Cytosolic dehydrogenase activity

Enzyme activities were detectable only in protein extracts derived from cell cultures after induction with the corresponding substrates. Two different enzyme activities were shown to be involved in this subpathway, one located in the microsomal fraction, and one with a typical cytosolic dehydrogenase activity. 

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. 

Because the metabolism of DEHP was catalyzed by so many fractions of the trout liver homogenate, these fractions were characterized by measurement of marker enzymes to determine which organelles actually were responsible for the observed DEHP metabolism. Succinic dehydrogenase activity was used as a marker for mitochondria, whereas glucose-6-phosphatase was used as a marker for microsomes. The distribution of DEHP oxidase activity (production of polar metabolites 1 and 2 with added NADPH) and of DEHP esterase activity (production of monoester without added NADPH) were also determined. It was found (Figure 2) that the distribution of DEHP oxidase activity parallels the distribution of microsomal activity and the distribution of DEHP esterase activity parallels the distribution of microsomal activity, but is also present in the cytosol fraction. 

Shimazaki, Y, Sugarawa, Y Activity and sequence structure analysis of cytosolic dehydrogenase by mass spectrometry after separation by nondenaturing two-dimensional electrophoresis. Ancd. Biochem. 2004, 328, 87-89. 

Meaden, P. G., Dickinson, F. M., Mifsud, A., Tessier, W., Westwater, J., Bussey, H., Midgley, M. (1997) The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic, Mg -activated acetaldehyde dehydrogenase. Yeast, 13, 1319-1327. 

A 3a-hydroxysteroid dehydrogenase active on 7a-hydroxy-5 -cholestan-3-one and 7a,12a-dihydroxy-5 -cholestan-3-one (cf. Fig. 3), was partially purified (about 300-fold) from rat Uver cytosol by Berseus [112,113]. NADPH was required as cofactor and hardly any activity was observed with NADH. The preparation was also active towards 3-oxo steroids of the Cjg, C21 and C24 series, and in these cases appreciable activity was obtained also with NADH. The mechanism of reduction involves a stereospecific transfer of a hydride ion from the 4A position of NADPH to the 3)S position of the steroid [114],... 

The stereochemistry of the reduction was elucidated by use of [4A- H]NADPH, [4B- H]NADPH or the comparable forms of NADH only the tritium from the [4A- H]NADPH or [4A- H]NADH was incorporated into the product, probably in the 3 position [170]. Whether more than one 3-hydroxysteroid dehydrogenase active on 3-oxo-bile acids exists in cytosol of liver cells remains to be determined. 

Two pathways have been described for formate metabolism a peroxidatic pathway via catalase and a folate dependent one carbon pathway. Treatment of rats with amino-triazole, an irreversible inhibitor of catalase, severely depressed a-oxidation activity but only slightly decreased the production of CO2 from exogenously added formate. " Whether the effect of aminotriazole is (solely) linked to the inhibition of catalase is unclear (see further). In addition to these above mentioned pathways our data point to a cytosolic NAD -dependent dehydrogenase activity that acts on the formate produced during a-oxidation. In peimeabilized cells or broken systems, suppUed with the qi-propriate cofactors (see further), almost no CO2 is formed, during a-oxidation unless NAD is added. ... 

The inset shows that if cells were maintained continuously in exponential phase, then shikimate dehydrogenase activity leveled off after eight generations to a lower activity than was ever reached in ordinary subcultures. It will be interesting to see whether stationary-phase cultures might yield a cytosolic form of shikimate dehydrogenase that has not been seen in extracts thus far studied from exponential-phase cells. 

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