Purification and properties of cytoplasmic malate dehydrogenase isolated from a larval crustacean, Artemia salina

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The cytoplasmic isozyme of malate dehydrogenase (s‐MDH) has been isolated from brine shrimp nauplii to a state of high purity. The purified enzyme is free of glutamate‐oxaloacetate transaminase and lactate dehydrogenase activities and has specific activities of 550–570 μM NAD+/min/mg protein (oxaloacetate reduction) or 115 μM NADH/min/mg protein (malate oxidation). Nondenaturing polyacrylamide gel electrophoresis resolved at least three catalytically active s‐MDH subforms with no observable non‐MDH protein present. The enzyme displays a single uniform boundary in sedimentation velocity centrifugation which yields a S20,w value of 4.4. From sedimentation equilibrium a molecular weight of 75,300 is calculated, and a break in the plot of In C versus r2 is evident for the low protein concentration portion of the boundary, suggesting a dimer–monomer dissociation and/or a low molecular weight microcontaminant. Sodium dodecyl sulfate polyacrylamide gels give an apparent molecular weight of 36,000–38,000 for the s‐MDH subunit. The cytoplasmic isozyme has a pH optimum of 8.0 for oxaloacetate reduction and exhibits low susceptibility to thermal denaturation, with less than 10% loss of catalytic activity observed at 48°C in 1 hour. An Arrhenius plot gives a linear relationship with no transitions and an estimated molar activation energy of 9.75 Kcal/mole for the overall catalytic step. Michaelis constants in 0.05 M Tris/HCL buffer, pH 8.0, at 25°C are 4.2 × 10−5 M for oxaloacetate and 1.5 × 10−5 M for NADH, and the molecular activity (kcat) is 42,000. Substrate inhibition by oxaloacetate or malate is not observed at low concentrations, but begins at 7–10 mM and 20–30 mM, respectively. Copyright © 1981 Wiley‐Liss, Inc., A Wiley Company

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Journal of Experimental Zoology

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