Mussel Gill Tissue Research Articles

Reaction rates as a function of pressure, temperature and concentration by heat-conduction differential scanning calorimetry

New equipment and procedures to examine reaction and metabolic rates as functions of temperature and pressure by scanning and isothermal calorimetry are described. Three applications are presented to illustrate the capabilities of the methods. The first demonstrates the effects of pressure and temperature on the metabolic rates of cultured plant cells. Pressures in the range from 7 to 16MPa shifted significantly the high-temperature inactivation of metabolic activities in tomato cells to higher temperatures. The second demonstrates the use of differential scanning calorimetry to investigate rapidly reaction kinetics and mechanisms. The chemical degradation rates of pineapple juice concentrate as a function of oxygen concentration and temperature were determined. The third demonstrates the use of the instrument in the isothermal mode to determine the effects of total pressure and oxygen partial pressure p o 2 on the metabolic rate in an ectothermic animal tissue. The metabolic rates of fresh-water mussel gill tissues were increased by both increased total pressure and increased p o 2

Cytosolic and mitochondrial l-malate dehydrogenases from ribbed mussel ( Modiolus demissus) gill tissue

1. 1. The cytosolic and mitochondrial malate dehydrogenases (cMDH and mMDH) from ribbed mussel gill tissue are dimeric (native M r = 60,000–62,000) and electrophoretically and immunologically distinct. 2. 2. The kinetics of the purified mMDH and the electrophoretically slow-moving cMDH form (C1) were studied. 3. 3. At pH 8, the apparent K ms for NADH and oxaloacetate (OAA) were low (5–20 μM). 4. 4. At pH 9.2, the apparent K ms for malate were high (0.4–1 mM) and the apparent K ms for NAD (40–150 μM) were higher then the K ms for NADH. 5. 5. Inclusion of 10 mM MgCl 2 in assay mixtures with both MDHs increased the apparent K ms of all substrates under all conditions studied. 6. 6. Hydroxymalonate was a competitive inhibitor of OAA with the mMDH and a mixed, non-competitive inhibitor of OAA with the cMDH at pH 8; these apparent K i values (0.25–1.2 mM) were larger (15–70x) than the apparent K i for hydroxymalonate inhibition of the malic enzyme from this tissue (Bishop and Brodey, 1992). 7. 7. ATP was a weak non-competitive inhibitor of the mMDH ( K i = 5.38 mM) and a strong competitive inhibitor of the cMDH ( K i = 0.77 mM).

Malic enzyme from ribbed mussel ( Modiolus demissus) gill tissue mitochondria

1. 1. NADP-malic enzyme was purified from ribbed mussel gill tissue mitochondria and found to be a tetramer composed of subunits with M r = 65,000 . 2. 2. At pH 7, with 1 mM MnCl 2, Michaelis-Menten kinetics were observed. 3. 3. The apparent K M values for NADPH, pyruvate, sodium bicarbonate malate and NADP were 129 μM, 8.5 mM, 19 mM, 43 μM and 4 μM, respectively. 4. 4. NADPH and pyruvate exhibited substrate inhibition at high concentrations ( NADPH > 100 μM ; pyruvate > 20 mM ). 5. 5. ATP was a mixed inhibitor of NADP utilization ( K i = 0.5 mM ). 6. 6. While the enzyme reaction is freely reversible when the substrate concentrations are optimized, reversibility (malate synthesis) in vivo is probably limited by low tissue bicarbonate (CO 2) and pyruvate concentrations.

Cadmium-induced changes in macromolecular synthesis at transcriptional and translational level in gill tissue of sea mussels, Mytilus edulis L.

1. Sea mussels, Mytilus edulis, were exposed to cadmium chloride at 0–500 μg Cd/l for 48 hr. The gills were excised and incubated with protein and RNA precursors. The exposure resulted in a concentration-dependent inhibition of the synthesis of proteins and of RNA. The inhibitory effect was most pronounced in RNA synthesis. 2. RNA was extracted from the gills as total RNA or as polyadenylated RNA. The translational activity of RNAs and the induction of mRNA for metallothionein-like proteins were studied by translation in a cell-free system. 3. Exposure of the animals to cadmium at 500 μg/l caused a 5-fold increase in proto-oncogene c- fos mRNA.

Mitochondrial malic enzyme from ribbed mussel ( Modiolus demissus) gill tissue: native molecular weight, divalent metal ion requirement and effect of hydroxymalonate

1. 1. Ribbed mussel gill mitichondrial malic enzyme (mME) was partially purified by ammonium sulfate precipitation and gel filtration. The mME had an apparent M r = 265,000, and used NAD at 2% of the rate with NADP. 2. 2. The pH optima for malate and pyruvate utilization were pH 8.5 and pH 6, respectively. The apparent K m s for malate were lower at physiological pHs (pH 7) than at the optimal pH. 3. 3. A divalent metal ion was required for activity. Using EDTA-divalent metal ions buffers at pH 7, S 0.5 values for metal ion-ME complexes were 18 pM, 2 pM and 140 μm with Mn 2+, Co 2+, and Mg 2+, respectively. Co 2+ or Mg 2+ could restore only 60% of the Mn 2+-dependent activity. 4. 4. Increasing the concentration of Mn 2+ decreased the apparent K m for malate and the cooperativity associated with malate reactivity. No cooperativity with malate was observed at pH 7 with 1 mM Mn 2+. 5. 5. Increasing the concentration of malate decreased the apparent K m for NADP. NADPH and pyruvate were competitive inhibitors of NADP and malate utilization, respectively. 6. 6. Alkali chloride salts and sodium acetate were inhibitory with apparent I 50s in the 40–80 mM range. 7. 7. Hydroxymalonate was a competitive inhibitor of malate utilization and a non-competitive inhibitor of pyruvate utilization. ATP was not inhibitory in the presence of 10 mM MnCl 2.

Changes in monoamine transmitter concentration in freshwater mussel tissues

Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated at nearly constant levels. Serotonin is an important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.

Phosphoenolpyruvate carboxykinase from ribbed mussel gill tissue: Reactivity with metal ions, kinetics, and action of 3‐mercaptopicolinic acid

AbstractPhosphoenolpyruvate carboxykinase (E.C. 4.1.32) from ribbed mussel gill cytosol exists as a monomer with Mr 70,000. Low levels of Mn2+ or Zn2+ were required for activity when assayed at high physiological Mg2+ levels. Mg2+ alone would not activate the enzyme. Optimal activity was observed at pH 6.6. The apparent Kms for phosphoenolpyruvate (PEP) and NaHCO3 were 396 μM and 13 mM, respectively, when assayed at pH 6.8 in the presence of 1 mM Mn2+, 1 mM Mg2+, and 1.5 mM IDP. GDP (Kmapp 24 μM) and IDP (Kmapp 46 μM) were both reactive; other nucleoside diphosphates were not. Under standard assay conditions at pH 6.8, the reaction was freely reversible with the rate of PEP carboxylation two to three times the rate of OAA decarboxylation. When assayed in the PEP synthesizing direction at pH 6.8 with 2 mM Mn2+ and Mg2+, the apparent Kms for GTP and OAA were 55 μM and 24 μM, respectively. Chloride ion was inhibitory (I50 120 mM); 3‐mercaptopicolinic acid (MPA) and 3‐aminopicolinic acid (APA) were competitive inhibitors with respect to PEP and noncompetitive with respect to IDP. MPA was a more effective inhibitor (Kiapp 8–34 μM) than APA (Kiapp 250–500 μM). Aminooxyacetate and N‐butylmalonate (BMA) were not inhibitory and hydroxymalonate was slightly inhibitory (Kiapp 15 mM). ATP showed mixed competitive inhibition with respect to PEP (Kiapp 0.54 mM); inhibition by ATP was not reversed by L‐alanine. Experiments employing BMA and MPA with gill tissue pieces subjected to hyperosmotic stress indicated that the PEPCK reaction in gill tissue was not significantly involved in the regulation of carbon flow for the synthesis of L‐alanine as an intracellular osmotic buffer.

Synthesis of stress proteins under normal and heat shock conditions in gill tissue of sea mussels ( Mytilus edulis) after chronic exposure to cadmium

1. Sea mussels were exposed to 16.5 μg Cd/l under semi-field conditions for almost one year. The isolated gills were incubated with 35S-methionine or -cysteine. 2. Chronic exposure to cadmium neither altered the rate of amino acid incorporation nor induced expression of heat shock proteins in the gills. 3. Heat shock imposed after chronic exposure to cadmium resulted in an increased synthesis of heat shock proteins, especially those of high molecular weight. 4. Synthesis of cadium-bincling, low molecular weight proteins was observed at any point of the exposure time. Their cadmium-bincling capacity and rate of synthesis, after the initial increase, remained unchanged throughout the exposure.

Malate dehydrogenase isoforms from ribbed mussel ( Modiolus demissus) gill tissue

1. 1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis. 2. 2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states. 3. 3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer. 4. 4. The identity of C5 is uncertain. 5. 5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K ms for oxaloacetate (OAA) than the cytosolic forms. 6. 6. For all isozymic forms, the apparent K ms for OAA increased as the pH increased between pH 6 and 9 7. 7. Increasing the salt concentration raised the K m for OAA for all forms. 8. 8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs. 9. 9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K is for substrate inhibition than the cMDH at each pH tested. 10. 10. Differences and similarities in K m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

The Effect of the Parasite Phyllodistomum Macrocotyle (Trematoda) On Heavy Metal Concentrations in the Freshwater Mussel Dreissena Polymorpha

The freshwater mussel Dreissena polymorpha can be parasitized by the digenetic trematode Phyllodistomum macrocotyle, which forms sporocysts in the gill tissue. The gill tissue of the mussel is seriously damaged by the parasite and infested animals loose about one-third of their total dry weight. Infested and non-infested mussels were analysed for the heavy metals Cu, Zn, Cd and Pb. In a number of infested and non-infested animals the gills were separated from the rest of the soft tissues. Concentrations of Cu, Zn, Cd and Pb in total soft tissues and in the gills were higher in parasitized than in non-parasitized animals. In soft tissues without gills only Cd and Pb concentrations were higher in infested than in non-infested animals.

Serotonin regulation of gill cAMP production, Na, and water uptake in freshwater mussels

AbstractActive Na transport in freshwater mussels was stimulated by addition of 10−4 M serotonin (5‐HT) in the bath. Carrier kinetics apply to serotonin‐stimulated Na uptake with no effect on the affinity (Km 10−4 M). The Jmaxi was increased 350% over controls to 5.83 μeq (g dry tissue · h)−1. Amiloride reversibly inhibited the 5‐HT‐stimulated Na influx. Serotonin also caused a net water uptake of 0.36 ml H2O (g dry tissue · h) −1, due, in part, to a reduction in renal filtration. When mussel gill tissue was incubated in pondwater containing 10−4 M 5‐HT, cAMP content increased 2.5 times control levels within the first minute and then declined toward control values by 20 minutes. Theophylline (10−3 M) in the incubation medium prevented this decline. Dopamine, epinephrine, or norepinephrine (10−4 M in pondwater) increased gill cAMP concentrations; L‐dopa did not. Both 5‐HT and dopamine significantly increased gill cAMP concentration at 10−5 M.

Aspartate aminotransferases from ribbed mussel gill tissue: Reactivity with β- l-cysteinesulfinic acid and other properties

1. 1. The β-form of the cytosolic aspartate aminotransferase (cAAT) and the mitochondrial aspartate aminotransferase (mAAT) from ribbed mussel ( Modiolus demissus = Gukensia demissa) gill tissue each exist as dimers of equal mol. wt (44,000). 2. 2. Both the cAAT and mAAT are reactive with β- l-cysteine sulfinic acid (CSA) as an amino donor substrate. 3. 3. The apparent K ms for CSA are very high (19–250 mM) and increase with decreasing pH.

Amino acid metabolism in euryhaline bivalves: regulation of glycine accumulation in ribbed mussel gills.

Glycine levels in isolated ribbed mussel (Modiolus demissus) gill tissue increased slightly and decreased markedly when incubated at high and low salinities, respectively. Low levels of the enzymes involved in the biosynthesis of serine from triose phosphate intermediates, the serine hydroxymethyltransferase, and serine dehydrase were detected in gill tissue homogenates. Experiments using gill tissue incubated with (U-14C)-glycine and (U-14C)-serine indicated interconversion between serine and glycine and transfer of label to alanine, asparate, glutamate, CO2, organic acids, and protein. Glyoxylate was metabolized more slowly than glycine and was probably converted to glycine for catabolism. Studies using (1-14C)-glycine and (2-14C)-glycine with isolated gill tissue and mitochondria indicated that the mitochondrial glycine cleavage enzyme was the major route of glycine catabolism. Metabolic controls activating or inhibiting the glycine cleavage enzyme regulate tissue glycine accumulation and catabolism during hypersalinity or hyposalinity stress.

Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves

1. 1. Aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), malic enzyme (ME), malate dehydrogenase (MDH), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (PEPCK) activities in cytosolic and mitochondrial fractions of gill tissue from Modiolus demissus (ribbed mussel), Mytilus edulis (sea mussel), Crassostrea virginica (oyster) and Mercenaria mercenaria (quahog) were determined using enzyme assay and starch gel electrophoresis combined with subcellular fractionation. 2. 2. AAT showed distinct mitochondrial and cytosolic isozymes in gills of all these animals. 3. 3. Although ALAT showed distinct mitochondrial and cytosolic isozymes in the gills of oysters, sea mussels and quahogs, only the mitochondrial ALAT was evident in ribbed mussel gill tissue. 4. 4. PK and PEPCK were cytosolic in all these preparations. 5. 5. ME was found only in the mitochondrial fraction of ribbed mussel and quahog gill tissue whereas sea mussel gills showed distinct cytosolic and mitochondrial ME isozymes. 6. 6. With oyster gills, the “cytosolic ME” was electrophoretically identical to the mitochondrial ME indicating that in vivo, the ME is probably mitochondrial. 7. 7. MDH showed distinct cytosolic and mitochondrial isozymes in all bivalve gills tested.

Cellular location and partial characterization of the alanine aminotransferase in ribbed mussel gill tissue

AbstractDifferential centrifugation of ribbed mussel gill tissue homogenates and extraction of the mitochondrial fraction demonstrated that most (72%) alanine aminotransferase (AlAT) activity was mitochondrial. Subsequent characterization of the cytosolic activity demonstrated properties identical to those demonstrated by the mitochondrial enzyme. Both enzyme fractions showed little variation in Vmax with pH, had low Km's for ketoacid substrates, and were inhibited by aminooxyacetate (AOA), L‐cycloserine, and β‐chloro‐L‐alanine. It appears that the AlAT in ribbed mussel gill tissue is strictly mitochondrial and that alanine production during hypoxia or hyperosmotic stress must be mitochondrial.

Partial characterization of the cytosolic and mitochondrial aspartate aminotransferase from ribbed mussel gill tissue

The cytosolic (cAAT) and mitochondrial (mAAT) aspartate aminotransferases were partially purified from gill tissue of the ribbed mussel, Modiolus demissus, and individually characterized with respect to heat stability, electrophoretic mobility, pH optimum, Km for substrates, rates in the forward vs. the reverse direction, and reactivity with aminooxyacetic acid (AOA). Isozyme analysis for this population of ribbed mussels indicated three alleles for the cAAT and a single all ele for the mAAT. The mAAT was a single isozymic form with a broad pH optimum and relatively low Kms for aspartate, oxaloacetate, and 2-oxoglutarate and high Km for glutamate substrates. The cAAT used for the kinetic studies was a single (homozygous) isozymic form, showed great variation in Km and Vmax with pH, and had high Kms for the amino acid substrates and low Kms for the ketoacid substrates. Both enzymes were inhibited to the same degree by AOA. It is hypothesized that the cAAT is involved more with aspartate synthesis and the mAAT with aspartate catabolism.

Preparation and some respiratory properties of coupled mitochondria from ribbed mussel (Modiolus demissus) gill tissue

AbstractPreliminary experiments with 2,4‐dinitrophenol (DNP), 2,4,6‐trinitrophenol (TNP), and sodium azide (NaN3) indicated that most of the oxygen consumption by ribbed mussel gill tissue is the result of mitochondrial respiration. A procedure utilizing isoosmotic sucrose, EGTA, defatted serum albumin, and HEPES as the isolation medium was devised for the preparation of fully coupled ribbed mussel gill mitochondria. Optimal rates of respiration and respiratory coupling required substrate, ADP, inorganic phosphate, and a fairly high KCl concentration (90 mM) in the assay medium. Glutamate, proline, malate, and succinate stimulated oxygen consumption with high respiratory control indices and P/O of 3, 3, 3 and 2, respectively. Pyruvate was a weak stimulator of mitochondrial respiration and showed a low respiratory control index with a low P/O. Preparation of gill mitochondria in isoosmotic solutions containing high KCl concentrations (150 mM) yielded mitochondria showing state 2 respiration, slow partially uncoupled ATP synthesis during state 3 respiration and no state 4 respiration. D‐mannitol was not used in the mitochondrial isolation or assay medium because of the probable presence of a D‐mannitol oxidase in these gill mitochondria.

3H]spiroperidol binding in crude membrane suspensions of gill tissue from the marine mussel, Mytilus californianus

1. Saturable, high-affinity binding sites for [ 3H]spiroperidol can be demonstrated in crude suspensions of mussel gill tissue. 2. This binding shows stereospecificity toward the d- and l-isomers of butaclamol. 3. Competition studies show that both dopamine and serotonin can displace [ 3H]spiroperidol from binding sites at nanomolar concentrations. 4. Evidence is presented that suggests that the [ 3H]spiroperidol-binding sites can be divided into two distinct groups: those with high affinity for dopamine and those with high affinity for serotonin.