Isocitrate dehydrogenase, activation

It may seem surprising that isocitrate dehydrogenase is strongly regulated, because it is not an apparent branch point within the TCA cycle. However, the citrate/isocitrate ratio controls the rate of production of cytosolic acetyl-CoA, because acetyl-CoA in the cytosol is derived from citrate exported from the mitochondrion. (Breakdown of cytosolic citrate produces oxaloacetate and acetyl-CoA, which can be used in a variety of biosynthetic processes.) Thus, isocitrate dehydrogenase activity in the mitochondrion favors catabolic TCA cycle activity over anabolic utilization of acetyl-CoA in the cytosol. 

Kulkybaev GA, Merkusheva NV. 1992d. Time course of NAD-dependant isocitrate dehydrogenase activity in homogenized rat liver exposed to toxic products of the phosphoms industry. Gig Tr Prof Zabol 0(l) 21-23. 

In the present experiment, you will determine the activity of isocitrate dehydrogenase extracted from pork heart muscle. The commercial preparation comes in powder form and it uses NADP+ rather than NAD+ as a coenzyme. The basis of the measurement of the enzyme activity is the absorption spectrum of NADPH. This reduced coenzyme has an absorption maximum at 340 nm. Therefore, an increase in the absorbance at 340 nm indicates an increase in NADPH concentration, hence the progress of the reaction. We define the unit of isocitrate dehydrogenase activity as one that causes an increase of 0.01 absorbance per min. at 340 nm. 

What other reactant or product concentration could be used to measure the isocitrate dehydrogenase activity ... 

If your powder extract contained 80% protein, what would be the average isocitrate dehydrogenase activity per mg protein (enzyme) ... 

Roy, S. O., and Packard, T. T. (2001). CO2 production rate predicted from isocitrate dehydrogenase activity, intracellular substrate concentrations and kinetic constants in the marine bacterium Pseudomonas nautica. Mar. Biol. (Berl.) 138, 1251-1258. 

Isocitrate dehydrogenase (Activated by ADP, inactivated by NADH) ot-Ketoglutarate dehydrogenase (Inhibited by Succinyl-CoA and NADH)... 

The isocitrate dehydrogenase activity in human serum has been determined by kinetic assay at 37°C, with isocitrate and NADP as substrates. The rate of formation of NADPH was amperometrically monitored and the selectivity was improved by covering the active surface of the electrode with a poly(o-phe-nylenediamine) film. The electroimmobilization of the enzyme monoamine oxidase into a polypyrrole film around a platinum electrode has been used to determine the antidepressant drug fluoxetine in pharmaceutical preparations. 

The first breakthrough came when Schneider and Hogeboom [81] directly measured NADP isocitric dehydrogenase activity and demonstrated that only 10% of the total enzyme activity is present in the mitochondrial fraction. According to their estimate, this amount is not sufficient to explain the normal oxidation rate of the Krebs cycle metabolites. Assuming that the NADP isocitric dehydrogenase in the mitochondria is a contaminant, they concluded that at least some of the enzymes involved in the Krebs cycle are extrami-tochondrial. 

The operation of the Krebs citric acid cycle is first of all regulated by the availability of ADP to the respiratory chain. When little or no ADP is present, the flow of reducing equivalents down the respiratory chain is limited. As a result, a marked fall in the flux of substrates along the cycle is observed. In mammalian mitochondria ADP could control the rate of the citric acid cycle also by virtue of its activating effect on isocitric dehydrogenase activity. 

The efficiency of this pathway appears doubtful, since no good correlation exists between isocitrate dehydrogenase activity and the intensity of lipogenesis (Young et al., 1964). This pathway is probably active in the locust (Walker and Bailey, 1969). 

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