Adenylate cyclase of Mytilus galloprovincialis posterior adductor muscle: basal properties and sensitivity to serotonin

The presence and characteristics of a membrane-bound adenylate cyclase from Mytilus galloprovincialis Lmk posterior adductor muscle have been investigated. The enzyme has a Michaelis constant (K _m) of 0.38 mM at 20°C and requires divalent cations (Mg²⁺/Mn²⁺) for its activity. Optimal GTP concentrations are between 10^-5 and 10^-4 M. The non-hydrolizable GTP-analogues GMPpNHp and GTPS increase the activity of the enzyme four- to ten-fold. Sodium fluoride stimulates the enzymatic activity seven- to eight-fold. Forskolin increases the enzymatic activity two- to three-fold. Serotonin stimulates the adenylate cyclase activity in a dose-dependent manner. These experiments were performed with mussels collected from the estuary of Muros, Spain, in 1990.     

NADP+-dependent glutamate dehydrogenase from Acropora formosa: purification and properties

As an initial step in our study of nitrogen metabolism in the coral/algal symbiosis we have purified glutamate dehydrogenase (EC 1.4.1.4) to homogeneity from polyp tissue of the staghorn coral Acropora formosa collected from Magnetic Island (North Queensland) in 1985–1986. The purified enzyme had a specific activity of 78 U mg^-1. The native enzyme had a relative molecular weight, M _r, of 360 000 (±20 000), and appears to be a hexamer with subunits of M _r=56000 (±3 000). Like the enzyme from other coelenterates, the coral glutamate dehydrogenase (GDH) was absolutely specific with respect to the coenzyme substrate (NADP⁺/NADPH), and was insensitive to allosteric regulation by nucleotides; unlike other coelenterate GDHs, the coral enzyme was absorlutely specific for ammonium as amino group donor in the reductive amination reaction, and major differences in kinetic properties were apparent. Linear Michaelis-Menten kinetics were observed for the substrates a-ketoglutarate, NADPH and NADP⁺, the K _m values being 0.93, 0.11 and 0.03 mM, respectively. However glutamate dehydrogenase displayed biphasic kinetics with respect to l-glutamate and ammonium, indicating two apparent K _m values (18 and 81 mM for l-glutamate and 9.2 and 416 mM for ammonium). The enzyme also exhibits Scatchard plots, Hill coefficients and cooperativity indices characteristic of enzymes displaying negative cooperativity.     

Effect of fructose 2,6-bisphosphate and ammonium ions on AMP-mediated activation of phosphofructokinase from the posterior adductor muscle of the sea musselMytilus galloprovincialis

The effects of different modulators on the phosphofructokinase (PFK) activity from the posterior adductor muscle of the sea musselMytilus galloprovincialis Lmk. were studied in mussels collected from N.W. Spain in spring/summer, 1988. Adenosine monophosphate (AMP), fructose 2,6-bisphosphate (Fru 2,6-P₂) and ammonium ions individually activated PFK. AMP and Fru 2,6-P₂ reduced ATP-mediated inhibition and the value ofS _0.5 [concentration of fructose 6-phosphate (Fru 6-P) for half-maximum velocity], whereas ammonium ions increasedV _max (the rate at the saturating concentrations of Fru 6-P). Fru 2,6-P₂ and ammonium ions both enhanced the effect of AMP, but in different ways: Fru 2,6-P₂ reduced apparentK _m for AMP (concentration of AMP for half-maximum activation) without affectingK _m , whereas ammonium ions only altered the latter. The sharp increases in the values ofS _0.5 for Fru 6-P and of apparentK _m for AMP that are caused by a drop in pH were reduced by the presence of Fru 2,6-P₂, but not by ammonium ions. The results shows that Fru 2,6-P₂ may contribute to maintain the PFK activity during hypoxia periods, whereas ammonium ions (in the presence of high AMP concentrations) may activate the enzyme during the first few hours of post-hypoxia re-immersion.     

Effect of Ca2+ on survival and development of metamorphosing bonefish (Albula sp.) leptocephali

Bonefish (Albula sp.) larvae (leptocephali) from the Gulf of California complete metamorphosis in ˜10 d in natural seawater (35‰S; Ca²⁺ conc = 10.5 mM). The increase in ossification that occurs near the end of the non-feeding metamorphic period, in addition to the ability of larvae to complete metamorphosis in dilute seawater (8‰ S) prompted the present study, where the effects of varying the external calcium ion concentration, [Ca²⁺]_e, of artificial seawater (ASW) on the survival, development and internal (whole-body) calcium ion content, (Ca²⁺)_i, of unfed metamorphosing larvae were investigated. Early-metamorphosing larvae placed in␣ASW, where [Ca²⁺]_e = 10.1 mM, survived for up to 10 d and developed normally without exogenous nutrients. In shorter-term experiments (4 to 5 d), no differences in survival were found for larvae in ASW with [Ca²⁺]_e rang-ing from 1.5 to 10.1 mM. However, in Ca²⁺-free ASW, most larvae died within 27 h and no larvae survived more than 42 h; the median lethal time (LT₅₀), and its 95% confidence limits, were 14.5 (10.0 to 20.9) h. High mortality (81% after 20 h) also occurred in 1.0 mM Ca²⁺ ASW, but 2 of 16 larvae tested survived for 96 h. The 96 h median tolerance limit (TL_M), corrected for control mortality, was 1.2 mM Ca²⁺. In natural seawater, larval (Ca²⁺)_i remained relatively constant ( = 0.419 mg larva⁻¹)␣in early- and intermediate-metamorphosing larvae, and then increased to a mean value of 0.739 mg larva⁻¹ in advanced larvae, indicating that Ca²⁺ was␣taken up from the medium at this stage; the increase in (Ca²⁺)_i corresponded to the period of ossification of the vertebral column. Internal (whole-body) magnesium ion content (Mg²⁺)_i showed no significant change during metamorphosis ( = 0.089 mg larva⁻¹). No significant differences in (Ca²⁺)_i were found in advanced larvae in natural seawater and those in ASW, with [Ca²⁺]_e ranging from 2.0 to 10.1 mM. However, clearing and staining revealed that ossification of the vertebral column had not yet occurred in advanced larvae from 2.0 to 10.1 mM Ca²⁺ ASW. Also, low [Ca²⁺]_e (1.0 to 2.0 mM) usually produced deformed larvae that swam erratically, at times showing “whirling” behavior. Received: 21 August 1996 / Accepted: 26 August 1996     

Photosynthetic carbon acquisition in the red algaGracilaria conferta

Photosynthetic properties of the common red algaGracilaria conferta, collected from the eastern Mediterranean Sea were investigated in 1989, in order to begin evaluating its adaptative strategies with regard to the inorganic carbon composition of seawater, and to test whether the alleged C₄ photosynthesis of anotherGracilaria species is common within the genus. Net photosynthetic rates ofG. conferta were, under ambient conditions of inorganic carbon (ca. 10µM, CO₂ and 2.2 mM HCO ₃ ^- ), not sensitive to O₂ over the range 10 to 300µM, and the CO₂ compensation point was low (ca. 0.005µM). Ribulose-1,5-bisphosphate carboxylase/oxygenase was the major carboxylating enzyme, with a crude extract activity of 175µmol CO₂ g^–1 fresh wt h^–1 while phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase were present at 70 and 20%, respectively, of that activity. No activities of the decarboxylases NAD-and NADP-malic enzyme could be detected. The¹⁴C pulse-chase incorporation pattern showed thatG. conferta fixes inorganic carbon via the photosynthetic carbon reduction cycle only, with no evidence for photosynthetic C₄ acid metabolism. Photosynthesis at the natural seawater pH of 8.2 was, at 25°C and saturating light, saturated at the ambient inorganic carbon concentration of 2.5 mM. It is proposed that, under ambient inorganic carbon conditions, a CO₂ concentrating system other than C₄ metabolism provides an internal CO₂ concentration sufficient to suppress the O₂ effect on ribulose-1,5-bisphosphate carboxylase/oxygenase and, thus, on photorespiration, in a medium where the external free CO₂ concentration is lower than theK _m(CO₂) of the carboxylating enzyme. Since inorganic carbon, under natural saturating light conditions, seems not to be a limiting factor for photosynthesis ofG. conferta, it likely follows that other nutrients limit the growth of this alga in nature.     

Role of glutamine synthetase in ammonia assimilation by symbiotic marine dinoflagellates (zooxanthellae)

The dinoflagellate symbionts (zooxanthellae) present in many reef corals aid in the survival of the symbiotic unit in nitrogen deficient tropical waters by providing additional routes of nitrogen uptake and metabolism. The enzymatic pathway of ammonia assimilation from seawater and the re-assimilation of coral ammonium waste by zooxanthellae was studied by examining the affinity of glutamine synthetase for one of its substrates, ammonia. Glutamine synthetase activity was measured in dinoflagellates of the species Symbiodinium microadriaticum found in symbiotic association with various marine coelenterates. Michaelis-Menten kinetics for the substrate ammonia were determined for freshly isolated dinoflagellates from Condylactis gigantea (apparent NH₃ K_m=33 M) and for cultured dinoflagellates from Zoanthus sociatus (apparent NH₃ K_m=60 M). On the basis of the low apparent K_ms for NH₃, it appears that ammonia assimilation by these symbiotic dinoflagellates occurs via the glutamine synthetase/glutamate synthase pathway. Additionally, the uptake of exogenous ammonium by an intact coelenterate-dinoflagellate symbiosis was strongly inhibited by 0.5 mM methionine sulfoximine, and inhibitor of glutamine synthetase.     

Inhibition of Na+/K+-ATPase and of active ion-transport functions in the gills of the shore crab Carcinus maenas induced by cadmium

Inhibition of Na⁺/K⁺-ATPase from gill plasma membranes of the shore crab Carcinus maenas by cadmium was investigated and compared with inhibitory effects by known antagonists (ouabain and Ca²⁺). For comparative considerations the Cd²⁺-inhibition of the enzyme from dog kidney was also tested. Na⁺/K⁺-ATPase from dog kidney and from crab gill differed greatly in sensitivity against ouabain. The inhibition constant K _i of the dog enzyme amounted to 9.1 × 10⁻⁷ mol l⁻¹, i.e. more than 300-fold smaller than the K _i of 2.9 × 10⁻⁴ mol l⁻¹ determined for the crab enzyme. Ca²⁺ inhibited the activity of Na⁺/K⁺-ATPase from crab gill plasma membranes with a K _i of 4.3 × 10⁻⁴ mol l⁻¹. The Na⁺/K⁺-ATPase from crab gill was inhibited by Cd²⁺ with a K _i of 9.1 × 10⁻⁵ mol l⁻¹. Cd²⁺ inhibited the Na⁺/K⁺-ATPase from dog kidney with a K _i (6.4 × 10⁻⁵ mol l⁻¹) comparable to that observed in the crab gill enzyme. Under experimental conditions Cd²⁺-inhibition of Na⁺/K⁺-ATPase was irreversible. Repeated washing, centrifugation and homogenization of the plasma membranes (four times) with Cd²⁺-free buffer did not restore any activity lost in the presence of 1 × 10⁻³ mol l⁻¹ Cd²⁺. Since ouabain-insensitive (nonspecific) ATPases in the plasma membrane fraction of crab gills were inhibited by Cd²⁺ in the same way as Na⁺/K⁺-ATPase, the heavy metal is considered as an unspecific ATPase inhibitor. Comparing these results with literature data on Cd²⁺-binding to electrophoretically separated proteins suggests that Na⁺/K⁺-ATPase is a Cd²⁺-binding enzyme. The results obtained on Na⁺/K⁺-ATPase were reflected by Cd²⁺-inhibition of the branchial ion-transport functions depending on this enzyme. The transepithelial short-circuit current of isolated gill half lamellae, a direct measure of area-specific active ion uptake, and the transepithelial potential difference of isolated, perfused whole gills, also indicative of active ion uptake, were inhibited by the heavy metal in a time- and dose-dependent mode. Remarkably these inhibitions were also irreversible. These findings are ecologically and biomedically significant: even when the actual environmental or tissue concentrations measured are low, biological microstructures such as Na⁺/K⁺-ATPase may accumulate the heavy metal by tight binding over prolonged periods until the first inhibitory effects occur. Received: 25 June 1997 / Accepted: 25 August 1997     

Purification and characterisation of octopine dehydrogenase from the marine nemertean Cerebratulus lacteus (Anopla: Heteronemerta): comparison with scallop octopine dehydrogenase

Octopine dehydrogenase from the nemertean Cerebratulus lacteus was purified over 1000-fold to almost homogeneity. The enzyme does not bind to arginine Sepharose 4B. It has a monomeric structure with a relative molecular mass of 40000. Two isoenzymes were identified with isoelectric points of 5.6 and 5.4, whereas the purified isoenzymes of Pecten jacobaeus adductor mucles (which bind to arginine Sepharose 4B) had lower IEP's of 4.9 and 4.7. Apparent K_m's of the nemertean ODH for arginine and pyruvate are dependent on the respective co-substrate concentration. This phenomenon may result in activation of ODH and, thus, production of octopine in locomotory highly active individuals while attacking food, especially when this takes place in a hypoxic habitat, such as decaying mud near the high-water mark. The apparent K_m's for octopine (0.22 mM) and NAD⁺ (14 M) are low. Octopine is a substrate inhibitor for the reverse reaction above 2 mM, and a product inhibitor of the forward reaction by 50% at 1.2 mM. Therefore, only small amounts of octopine are likely to accumulate in vivo. Amino acid substrate specificity is limited to guanidino amino acids. We believe that the amino acid substrate specificity is not an evolutionary modification, but rather that it is narrowed to guanidino amino acids (or even specificity to arginine) in those species where ODH has a physiological function in maintaining redox balance during exercise. The specificity for keto acids is dependent on chain length, (-ketobutyrate>-ketocapronate); a second carboxyl group inactivates the enzyme.     

Antioxidant enzymes in the digestive gland of the common musselMytilus edulis

Antioxidant enzymes function to remove deleterious reactive oxygen species, including the superoxide anion radical and H₂O₂. Subcellular distributions and optimal and other properties of catalase (EC. 1.11.1.6), superoxide dismutase (SOD; EC. 1.15.1.1), selenium-dependent glutathione peroxidase (Se-GPX; EC. 1.11.1.9) and total glutathione peroxidase (GPX) activities were determined in the digestive gland of the common musselMytilus edulis L. by spectrophotometric and cytochemical/electron microscopic (catalase) techniques. Assay conditions for Se-GPX and total GPX activities were determined which optimized the difference between the non-enzymic and enzymic rates of reaction. General peroxidase activity (guaiacol as substrate) (EC. 1.11.1.7) was not detectable in any subcellular fraction. Catalase was largely, if not totally, peroxisomal, whereas SOD and GPX activities were mainly cytosolic. Distinct mitochondrial (Mn-SOD) and cytosolic (CuZn-SOD) SOD forms were indicated. Catalase properties were consistent with a catalase, rather than a catalase-peroxidase. The pH-dependence and temperature-dependence of GPX activity were different with H₂O₂ or CHP as substrate, and these and other observations indicate the existence of a distinct Se-GPX. Under saturating or optimal (GPX) assay conditions, the apparent Michaelis constantsK _m (mM) were: catalase, 48 to 68 (substrate, H₂O₂); Se-GPX, 0.11 (H₂O₂) and 2.0 (glutathione); and total GPX, 2.2 (eumene hydroperoxide) and 1.2 (glutathione). Calculated catalase activity was 2 to 4 orders of magnitude greater than Se-GPX activity over an [H₂O₂] of 1 to 1000 M. The results are discussed in relation to theoretical calculations of in vivo oxyradical production and phylogenetic differences in antioxidant enzyme activities.     

Metabolic specialization of mitochondria from scallop phasic muscles

In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol O₂ min^-1 mg^-1 mitochondrial protein at 25°C, while malate and glutamine were oxidized at 75% and proline at 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent K _{m, app}=0.013 mM) than at pH 7.0 (K _{m, app}=0.026 mM) while the affinity for ADP (K _{m, app}=0.015 mM) and that for glutamate (K _{m, app}=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.     

Photosynthetic utilization of CO2(aq) and HCO 3 - in Thalassia testudinum (Hydrocharitaceae)

The effects of total dissolved inorganic carbon (DIC), free carbon dioxide [CO_2(aq)], and bicarbonate (HCO ₃ ^- ) concentrations on net photosynthetic oxygen evolution of the marine angiosperm Thalassia testudinum Banks ex König collected from Biscayne Bay (1988) and from Tampa Bay (1990), Florida, USA, were examined. Rates of photosynthesis declined by 85% from pH 7.25 to 8.75 in buffered seawater media with constant DIC concentration (2.20 mM), suggesting a strong influence of CO_2(aq) concentration. A plateau in the pH-response curve between pH 7.75 and 8.50 indicated possible utilization of HCO ₃ ^- . Responses of photosynthesis measured in buffered seawater media of varying DIC concentrations (0.75 to 13.17 mM) and pH (7.8 to 8.61) demonstrated that photosynthesis is rate-limited at ambient DIC levels. Photosynthesis increased in media with increasing HCO ₃ ^- concentrations but near-constant CO_2(aq) levels, confirming HCO ₃ ^- assimilation. Calculated half-saturation constants (K _s )for CO_2(aq) and HCO ₃ ^- indicated a high affinity for the former [K _s (CO₂)=3 to 18 M] and a much lower affinity for the latter [K _s (HCO ₃ ^- )=1.22 to 8.88 mM]. Calculated V _max values for HCO ₃ ^- were generally higher than those for CO_2(aq), suggesting relatively efficient HCO ₃ ^- utilization, despite the apparent low affinity for this carbon form.     

Characterization of two glutamate dehydrogenases from the symbiotic microalga Symbiodinium microadriaticum isolated from the coral Acropora formosa

In view of their possible involvement in ammonium assimilation in the coral/algal symbiosis, we have purified two distinct glutamate dehydrogenase isoenzymes from the symbiotic dinoflagellate Symbiodinium microadriaticum (Freudenthal) extracted from the staghorn coral Acropora formosa collected from Magnetic Island, North Queensland, Australia, in 1986–1987. An NADPH-specific glutamate dehydrogenase (GDH) displayed biphasic kinetics with respect to ammonium as the variable substrate; at low substrate concentrations the apparent K _m was below 1 mM, whereas at high substrate concentrations the corresponding value was approximately 200 mM. The NADPH-GDH displayed extremely low activity in the direction of glutamate oxidation; together with the kinetic data this suggests a probable role in ammonium assimilation. A second (NADH-specific) GDH was found to have both amination and deamination activities, and presumably functions in vivo in glutamate oxidation. Kinetic constants are reported for both GDH isoenzymes.     

Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri

Phenoloxidase (PO) from the colonial ascidian Botryllus schlosseri was purified using two different chromatographic strategies. A three-step purification was developed in order to maintain enzyme activity, whereas an easier purification procedure was adopted to obtain enough PO for the production of specific polyclonal antibodies. The enzyme showed optimal pH and temperature values of 7.0 to 7.5 and 35 °C, respectively, and a K _m value of 4.62 ± 0.76 mM was estimated using l-DOPA as substrate. A molecular weight of 160 kDa was determined after SDS-PAGE under non-reducing conditions. The addition of the reducing agent β-mercaptoethanol caused the disappearance of the 160 kDa band and the appearance of a new band at 80 kDa, suggesting that active PO is a dimer and the two subunits are linked by disulphide bridges. Received: 14 December 1998 / Accepted: 24 August 1999     

Characteristics of the uptake system for L-lysine and L-arginine inPhaeodactylum tricornutum

Cells ofPhaeodactylum tricornutum Bohlin develop the ability to take up L-lysine when they are deprived of nitrogen (illuminated in nitrogen-free medium), carbon (incubated in darkness) or both. Cells with a developed uptake system take up and accumulate lysine in an unchanged form. Uptake occurs under either aerobic or anaerobic conditions and is dependent on the presence of sodium⁺ ions (K _s ^{Na +}=,ca. 10 mM). Some potassium⁺ ions are necessary for uptake, presumably within the cells, but with potassium⁺-replete cells, increasing K⁺ concentration depresses lysine uptake. The lysine-uptake porter also transports L-arginine.K _s values are about 1.5 M for lysine and 0.5 M for arginine. It is, however, possible that the uptake system developed by incubating cells in darkness differs from that produced in light; it shows a pronounced pH optimum at pH 8.5, whereas the activity of the light-developed system declines from pH 6.5 to pH 9.0 and correlates well with the concentration of lysine⁺. The uptake system developed in darkness may also have a higher affinity for lysine. Lysine uptake is not inhibited by 1 mM concentrations of nitrate, nitrate, ammonium, or urea nor by similar concentrations of amphoteric or acidic amino acids.     

The mechanism of crustacean salinity tolerance: Cell volume regulation by K+ and glycine effluxes

The regulation of muscle fiber K⁺ and free amino acid (FAA) concentrations during hypoosmotic stress was investigated in the moderately euryhaline crab Cancer irroratus. After 6 h of exposure to 60% ASW, muscle fiber K⁺ concentration declined from 185 mM to 140 mM. Following this, the blood glycine levels began to increase, indicating an FAA efflux from the cells. These data indicate that both muscle fiber K⁺ and FAA contribute to cell volume regulation in C. irroratus. The early release of K⁺ limits the initial rate of cell hydration. The subsequent efflux of glycine accounts for the volume regulation response of the muscle fibers. The cell volume regulatory system of C. irroratus is a coordinated use of both inorganic ions and FAA.     

Photosynthetic utilisation of inorganic carbon by seagrasses from Zanzibar, East Africa

Photosynthetic rates of eight seagrass species from Zanzibar were limited by the inorganic carbon composition of natural seawater (2.1 mM, mostly in the form of HCO₃ ⁻), and they exhibited more than three time higher rates at inorganic carbon saturation (>6 mM). The intertidal species that grew most shallowly, Halophila ovalis, Halodule wrightii and Cymodocea rotundata, showed the highest affinity for inorganic carbon (K _1/2 = ca. 2.5 mM), followed by the subtidal species (K _1/2 > 5 mM). Photosynthesis of H. wrightii, C. rotundata, Cymodocea serrulata and Enhalus acoroides was >50% inhibited by acetazolamide, a membrane-impermeable inhibitor of carbonic anhydrase, indicating that extracellular HCO₃ ⁻ dehydration is an important part of their inorganic carbon uptake. Photosynthetic rates of H. wrightii, Thalassia hemprichii, Thalassodendron ciliatum, C. serrulata and E. acoroides were strongly reduced by changing the seawater pH from 8.2 to 8.6 in a closed system. In H. ovalis, C. rotundata and Syringodiumisoetifolium, photosynthesis at pH 8.6 was maintained at a higher level than could be caused by the ca. 30% CO₂ concentration which remained in the closed experimental systems at that pH, pointing toward HCO₃ ⁻ uptake in those species. It is suggested that the ability of H. ovalis and C. rotundata to grow in the high, frequently air-exposed, intertidal zone may be related to a capability to take up HCO₃ ⁻ directly, since this is a more efficient way of HCO₃ ⁻ utilisation than extracellular HCO₃ ⁻ dehydration under such conditions. The inability of all species to attain maximal photosynthetic rates under natural conditions of inorganic carbon supports the notion that seagrasses may respond favourably to any future increases in marine CO₂ levels. Received: 19 March 1997 / Accepted: 31 March 1997     

Physiological role of branchial carbonic anhydrase in the shore crabCarcinus maenas

Excretion of total CO₂ and uptake of sodium and chloride ions across the branchial epithelium of the posterior gills of the shore crabCarcinus maenas, collected from Kiel Bay (Baltic Sea) in 1989, were measured using isolated perfused gill preparations. Total CO₂ effluxes depended on the HCO ₃ ^- concentration of the internal perfusate in a saturable mode and were inhibited by internally and externally applied acetazolamide at 10^–4 M. Potential differences between hemolymph space and medium did not change significantly during experimental treatments. Neither a bicarbonate gradient (6 mM) directed from the internal perfusate to external bath solution nor symmetrically applied 10^–4 M acetazolamide significantly influenced the influxes of Na⁺ and Cl^–. Results confirmed the role of carbonic anhydrase in CO₂ excretion but called into question the assumed functioning of the enzyme in branchial ion transport processes.     

Heavy-metal adsorption by non-living biomass

The adsorption of some heavy metals onto the walls of harvested, washed, and dried non-living biomass cells of different Pseudomonas strains was studied at optimum experimental conditions using a simplified single component system. The Langmuir adsorption model was found to be a suitable approach to describe the system via multi-step processes. Isotherms measured at 30.0°C and pH 5.5 with [M]_total?=?10–100 mM for tight, reversible Cr⁶⁺(aq), Ni²⁺(aq), Cu²⁺(aq) and Cd²⁺(aq) binding by the cell walls of the investigated biomass fit the Langmuir model and give the pH-independent stoichiometric site capacities ν _i and equilibrium constants K _i for metal binding at specific biomass sites i?=?A, B, C, and D. Tight binding sites A, B, and D of the non-living biomass are occupied by Cr^VI, sites A and C by Ni^II, sites A and D by Cd^II, and only site B by Cu^II. It is concluded that ν _i is a stoichiometric parameter that is independent of the magnitude of K _i for binding site i and that the studied heavy metals selectively and tightly bind at different biomass sites.     

Growth rate and carbon affinity ofUlva lactuca under controlled levels of carbon,pH and oxygen

We examined the growth rate (µ) ofUlva lactuca L. (collected from Roskilde Fjord, Denmark in 1987) at different levels of dissolved inorganic carbon (DIC), pH and oxygen in two culture facilities. Growth was faster in Facility A (µ _max ca 0.3 d^–1) than in B (µ _max ca 0.2 d^–1), probably because of more efficient stirring and higher light intensity. The growth-DIC response curve exhibited low half-saturation constant (K _1/2) values (0.35 mM DIC in A, 0.55 mM in B) and growth rates close toµ _max at natural seawater concentration of 2 mM DIC. Growth rate showed a low sensitivity to oxygen over a wide range of DIC and oxygen concentrations. Collectively, the results demonstrated an efficient mechanism for DIC use, unaffected by acclimatization to DIC concentrations between 0.2 and 3 mM. The growth rate decreased little between pH 7.5 and 9 at 2 mM DIC, but steeply above pH 9 approaching zero just above pH 10. The decline of growth at high pH may result from direct pH effects on cell pH, reduced HCO ₃ ^- availability and impaired operation of the carbon uptake process. The growth responses ofU. lactuca to DIC, pH and oxygen resembled those observed in previous short-term photosynthetic experiments. This similarity is probably due to the fast growth ofU. lactuca which means that photosynthetic products are rapidly converted into cell growth. Based on the culture experiments we argue that field plants ofU. lactuca not exposed to stagnant water and DIC depletion are likely to be limited in growth by environmental factors other than DIC (e.g. light and nutrients). Dense mats ofU. lactuca, however, may show reduced growth as a result of DIC depletion, high pH and self-shading.     

Inorganic carbon uptake by photosynthetically active protoplasts of the red macroalga Chondrus crispus

Photosynthetically active protoplasts were isolated from Chondrus crispus Stackh. by treating thalli with -carrageenase produced from batch culture of Pseudomonas carrageenovora. Using the silicone oil centrifugation technique, it was found that the protoplasts: (1) did not generally accumulate inorganic carbon (C_i) above the concentration in their incubation medium; (2) were saturated at C_i concentrations of 3 to 4 mM; (3) had an intracellular pH of 7.50 when incubated at pH 7.5; and (4) their initial carbon fixation rate was reduced by carbonic anhydrase inhibitors. Although the carbon fixation rate of the protoplasts was about 30% that of thallus fragments, presumably due to the relatively harsh protoplast isolation treatment, the behavior of the protoplasts was similar to that of fragments. This similarity indicates that the protoplasts are photosynthetically active and behave as thallus fragments. Further, the data are consistent with the hypothesis that C. crispus acquires C_i for photosynthesis by the diffusion of CO₂ across the plasma membrane.