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

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

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 Km = 28±2.1mM) than for glycine (apparent Km = 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 Kmvalues 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 (Kmvalues 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