Malate Dehydrogenase (MDH, EC 1.1.1.37）is widely exist in animal, plant, microbe and cells culture.
MDH in mitochondria is one of the key enzymes of TCA cycle, which catalyzes the formation of
oxaloacetic acid from malic acid. In contrast, MDH in serum catalyzes the formation of malic acid from
oxaloacetic acid. Oxaloacetic acid is an important intermediate that connects several important metabolic
pathways. MDH plays an important role in many physiological activities of cells, including mitochondrial
energy metabolism, malic acid-aspartic acid shuttle system, reactive oxygen species metabolism and
disease resistance. According to the different coenzyme specificity, MDH is divided into NAD- dependent
MDH and NADP- dependent MDH. Bacteria usually only contain NAD-MDH. NAD-MDH is distributed
in the cytoplasm and mitochondria in eukaryotic cells.
NAD-MDH catalyzes NADH to reduce oxaloacetic acid into malic acid, which resulting in a decrease in
absorption at 340nm.
Required but Not Provided:
Ultraviolet spectrophotometer,desk centrifuge, water-bath, adjustablepipette, 1mL quartzcuvette, distilled
water.
Protocol
I. Preparation:
1. Cells or bacterial
Collect bacteria or cells into the centrifuge tube. The liquid in the upper layer is discarded after
centrifugation. It is suggested to take about 2 million bacteria/cell and add 400μLof Extractsolution.
Bacteria/cell is splitby ultrasonic(power 20%, ultrasonic 3s, interval 10s, repeat for 30 times). Centrifuge at
8000 ×g and4℃ for 10minutes. Take the supernatant and placed it on ice for testing.
