Mitochondria enzyme activity

In addition to the foregoing more general concerns are questions concerning the localization of an enzyme activity. The location of an enzyme can determine the type of cell lysis, since it could be more advantageous to lyse the cell completely or in such a manner that the organelles are left intact. For example, some lysis methods such as sonication completely disrupt mitochondria, nuclei, and Golgi systems. If an activity is localized in an organelle such as a mitochondrion, it would seem sensible to adopt a method that leaves these structures intact, to facilitate their separation from the rest of the cellular debris. Thus, for the isolation of mitochondrial enzymes, sonication is not the method of choice for cell lysis. 

In Vitro Studies. An assay for measuring taurine N-acyltransferase activity with C-phonylacetyl CoA as substrate has boon described (79). Enzyme activity was located In the matrix of the mitochondrion and was usually higher In kidney than liver for the nine fish species studied. Studies with Isolated renal tubules from the winter flounder and the southern flounder (84 and James and Pritchard, unpublished) have shown that added phenylacetlc acid and benzoic acid were extensively metabolized to their taurine conjugates during a 60 minute Incubation. 

As we noted in Chapter 16, the enzymes of many metabolic pathways are clustered (p. 605), with the product of one enzyme reaction being channeled directly to the next enzyme in the pathway. In the urea cycle, the mitochondrial and cytosolic enzymes appear to be clustered in this way. The citrulline transported out of the mitochondrion is not diluted into the general pool of metabolites in the cytosol but is passed directly to the active site of argininosuccinate synthetase. This channeling between enzymes continues for argininosuccinate, arginine, and ornithine. Only urea is released into the general cytosolic pool of metabolites. 

In bacteria and green plants PEP carboxylase (Eq. 13-53), a highly regulated enzyme, is responsible for synthesizing oxaloacetate. In animal tissues pyruvate carboxylase (Eq. 14-3) plays the same role. The latter enzyme is almost inactive in the absence of the allosteric effector acetyl-CoA. For this reason, it went undetected for many years. In the presence of high concentrations of acetyl-CoA the enzyme is fully activated and provides for synthesis of a high enough concentration of oxaloacetate to permit the cycle to function. Even so, the oxaloacetate concentration in mitochondria is low, only 0.1 to 0.4 x 10-6 M (10-40 molecules per mitochondrion), and is relatively constant.65 79... 

There are also inhibitors that affect enzyme synthesis. Inhibitors of transcription (e.g., dibromothymoquinone [DBMIB]) and inhibitors of translation (e.g., cyclo-heximide [CHX]) are available but these are not specific to particular enzymes. In addition, because protein synthesis takes place within the chloroplast and mitochondrion in eukaryotes, prokaryotic protein synthesis inhibitors (e.g., chloramphenicol [CAP]) may be necessary to distinguish prokaryotic versus eukaryotic activity (e.g., Segovia and Berges, 2005). 

The malic enzyme/citrate lyase pathway is shown in Figure 5.10. The 2-carbon units acetyl groups) for fatty acid synthesis are supplied by the activity of citrate lyase, which may be considered an enzyme of fatty acid biosynthesis. The reduced NADP is Supplied at the point of malic enzyme. Figure 5.10 reveals no net production or utilization of NAD in the cytoplasm. The NADPH + H generated in the cytoplasm is used for fatly acid synthesis, which regenerates NADP. One molecule of CO is produced in the cytoplasm. The diagram reveals no net production or utilization of CO in the mitochondrion. One molecule of NAD is... 

B. This compound is acetoacetate, which is synthesized in the liver when blood insulin levels are low. HMG CoA synthetase is the key regulatory enzyme for synthesis, not oxidation. Acetoacetate is transported to tissues, such as muscle, where it is activated in the mitochondrion by succinyl CoA (not ATP), cleaved to 2 acetyl CoA, and oxidized via the TCA cycle, which requires the vitamin thiamine as thiamine pyrophosphate, a cofactor for a-ketoglutarate dehydrogenase. Biotin is not required. 

Actively respiring fungal cells possess a distinct mitochondrion, which has been described as the power-house of the cell (Fig. 4.2). The enzymes of the tricarboxylic acid cycle (Kreb s cycle) are located in the matrix of the mitochondrion, while electron transport and oxidative phosphorylation occur in the mitochondrial inner membrane. The outer membrane contains enzymes involved in lipid biosynthesis. The mitochondrion is a semiindependent organelle as it possesses its own DNA and is capable of producing its own proteins on its own ribosomes, which are referred to as mitoribosomes. 

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