Studies on the cytoplasmic malate dehydrogenase from a larval halophilic crustacean, Artemia salina Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/th83m198s

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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 with a protocol involving ammonium sulfate fractionation and chromatography on DEAE-cellulose, followed by sequential passes over cellulose phosphate columns at pH 5.8 then 5.0. The purified enzyme is free of glutamateoxaloacetate transaminase and lactate dehydrogenase activities and has specific activities of 550-570 uM NAD⁺/min/mg protein (oxaloacetate reduction) or 115 uM NADH/min/mg protein (malate oxidation). Holoenzyme polyacrylamide gel electrophoresis resolved at least 3 catalytically active s-MDH subforms with no observable non-MDH protein present on the gels. 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 75,000 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 h. Michaelis constants in 0.05 M tris buffer, pH 8.0, at 25°C are 4.2 X 10⁻⁵ M for oxaloacetate and 1.5 X 10⁻⁵ M for NADH, and the molecular activity is 41,000. Substrate inhibition by oxaloacetate or malate is not observed at low concentrations, but begins at 7-10 mM and 20-30 mM, respectively. Monospecific rabbit antiserum was produced against purified s-MDH and used for rocket IEP as a quantitative assay for the cytoplasmic isozyme. Since cross-reactivity is not observed against the mitochondrial MDH (m-MDH), the assay allows specific measurement of the s-MDH in crude naupliar supernatants in the presence of contaminating m-MDH. The assay has a sensitivity of approximately 100 nanograms s-MDH protein with a 4% standard deviation using either the purified enzyme or supernatant preparations. Catalytic inhibition studies using the monospecific antiserum gave 85% inhibition of s-MDH but no significant inhibition of the brine shrimp m-MDH. Lack of complete s-MDH inhibition by the antiserum suggests a difference between the enzyme active site and immunological binding site. Porcine s- and m-MDH, and beef and pigeon m-MDH were also tried as antigens against the brine shrimp s-MDH antiserum, and reactivity was not achieved with the enzymes during rocket IEP or catalytic inhibition experiments. Brine shirmp cytoplasmic MDH is a component of an energy-yielding glycolytic pathway which may play a role in meeting the energy demands imposed on nauplii in high salt environments. To investigate this possibility, it is necessary to quantitatively measure s-MDH levels under varying developmental and environmental conditions. Brine shrimp nauplii challenged with fortified sea water (2.5 M NaC1) maintain significantly higher levels of cytoplasmic malate dehydrogenase (s -MDH) than larvae incubated in sea water. Eight to ten hours after emergence of freeswimming nauplii in sea water, s-MDH exhibits a steady decline for 20 to 40 hours; the decrease is smaller and stabilizes sooner in nauplii incubated in fortified sea water. Incorporation of H¹⁴C0₃ into s-MDH protein was assayed using quantitative rocket immunoelectrophoresis (IEP) with monospecific antiserum prepared against purified brine shrimp s-MDH. A 40% faster rate of enzyme biosynthesis is observed in high salt, and together with the rapid s-MDH turnover (half-life of approximately one day), probably accounts for the difference in level between salt treatments. In contrast, H¹⁴C0₃ incorporation into total TCA-precipitable protein in supernatant preparations decreases slightly in high salt, indicating a preferential synthesis of s-MDH. The results are discussed in relation to the bioenergetics and temporal development of water and electrolyte regulation in hypersaline environments.
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