On the role of fumarate reductase in anaerobic carbohydrate catabolism of Mytilus edulis L

• 1.1. The role of the fumarate:NADH oxidoreduction in the anaerobic glycolysis of the sea mussel is examined and discussed.
• 2.2. Fumarate reductase activity is present in submitochondrial particles especially from adductor muscle, digestive gland and mantle.
• 3.3. The pH optimum of the enzyme complex is 7.9; the approx K_m's for NADH and fumarate are 4.0 × 10⁻⁵ M and 6.3 × 10⁻⁵ M, respectively.
• 4.4. The enzyme complex is inhibitied by amytal, antimycin, ethanol, malonate, phosphate, rotenone, and succinate, and stimulated by Mg²⁺.
• 5.5. It is concluded that part of the mitochondrial respiratory chain is involved in the reduction of fumarate by NADH, comprising site 1 of the oxidative phosphorylation.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Organ specific changes in energy metabolism due to anaerobiosis in the sea mussel Mytilus edulis (L.)

• 1.1. Anaerobic energy metabolism was investigated in different organs of Mytilus edulis and the whole animal.
• 2.2. Succinate accumulates to high levels in most organs but remains low in the hemolymph.
• 3.3. After 16 hours propionate accumulation is observed in all organs. Experimental evidence is not sufficient yet to point out organs that produce more propionate than others.
• 4.4. Acetate is a minor end product.
• 5.5. Acetate and propionate are found in the hemolymph in amounts equal to those in the organs.
• 6.6. Animals incubated in oxygen-free seawater accumulate more end products than animals exposed to air, in the form of volatile fatty acids that are excreted into the incubation water.
• 7.7. Alanine and glutamine increase in the posterior adductor muscle. Aspartate decreases in the total animal, posterior adductor muscle and gills, while in the hemolymph decrease in alanine, asparagine, serine, threonine and proline are observed.

     

Varying effects of calcium on the oxidation of palmitate and α-ketoglutarate in isolated rat liver mitochondria incubated in KCl-based and sucrose-based media

• 1.1. Isolated mitochondria from rat liver were incubated in the presence of [U-¹⁴C]palmitate, ATP, CoA, carnitine, EGTA (ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid) and varying amounts of calcium.
• 2.2. When a KCl-based incubation medium was used, the oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1–10μM.
• 3.3. When a sucrose-based incubation medium was used, the basal rate of palmitate oxidation was about half of that observed with the KCl-medium and calcium had a stimulatory effect.
• 4.4. With the KCl-medium the rate of oxygen consumption was inhibited by calcium with α-ketoglutarate as well as palmitate as the respiratory substrate.
• 5.5. No inhibitory effect of calcium was observed with succinate or β-hydroxybutyrate.
• 6.6. With the KCl-medium and with α-ketoglutarate as the respiratory substrate, state 3 respiration but not state 4 respiration was inhibited by calcium.
• 7.7. When the sucrose-medium was used, state 3 respiration was first inhibited by calcium, but this inhibition was gradually relieved and the respiratory rate finally became higher than it was before calcium addition.

     

The mechanism of calcium-dependent activation of respiration of liver mitochondria from hibernating ground squirrels,Citellus undulatus

• 1.1. It is shown that Ca²⁺-dependent activation of respiration of liver mitochondria from hibernating ground squirrels is accompanied by mitochondrial swelling.
• 2.2. The swelling of mitochondria from hibernating ground squirrels, as well as the activation of mitochondrial respiration, is precluded by cyclosporin A, p-bromphenacylbromide and oligomycin. Carboxyatractiloside, on the contrary, under these conditions favors the swelling and the acceleration of respiration.
• 3.3. It was concluded that Ca²⁺-dependent activation of hibernating ground squirrel liver mitochondrial respiration resulted from the appearance of a non-specific permeability pathway and from swelling of mitochondria.

     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

The effects of sodium chloride concentration on electrophoretic patterns of adductor muscle proteins from bivalve molluscs

• 1.1. An extraction medium was sought that would produce the highest quality electrophoretic patterns of proteins from bivalve molluscan adductor muscles.
• 2.2. Cellulose acetate electrophoresis was conducted on adductor muscle proteins extracted with distilled water or various concentrations of saline, with and without dialysis, from three bivalve molluscan species: Pacific oysters, Crassostrea gigas; Japanese littleneck clams, Prototheca semidecussata; and Hawaiian mussels (Mahawele), Isognomon constellateum.
• 3.3. The protein patterns obtained by electrophoresis revealed that muscle extracted and dialyzed in 0.030 g% NaCl solution produced superior patterns.

     

Seasonal changes of lipids and fatty acids in oyster tissues (Crassostrea virginica) and estuarine particulate matter

• 1.1. Lipid concentration in adductor muscle ranged from 2–68, in visceral mass from 5–28, in mantle and gill from 5–20 and in heart from 27.8–79 mg/g wet tissue. Particulate matter lipids varied from 1.0–2.6 mg/1 of estuarine water.
• 2.2. Neutral and polar lipids ranged from 25–38% of the total lipids in the oyster tissue and from 62–75% of the estuarine particulate organic matter.
• 3.3. Seasonal maxima of lipid concentrations varied among oyster tissues. Peak particulate lipids occurred in November.
• 4.4. It is proposed that seasonal variation in oyster lipids was more related to reproductive cycles than to food lipid supply.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Evidence for target site insensitivity to cyanide in Spodoptera eridania larvae

• 1.1. Isolated mitochondria from the whole southern armyworm larvae, Spodoptera eridania, show all of the characteristics of mammalian liver mitochondria, except for target site sensitivity to cyanide.
• 2.2. The armyworm larval mitochondria are 17 times less sensitive to cyanide when compared to rat liver mitochondria and cannot be completely inhibited with extremely large doses.
• 3.3. These data suggest the presence in the southern armyworm of either a cyanide-insensitive cytochrome oxidase, or the elaboration of cyanide-insensitive oxidative pathway reminiscent of an alternative oxidative pathway that is known to coexist in plants alongside the cyanide-sensitive pathway.

     

Transport of cholesterol in the hemolymph of the mollusc Diplodon delodontus

• 1.1. The dietary and inter-organ cholesterol transport in the hemolymph of the bivalve mollusc Diplodon delodontus, was studied. Plasma and hemocytes were obtained after feeding labeled cholesterol to animals or injecting it into the posterior adductor muscle.
• 2.2. In both cases, cholesterol was incorporated either into plasma or hematic cells.
• 3.3. Two plasmatic fractions differing in their hydrated densities were recognized as cholesterol carriers and were isolated. They have characteristics of high density (HDL) and very high density (VHDL) lipoproteins, respectively.
• 4.4. The major lipids in the different classes of lipoproteins were free sterols in HDL and phospholipids in VHDL.
• 5.5. Neither low nor very low density lipoprotein transporting cholesterol was detected.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

Aerobic metabolism,octopine production and phosphoarginine as sources of energy in the phasic and catch adductor muscles of the giant scallop placopecten magellanicus during swimming and the subsequent recovery period

• 1.1. The energy contributions of aerobic metabolism, phosphoarginine, ATP and octopine in the adductor muscles of P. magellanicus were examined during swimming and recovery.
• 2.2. A linear relationship was observed between the size of the phosphoarginine pool and the number of valve snaps. A linear increase in arginine occurred during the same period.
• 3.3. Octopine was formed during the first few hours of recovery, particularly in the phasic muscle.
• 4.4. The restoration of the phosphoarginine pool appeared to be by aerobic metabolism.
• 5.5. It is concluded that the role of octopine formation is to supply energy when the tissues are anoxic and to operate at such a rate as to maintain the basal rate of energy production.

     

Glycogen synthetase in the sea mussel Mytilus edulis L.—II. Seasonal changes in glycogen content and glycogen synthetase activity in the mantle tissue

• 1.1. The glycogen content of the mantle tissue reached a maximum in the summer (May–July) with levels of 41.0–53.5% of the dry tissue weight.
• 2.2. Seasonal changes in glycogen synthetase activity showed that the I-activity (independent of G6P) increased up to 10-fold in June as compared with December. The measured I-activity of glycogen synthetase was sufficient to account for the accumulation of mantle glycogen in the summer.
• 3.3. The I-activity of glycogen synthetase declined rapidly in July of each year. A possible role for the inhibition of glycogen synthetase by high levels of tissue glycogen is suggested.
• 4.4. The I-activity in the mantle tissue of mussels on the shore was higher than that for animals starved in the laboratory for 2–3 days. The differences were minimal in early May but increased markedly in late May–July. Starved mussels returned to the shore showed an increase in I-activity of glycogen synthetase.
• 5.5. Injection of 30 μmol glucose into the adductor muscle increased the concentration of glucose in the mantle fluid to 2.0–2.5 mM. A similar injection of 60 μ mol glucose resulted in a time-dependent increase in the I-activity of glycogen synthetase.
• 6.6. Injection of mussels with mammalian insulin or anti-insulin serum had no effect on the activity of glycogen synthetase. Our results are at variance with those of other workers who have used the mammalian hormone in molluscan studies (see Discussion).

     

Anaerobic metabolism in sublittoral living Mytilus galloprovincialis in the mediterranean—IV. Role of amino acids in adaptation to low salinities during anaerobiosis and aerobiosis

• 1.1. When Mytilus galloprovincialis were transferred from 38 to 19%. sea water (S), the metabolism became anaerobic for at least 8 hr. After 24 hr the animals were entirely aerobic again.
• 2.2. Upon transfer to 19%. S, the total free amino acid concentration in haemolymph doubled within 4 hr, remaining nearly constant thereafter, up to 48 hr.
• 3.3. In the posterior adductor muscle a strong decrease of alanine and glycine occurred at 48 hr exposure to 19%. S, and a smaller decrease of glutamate; taurine remained relatively constant. When transferred again to 38%. S after 14 days, a strong overcompensation occurred in the concentrations of alanine and proline, and a smaller overcompensation in those of threonine and serine.
• 4.4. In the gill no distinct change in the amino acid pool occurred during 14 days of exposure, with the exception of a decrease in serine. When transferred again to 38%. S, a strong overcompensation occurred in alanine, proline, glycine and serine, and a smaller in glutamate and threonine.
• 5.5. No evidence for anaerobic metabolism in the decrease of the amino acid pool was found.
• 6.6. M. galloprovincialis is less able to adapt to low salinities than the more euryhaline M. edulis.

     

Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulations from cardiac muscle

• 1.1. Subsarcolemmal and interfibrillar mitochondria were prepared with complete recovery from rabbit and porcine heart muscle by upward-flotation during 60 sec of Percollö density gradient centrifugation.
• 2.2. Mitochondrial subpopulations were identified and characterized according to buoyant density, electron-microscopy, marker enzyme activities and respiratory performance.
• 3.3. ADP-induced state 3-respiration related to latent citrate synthase activity as a marker for structurally intact mitochondria was not significantly different in both mitochondrial subtypes.

     

A halogenated amino acid-containing sperm activating peptide and its related peptides isolated from the egg jelly of sea urchins,Tripneustes gratilla,Pseudoboletia maculata,Strongylocentrotus nudus,Echinometra mathaei and Heterocentrotus mammillatus

• 1.1. Twenty-eight peptides were isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Strongylocentrotus nudus, Echinometra mathaei (type A and B) and Heterocentrotus mammillatus and their amino acid sequences were determined.
• 2.2. Two of the peptides obtained from T. gratilla egg jelly possessed a bromophenylalanine (Br-Phe) residue in their sequences (Gly-(Br-Phe)-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly and Gly-(Br-Phe)-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly).
• 3.3. All of the peptides elevated cyclic GMP concentrations in the spermatozoa of the respective sea urchin and caused a shift in the apparent mol. wt of a major sperm protein of the respective sea urchin.
• 4.4. They stimulated respiration rates of the spermatozoa of Hemicentrotus pulcherrimus as well as their own species.
• 5.5. One-half maximal concentrations of the peptides for respiratory stimulation of H. pulcherrimus spermatozoa were between 10⁻¹¹ M and 10⁻⁹ M except a methionine-containing peptide which was about 10⁻⁷ M.

     

Characterisation of the electron transport chain of an obligate methylotroph,strain 4025

• 1.1. The obligate methanol-utilising bacterium strain 4025 contains cytochromes b and c. Cytochrome a is never present.
• 2.2. The soluble cytochrome c is similar to that from other methylotrophs in reacting (slowly) with carbon monoxide and it can be separated into two types, differing markedly in their isoelectric points.
• 3.3. Some of the cytochrome b reacts rapidly with carbon monoxide and is thus the likely cytochrome oxidase (cytochrome o).
• 4.4. The partially purified, NAD⁺-independent methanol dehydrogenase is similar to such enzymes from the other methanol-utilising bacteria in respect of its prosthetic group, dependence on ammonia or methylamine for activity and its wide substrate specificity.
• 5.5. The fluorescence seen in colonies of this organism is probably due to a flavin derivative.
• 6.6. This study of electron transport components does not shed any light on the unusually high copper requirement shown by this methylotroph.

     

The response of Mytilus edulis to short duration hypoosmotic stress

• 1.1. Valve movements monitored from Mytilus edulis acclimated to 34‰ SW during exposure to low salinity indicate that the shell is not used to isolate the mussel from hypoosmotic SW when the salinity is 17‰ or greater.
• 2.2. The water content of the whole animal, gill and posterior adductor muscle tissue; hemolymph osmotic pressure, Na +, and Cl⁻ concentrations were determined hourly for 9 hr in mussels with valves propped open and exposed to five hypoosmotic salinities from 25.5 to 3.4‰.
• 3.3. Water loading was essentially complete within the first 2hr of exposure: whole animal water content increased 26.51 g H₂O/100g wet tissue in 11.2‰ while the gill tissue water content increased only 2.48 g H₂O/100g wet tissue. During the first 2hr of exposure hemolymph osmotic pressure decreased 200–250 mOsm, regardless of salinity, and at all salinities below 25.5‰ the hemolymph remained hyperosmotic to the medium for the 9 hr of the experiment.
• 4.4. When taken together, these data suggest physiological mechanisms other than valve closure are responsible for tolerance of short duration hypoosmotic stress in Mytilus edulis.