Abstract
Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate using Nicotinamide Adenine Dinucleotide (NAD+) as coenzyme. This paper communicates the results of first ever study on the large scale purification, characterization, cDNA cloning and sequence analysis of mitochondrial isozyme of malate dehydrogenase from the heart of River Buffalo (Bubalus bubalis). The enzyme was purified up to 1050 folds with 17.6% recovery by selective ammonium sulfate and acetone precipitations followed by cation exchange chromatography with Carboxymethyl Sephadex (CM-Sephadex). The purified sample displayed specific activity of 3250 units per mg of the enzyme and Km value of 32 mu M for oxaloacetate. Optimum activity of purified enzyme was measured at 35 degrees C and pH 7.5. The enzyme is a dimmer of 66kDa as determined by gel filtration chromatography. MALDI-TOF analysis has shown the subunit molecular weight of 33873.7 Da. The cDNA consisting of 1017 bp was generated by in vitro reverse transcription that encodes for 338 amino acids. The nucleotide sequence of cDNA encoding B. bubalis mitochondrial malate dehydrogenase has been compared with that of various mammalian species. Present study is a useful beginning towards cataloguing the genetic makeup and characteristics of encoded enzymes from B. bubalis, an unexplored species of great livestock importance in Asia.