acid transport. [3H]-L-histidine influx was mildly stimulated by Fe(2+) (0.5 microM), but was unaffected by either Ag(+) or Al(3+) (both at 0.5 microM). These results suggest that [3H]-L-histidine uptake into worm integument may take place by the classical Na(+)-independent L-transport system shared by L-leucine and regulated by exogenous calcium and other divalent metal concentrations.

Animals↗
PubMed2003

Efficiency of particle retention and clearance rate in the polychaete Sabellaria alveolata L.

The development of Sabellaria alveolata, a gregarious reef-building polychaete species, is maximal in Mont-Saint-Michel Bay (France), where trophic capacity is now threatened by increasing shellfish farming. As no data are available concerning the ecophysiological response of this species, the purpose of the present study was to obtain clearance rate and retention efficiency values to provide a first order of magnitude for the trophic role of this species. Data were obtained using a flow-through system with novel troughs suitable for 225 cm2 reef blocks containing a mean number of 940 +/- 102 (S.E.) individuals. The experimental diet used consisted of a mixture of two live microalgae, Skeletonema costatum (3800 cell ml-1) and Isochrysis galbana (23,700 cell ml-1), chosen to cover a broad size range (2 to 16 microns equivalent spherical diameter, ESD), as determined by a particle counter. On the basis of a mean clearance rate of 0.7 lh-1 obtained with reef blocks, the mean rate for an individual was estimated at 7.5 x 10(-4) L h-1. Particles larger than 6 microns ESD were cleared with 100% efficiency, but S. alveolata was unable to retain particles smaller than 2 microns ESD. The results are compared with data obtained for other polychaete species, and clearance rate values are extrapolated to an entire reef.

Animals↗
PubMed2000

The life-cycle of the flatfish nematode Cucullanus heterochrous.

Mature specimens of Cucullanus heterochrous Rudolphi, 1802 (Nematoda: Cucullanidae) were obtained from the intestine of the flounder, Platichthys flesus, from Danish waters. Eggs embryonate in seawater but do not hatch. Fully developed larvae pressed out of eggs are 430 microm long with amphids and dereids and enclosed within the cuticle of a previous larval stage. Infective larvae are believed to be in their third stage. Experimental studies showed that the polychaetes, Nereis spp., Scoloplos armiger, Brada villosa and Capitella sp., may act as intermediate hosts. In N. diversicolor the larvae increase their length to 1 mm within four weeks (15 degrees C) without moulting. Experimental infections showed that larvated eggs are not infective to fish, whereas >550 microm long larvae from polychaetes survived in 4-24 cm long flounders and plaice, Pleuronectes platessa. Third-stage larvae 550 microm to 1.1 mm long were found in the submucosa of the intestine one week post infection. At a length of about 800 microm to 1.4 mm they moult to fourth-stage larvae. Fourth-stage larvae, immature and mature worms occur in the intestine and rectum. Fourth-stage larvae and adults survived experimental transfer from one flounder to another. Similar developmental stages survived for two weeks in the intestine of experimentally infected cod, Gadus morhua.

Animals↗
PubMed1993

Cation binding and conformation of tryptic fragments of Nereis sarcoplasmic calcium-binding protein: calcium-induced homo- and heterodimerization.

Nereis sarcoplasmic calcium-binding protein (NSCP) is a compact 20-kDa protein that competitively binds three Ca2+ or Mg2+ ions and displays strong positive cooperativity. Its three-dimensional structure is known. It thus constitutes a good model for the study of intramolecular information transduction. Here we probed its domain structure and interaction between domains using fragments obtained by controlled proteolysis. The metal-free form, but not the Ca2+ or Mg2+ form, is sensitive to trypsin proteolysis and is preferentially cleaved at two peptide bonds in the middle of the protein. The N-terminal fragment 1-80 (T1-80) and the C-terminal fragment 90-174 (T90-174) were purified to electrophoretic homogeneity. T1-80, which consists of a paired EF-hand domain, binds one Ca2+ with Ka = 3.1 x 10(5) M-1; entropy increase is the main driving force of complex formation. Circular dichroism indicates that T1-80 is rich in secondary structure, irrespective of the Ca2+ saturation. Ca2+ binding provokes a difference spectrum which is similar to that observed in the intact protein. These data suggest that this N-terminal domain constitutes the stable structural nucleus in NSCP to which the first Ca2+ binds. T90-174 binds two Ca2+ ions with Ka = 3.2 x 10(4) M-1; the enthalpy change contributes predominantly to the binding process. Metal-free T90-174 is mostly in random coil but converts to an alpha-helical-rich conformation upon Ca2+ binding. Ca2+ binding to T1-80 provokes a red-shift and intensity decrease of the Trp fluorescence but a blue-shift and intensity increase in T90-174.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Ligand binding and slow structural changes in chlorocruorin from Spirographis spallanzanii.

Chlorocruorin is a cooperative respiratory pigment found in the blood of polychaete worms; its prosthetic group is a derivative of the iron protoporphyrin IX, in which the vinyl group at position 2 is substituted by a formyl group. The quaternary structure of chlorocruorins is complex: myoglobin-like subunits are grouped in tetramers and tetramers in dodecamers; 12 dodecamers are assembled in the 3500-kDa particle. Chlorocruorin from Spirographis spallanzanii displays the following overall functional properties: (i) the oxygen affinity is lower than in human hemoglobin, while that of CO is similar if not higher; (ii) the rates of combination with oxygen and carbon monoxide are low; and (iii) the off rate of oxygen is comparable to that of human hemoglobin, while the off rate of CO is 10 times smaller. When CO is partially photolyzed with a long and powerful light flash (70 microseconds), rebinding is biphasic as in mammalian hemoglobins; however, the slowest rate is faster than that observed by stopped flow, suggesting that the unliganded protein decays from the liganded high affinity state (R) to an intermediate state before reaching the low affinity (T) state. Oxygen binding was followed by stopped-flow and flash photolysis. While partial photolysis yields a fast, second-order time course, stopped-flow experiments yield slow, biphasic, and non-second-order time courses. This pattern of reactivity was attributed to a slow conformational transition(s) which is (are) rare limiting with oxygen, but not with CO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cement precursor proteins of the reef-building polychaete Phragmatopoma californica (Fewkes).

Two distinctive 3,4-dihydroxyphenyl-L-alanine-(DOPA-) containing proteins (Pc-1 and Pc-2) have been isolated and partially characterized from the thorax of the reef-building sabellariid Phragmatopoma californica. They are the first such reported from the phylum Annelida. The proteins are presumed to be soluble precursors of the quinone-tanned cement used to bind particulate materials in the construction of the tubes that serve as habitats for the worms. The proteins have apparent molecular weights ranging from 18,000 to 20,000 and isoelectric point greater than or equal to 8.0. Both proteins consist of repeated sequence motifs in their primary structure. Pc-1 has repeats of (XGGY*GY*GAK) where X = V, L, I, AA, or KV, and Y* is DOPA or tyrosine. Pc-2, in contrast, appears to have repeats of (X1-[GGY*]n-[GA]m-X2-[HP(A)V]p-HK) where X1 can be AL, A, or F; X2 can be WG or absent; n and m can be 1 or 2, and p = 0-2. Both protein families appear to share the same C-terminal sequence ALGGY*GAGA. Of the DOPA-containing proteins characterized from other phyla, Phragmatopoma cement precursors most resemble those from the liver fluke Fasciola hepatica and the mussel Trichomya hirsuta.

Amino Acid Sequence↗
PubMed1986

Nitrosyliron(III) hemoglobin: autoreduction and spectroscopy.

Nitrosyl complexes of the iron(III) forms of myoglobin, human hemoglobin, Glycera dibranchiata hemoglobins (Hbm and Hbh), and model iron(II) and iron(III) synthetic porphyrins including octaethylporphyrin (OEP) have been prepared. The iron(III) heme proteins are electron spin (paramagnetic) resonance (ESR) silent, while hexacoordinate solution structures are indicated for [Fe(OEP)(NO)2]ClO4 and for Hbm(II)NO, which has an ESR spectrum similar to that of Mb(II)NO and the hexacoordinate iron(II) model complex Fe(OEP)NO(BzIm). The splitting of the alpha- and beta-bands in the optical spectrum of Mb(III)NO and Hbh(III)NO contrasts markedly with the sharp, single bands observed in that of Hbm-(III)NO. The nondegeneracy of the dxz and dyz orbitals in Mb(III)NO and Hbh(III)NO is attributed to the influence of the distal histidine. Circular dichroism spectra were obtained for Hbm(III)NO, Hbm(II)NO, Hbh(III)NO, Hbh(II)NO, Mb(II)NO, and Mb(III)NO. The vicinal chiral center contribution that governs the heme protein CD leads to low Kuhn anisotropies, which have been used to assign certain electronic transitions. The Hb(III)NO spectrum is not stable but transforms into that of Hb(II)NO. This autoredox process follows kinetics that are first order in FeIIINO. The relative rates of autoreduction (25 degrees C, 1 atm NO) are Mb(III)NO less than Hbm(III)NO less than Hb alpha(III)NO less than HbA(III)NO. At high NO partial pressure or after "recycling" of HbA, the rates of reduction decrease. The first step in the reaction of NO with the ferric heme is the reversible formation of the formally iron(III) adduct. This reacts with another molecule of NO, generating the final heme(II)-NO via nitrosylation of NO itself or of an endogenous nucleophile. Kinetic and spectroscopic evidence shows involvement of trans-heme-(NO)2 in the reaction. The activation parameters delta H and delta S were determined. The overall reaction is photoenhanced.

Animals↗
PubMed1988

Anomalously slow cyanide binding to Glycera dibranchiata monomer methemoglobin component II: implication for the equilibrium constant.

In comparison to sperm whale metmyoglobin, metleghemoglobin a, methemoglobins, and heme peroxidases, the purified Glycera dibranchiata monomer methemoglobin component II exhibits anomalously slow cyanide ligation kinetics. For the component II monomer methemoglobin this reaction has been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0, employing 100-250-fold mole excesses of potassium cyanide at each pH. At 20 degrees C, with micromolar protein concentrations, kobsd varies between 9.11 x 10(-5) s-1 at pH 6.0, 100-fold KCN mole excess, and 1.12 x 10(-2) s-1 at pH 9.0, 250-fold KCN mole excess. Our analysis shows that the concentration-independent bimolecular rate constant (k1app) is small in comparison to those of the other heme proteins. For example, at pH 7.0 it is 0.491 M-1 s-1, compared to 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase; 111 M-1 s-1 for guinea pig methemoglobin; approximately 400 M-1 s-1 for sperm whale metmyoglobin; and 692 M-1 s-1 for soybean metleghemoglobin a, at the same pH and similar temperatures. Furthermore, our results show that the dissociation rate is extremely slow, with k-1app no larger than 10(-6) s-1. Separation of the bimolecular rate constant into contributions from kCN- (the rate constant for CN- binding) and from kHCN (the rate constant for HCN binding) shows that the former is approximately 90 times greater. These results indicate that cyanide ligation reactions are not instantaneous for this protein, which is important for those attempting to study the ligand-binding equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV.

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

cDNA cloning and predicted amino acid sequence of Glycera dibranchiata monomer hemoglobin IV.

The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins.

Amino Acid Sequence↗
PubMed2001

Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate. The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue. This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP. We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution. As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase. The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit. Many buried solvent molecules mediate subunit contacts at the interface. Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket. The structure reveals a mobile loop positioned over the active site. This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme. Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands. Rather, it appears to assist in the orientation of residues that do interact with active-site ligands. Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.

Adenosine Triphosphate↗
PubMed2005

Enzyme function of the globin dehaloperoxidase from Amphitrite ornata is activated by substrate binding.

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.

Animals↗
PubMed2006

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase.

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.

Animals↗
PubMed2006

Resonance Raman study of ferric heme adducts of dehaloperoxidase from Amphitrite ornata.

The study of axial ligation by anionic ligands to ferric heme iron by resonance Raman spectroscopy provides a basis for comparison of the intrinsic electron donor ability of the proximal histidine in horse heart myoglobin (HHMb), dehaloperoxidase (DHP), and horseradish peroxidase (HRP). DHP is a dimeric hemoglobin (Hb) originally isolated from the terebellid polychaete Amphitrite ornata. The monomers are structurally related to Mb and yet DHP has a peroxidase function. The core size marker modes, v2 and v3, were observed using Soret excitation, and DHP-X was compared to HHMb-X for the ligand series X = F, Cl, Br, SCN, OH, N3, and CN. Special attention was paid to the hydroxide adduct, which is also formed during the catalytic cycle of peroxidases. The Fe-OH stretching frequency was observed and confirmed by deuteration and is higher in DHP than in HHMb. The population of high-spin states of the heme iron in DHP was determined to be intermediate between HHMb and HRP. The data provide the first direct measurement of the effect of axial ligation on the heme iron in DHP. The Raman data support a modified charge relay in DHP, in which a strongly hydrogen-bonded backbone carbonyl (>C=O) polarizes the proximal histidine. The charge relay mechanism by backbone carbonyl >C=O-His-Fe is the analogue of the Asp-His-Fe of peroxidases and Glu-His-Fe of flavohemoglobins.

Animals↗
PubMed1997

Notomastus lobatus chloroperoxidase and Amphitrite ornata dehaloperoxidase both contain histidine as their proximal heme iron ligand.

Two novel heme-containing peroxidases, one able to incorporate halogens into aromatic substrates and the other able to remove them, have recently been isolated from marine sources and initially characterized by Chen et al. [(1991) J. Biol. Chem. 266, 23909-23915; (1996) J. Biol. Chem. 271, 4609-4612]. The haloperoxidase Notomastus lobatus chloroperoxidase (NCPO) is unusual in requiring a flavoprotein component for peroxidase activity. The dehaloperoxidase (DHP), isolated from Amphitrite ornata, is the only heme-containing peroxide-dependent dehalogenase known to be capable of removing halogens including fluorine. Both enzymes are also quite atypical in that the molecular weights of their heme-containing subunits are less than 16,000, approximately one-half to one-fifth the size of typical heme-containing peroxidases. Interestingly, we have also found that both enzymes are isolated in their oxyferrous states even though all protein purification was done in the absence of any reductant. In the present study, we have examined these two enzymes with magnetic circular dichroism and UV-visible absorption spectroscopy in order to determine the identity of their proximal heme iron ligand. Four derivatives of each enzyme, cyanoferric, deoxyferrous, oxyferrous, and (carbonmonoxy)ferrous, have been examined and spectroscopically compared to parallel derivatives of myoglobin, a well-studied histidine-ligated heme protein. The spectra observed for each derivative of the two new enzymes are very similar to each other and, in turn, to the spectra of the same derivatives of myoglobin. We conclude that both new heme enzymes contain histidine as their proximal heme iron ligand. This makes NCPO the first histidine-ligated heme-containing peroxidase capable of chlorinating halogen acceptor substrates using chloride as the halogen donor. Further, the novel reactivity of DHP is not the result of an unusual proximal ligand. The present results with NCPO and DHP challenge the current dogma of how heme-containing peroxidases function: one chlorinates substrates without having a thiolate proximal ligand, and the other both oxygenates and dehalogenates haloaromatics and yet has a histidine proximal ligand like numerous other peroxidases that are not capable of such a combined reactivity.

Animals↗