ISSN 2756-3855
International Journal of Urology and Nephrology ISSN 2091-1254 Vol. 7 (6), pp. 001-008, June, 2019. © International Scholars Journals
Full Length Research Paper
Purification and characterizations of NAD dependent isocritrate dehydrogenase from human kidney mitochondria
Mukaram Shikara
Biotechnology Division, Department of Applied Sciences, University of Technology, Baghdad, Iraq. E-mail: [email protected].
Accepted 12 February, 2019
Abstract
NAD -IDH (Nicotinamide adenine dinucleotide socitric dehydrogenase) has been purified in 1216-fold with a total recovery of 7.5% from the mitochondria of human kidney by using a combination of affinity chromatography with the anion-exchange matrix that allowed obtaining a preparation of high purity. The shape of the peak from Sephacryl S-100 and the results from SDS-PAGE confirm that the enzyme is a tetramer with subunits of 80,000 each, and a native molecular mass of about 320,000. The enzyme shows activity in the absence of any divalent metal ions, but Mn +2 is a better activator than Mg+2 at lower concentrations (0.5 mM), but it will inhibit the enzyme at higher concentrations (2 mM). ATP (Adenosine triphosphate) and NADH inhibit the enzyme competitively according to Lineweaver-Burk plot. The NAD-IDH does not indicate a homotropic cooperative effect of isocitrate in either the absence or presence of ADP. Increasing concentrations of NAD decrease the Km of isocitrate while increasing levels of isocitrate lower the Km of NAD. Km of either isocitrate or NAD is lowered further in the presence of ADP. The inhibition by ATP (or NADH) cannot be counteracted by ADP in the presence of isocitrate, so ADP cannot enhance NAD-IDH activity nor reverse inhibition by ATP (or NADH), while isocitrate will bind to the enzyme and prevent it from interacting with ADP. The activity of NAD-IDH in mitochondria is probably controlled in a complex way by NADH, ATP and divalent ions.
Key words: Nicotinamide adenine dinucleotide socitric dehydrogenase, isocitrate, Adenosine triphosphate, Nicotinamide adenine dinucleotide hydrogenase, human kidney, purification.