Jeremy H. A. Fields

Alanopine and strombine are novel imino acids produced by a dehydrogenase found in the adductor muscle of the oyster, Crassostrea gigas

Abstract Previous work on anaerobic metabolism in the heart of the oyster indicated the formation of an unidentified compound as a product of amino acid utilization during anoxia. A dehydrogenase, present in the adductor muscle and heart of the oyster, was found to utilize reduced nicotinamide adenine dinucleotide, pyruvic acid, and alanine or glycine as substrates. The stoichiometry of the reaction suggested the enzyme was similar to octopine dehydrogenase (EC 1.5.1.11), which is known to form the imino acid N 2 -(1-carboxyethyl)-arginine (octopine). Incubation of the enzyme with reduced nicotinamide adenine dinucleotide, alanine, and pyruvic acid gave a product identified as 2,2′-iminodipropionic acid (alanopine) on the basis of both physical and chemical data. Either synthetic alanopine or strombine reacted with the enzyme and nicotinamide adenine dinucleotide, which indicated the free reversibility of the reaction. This enzyme probably functions in the maintenance of a redox balance during anaerobiosis in the adductor muscle and heart of the oyster.

Some theoretical considerations on cytosolic redox balance during anaerobiosis in marine invertebrates

Abstract Studies on anaerobiosis in marine invertebrates have shown that many rely on malate, octopine, or alanopine dehydrogenases, rather than lactate dehydrogenase, for cytosolic redox balance. These systems were studied by computer simulations with the assumption that these dehydrogenases maintain their substrates and products at instantaneous equilibrium. The simulations permit a study of the redox ratio (NADH/NAD+) as a function of the concentration of lactate, malate, or octopine. The redox ratio was found to increase as these products accumulated. It was substantially less in the simulations of malate and octopine dehydrogenases when compared to those of lactate dehydrogenase. This factor may be important for maintaining glycolysis in these organisms, and suggests an advantage for the use of octopine dehydrogenase rather than its analogue lactate dehydrogenase.

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa

1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.

Tissue-specific alanopine dehydrogenase from the gill and strombine dehydrogenase from the foot muscle of the cherrystone clam Mercenaria mercenaria (Linn.)

Abstract Tissue-specific forms of alanopine and strombine dehydrogenases were detected in the bivalve mollusc, Mercenaria mercenaria (Linn.), by isoelectrofocusing. Alanopine dehydrogenase had a pI of 5.0–5.25 and predominated in the gill and mantle while strombine dehydrogenase had a pI of 4.75–4.80 and predominated in the adductor and foot muscles. The enzymes from gill and foot muscle were partially purified, about 20-fold each, to final specific activities of 3 and 37μmol·min −1 ·mgprotein −1 , respectively. The alanopine dehydrogenase from gill had a much higher affinity for alanine (apparent K m = 28±2.1mM) than for glycine (apparent K m = 291±40 mM) and showed substantial activity with a variety of other neutral L-amino acids. The strombine dehydrogenase from foot muscle had similar affinities for alanine and glycine (apparent K m values were 242±11 and 173±1.3 mM, respectively) but apart from these two amino acids and L-2-aminobutyrate showed a restricted use of other L-amino acids. Affinities for pyruvate were comparable for both enzymes ( K m values were0.38±0.05mM and 0.32±0.04 mM for the gill alanopine dehydrogenase and foot muscle strombine dehydrogenase, respectively). Both enzymes function as the terminal dehydrogenase of glycolysis in their respective tissues and have roles in maintaining energy production under the stresses of environmental (low-tide exposure) or functional (burst muscle work) anoxia. The distribution of the two enzyme types, coupled with the glycine (8.0 and 2.9μmol·gwetwt −1 in foot and gill) and alanine (18.9 and 3.8μmol·g − in foot and gill) contents of the two tissues, accounts for the dominance of alanopine in soft tissues of the clam and the appearance of near equal amounts of the two imino acids in muscles.

NAD(+)-linked isocitrate dehydrogenase in fish tissues.

NAD+-linked isocitrate dehydrogenase was found in the brain, heart, gills, kidney, liver and muscle of trout, and in the liver and muscle of eel. A complex homogenization buffer containing 1 mM ADP, 5 mM MgSO4, 5 mM citrate and 40% glycerol is required for retrieval of significant amounts of stable enzyme. The highest activities were found in brain of trout and the lowest in white muscle of trout and eel. The enzyme was partially purified from frozen trout heart to a final activity of 0.04 μM/min/mg protein, and the kinetic properties of this partially purified enzyme were studied. The enzyme requires either Mn2+ or Mg2+ for activity, higher activities being observed with Mn2+. Saturation kinetics for DL-isocitrate were sigmoidal, apparent S0·5=8.2±0.6 mM and nH=1.8±0.2, in the absence of ADP, changing to hyperbolic, apparent S0·5=1.4±0.3 mM and nH=1.0, with 1 mM ADP added. Citrate and Ca2+ were found to activate the enzyme to a small extent. NADH strongly inhibited the enzyme, I50=3.7±0.5 μM. ATP was also found to be an inhibitor, I50=7.2±1.4 mM. These properties are consistent with the role of the enzyme as a major control site of the tricarboxylic acid cycle.

A NOTE ON ANAEROBIC METABOLISM IN CALLINECTES SAPIDUS DURING THE INTERMOLT CYCLE

ABSTRACT Several tissues excised from stage A individuals of Callinectes sapidus accumulated L-lactate and, to a limited extent, alanine when exposed to hypoxia in vitro. Succinate did not accumulate, suggesting that alternative pathways of anaerobic metabolism are not used in early postmolt stages. In premolt animals hexokinase activity rose in the epidermis but not in the swimming and heart muscles. Pyruvate kinase and lactate dehydrogenase decreased in swimming muscle but not in other tissues of premolt crabs. No changes in glucokinase activity, which was very low, were detected in any premolt tissues. The kinetic properties of the pyruvate kinase and lactate dehydrogenase found in swimming muscle of stage C animals were similar to those reported for other crustaceans. The lactate dehydrogenase is more suited for lactate production than utilization, which is correlated with the dependence on anaerobic metabolism during sustained motor activity.

Effects of anoxia and quiescence on pyruvate kinase from the foot of the mangrove snail Littorina scabra angulifera Lamarck

Adult mangrove snails, Littorina scabra angulifera Lamarck, live above the high water mark whereas the juveniles are found intertidally. The adults have behaviors similar to those of terrestrial snails, particularly a quiescent phase in response to dry conditions. This study reports the changes observed in some kinetic parameters of pyruvate kinase during quiescence and exposure to anoxia. The apparent Km(PEP) of pyruvate kinase from the foot increased from 0.080 ± 0.02 mmol·l−1 to 0.17 ± 0.04 mmol·l−1 during 7 days of quiescence. There was no change in the activity of the enzyme in the tissue during the quiescent period. Exposure to anoxia for 12 h increased the apparent Km(PEP) from 0.080 ± 0.02 mmol·l−1 to 1.2 ± 0.4 mmol·l−1 while the activity decreased from 13.6 μmol·min−1·g wet wt.−1 to 4.7 μmol·min−1·g wet wt.−1. The changes induced by quiescence were readily reversed on forcible immersion for 1 h; those induced by anoxia were reversed when the animals were placed in air for 4 h. Alkaline phosphatase treatment could reverse the changes induced by anoxia, but had no effect on the enzyme from quiescent animals. This would suggest that anoxia induces a phosphorylation of the enzyme, but the mechanism underlying the changes during quiescence is different in some way.