The purification and steady state kinetic characterisation of rat liver mitochondrial malate dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Thumbnail Image
Open Access Location
Journal Title
Journal ISSN
Volume Title
Massey University
The Author
Mitochondrial malate dehydrogenase (mMDH) is one of the enzymes in the malate-aspartate shuttle system which facilitates the movement of reducing equivalents across the mitochondrial membrane. The metabolism of ethanol in the cytosol of liver tissue requires large amounts of NAD+ and produces NADH, which must be oxidised back to NAD+ in the mitochondria. The transfer into the mitochondria is carried out by the shuttle system which may thereby contribute significantly to the control of ethanol metabolism under certain circumstances. In the shuttle system mMDH acts in the opposite direction to the cytosolic form of the enzyme. Is this due to differences in the functioning of the two isozymes or to differences in their environments? In this study a new method for the purification of mMDH from rat liver has been developed. This method was developed to be quick and to cause as little disruption to the protein structure of the enzyme as possible. The scheme involves the partial purification of mitochondria by centrifugation followed by elution, by NADH, off carboxymethyl cellulose chromatography resin. The whole process takes two days, because of the need to dialyse the sample to remove NADH, before any kinetic studies can be carried out. The specific activity of the purified enzyme compares favourably with other published values and the enzyme shows up as a single band on an SDS polyacrylamide electrophoresis gel. The major problem with the enzyme produced was its lack of stability during storage. The second part of the project was to carry out steady state kinetic experiments with the purified enzyme under conditions that simulate the in vivo state. These are 37°C, pH 7.4, and 0.1 M phosphate buffer. The dissociation and Michaelis constants, for other tissues, have been published before but not those for rat liver. Very few of these experiments have been carried out under these near physiological conditions. The kinetic parameters produced are (µM): Ka, 72; Kia, 12; Kb, 86; Kib, 18; Kp, 1900; Kip, 15000; Kq, 170; Kiq, 1100; where a = NADH, b = oxaloacetate, p = malate, and q = NAD+. Some of these values have extremely high standard errors and so need further refining. The values produced in this work can be compared to those for cytosolic malate dehydrogenase (Crow et al., 1982) and because the two sets of constants are quite similar it appears that the different modes of action in vivo are mostly due to differences in the environment of the two isozymes.
Malate dehydrogenase, Purification