Malic dehydrogenase

Malic dehydrogenase catalyzes the reversible conversion of oxaloacetate and malate and is closely associated with the oxidation and reduction of dinucleotide coenzymes. Malic dehydrogenase serves as a fundamental enzyme in cellular metabolism and is widely utilized in research involving energy metabolism, mitochondrial function, redox homeostasis, and enzymatic reaction networks.

Method: Partially purified pig-heart mitochondrial malic dehydrogenase was studied at 25°C in Tris-acetate buffer of ionic strength 0.05 at pH 8.0. Initial-rate and product-inhibition experiments compared oxaloacetate with the alternative substrate ketomalonate; kinetic constants were extrapolated to zero-order conditions for substrate and coenzyme.
Rresult: With oxaloacetate versus ketomalonate, maximum initial velocity was 6.3±0.5×10⁴ versus 2.6±0.2×10⁴ min⁻¹; the DPN dissociation constant was 4.9±0.3×10⁻⁴ versus 2.2±0.1×10⁻⁴ M; the DPNH dissociation constant was 3.9±1.0×10⁻⁶ versus 2.4±0.2×10⁻⁶ M; and the DPNH Michaelis constant was 1.4±0.2×10⁻⁵ versus 1.2±0.2×10⁻⁵ M. The proportional reductions in velocity and DPN dissociation supported a reciprocating compulsory-order mechanism involving interacting enzyme subunits.[1]
Method: L-(−)-Malate was determined enzymatically using malic dehydrogenase and diphosphopyridine nucleotide. An alkaline medium was used to bind generated protons, and oxaloacetate was trapped as its hydrazone to shift the equilibrium toward malate oxidation. DPNH formation was monitored spectrophotometrically. The recommended MDH preparation had a specific activity of at least 2,000 units/mg according to the Bücher definition or 36 units/mg according to the Racker definition; contamination with lactate dehydrogenase or glycerol-1-phosphate dehydrogenase was required to be below 0.05% of MDH activity.
Rresult: Relatively high concentrations of DPN and malic dehydrogenase, together with oxaloacetate trapping, enabled quantitative and sufficiently rapid oxidation of L-(−)-malate for spectrophotometric determination. [2]

H2O: 80 mg/mL, Sonication is recommended.