The Chemical Activities of Mitochondria

Throughout the course of work on the vitamin D3 metabolism, parallel work has occasionally been carried out with the vitamin D2 series. 25-OH-D2 has also been isolated and chemically identified ° Furthermore, 1,25-(OH)2D2 has been prepared in vitro with chick kidney mitochondria and its structure unequivocally elud-dated" The biological activity of the vitamin D2 compounds has been assessed in the rat and found to be identical in every respect to the vitamin D3 compounds. In the bird, however, as described above, the vitamin D2 compounds, including 1,25-(0H)2D2, are approximately 1/5 to 1/10 as active as their vitamin D3 counterparts Of some interest is that Upjohn chemists have successfully synthesized the 25-OH-D2 and the 25-OH-24-epiD2. The 25-OH-24-epiD2 much less active than 25-OH-D2 (J. A. Campbell, L. Reeve and H. F. DeLuca, unpublished results). 

The chemical compositions of melanosoma and mitochondria have been determined and compared, using differential and density-gradient techniques 241). The data show that melanosomal fractions contain a high percentage of Zn and a low content of RNA and phospholipid-P as compared with mitochondrial fractions. The lack of succinoxidase and glutamate oxidase activities support the idea that melauosomes are sub-cellular particles, different from mitochondria, and contain a particular specialized metabolic pathway through which tyrosine or dopa is converted into melanin (259). 

Some of the chemical properties of the phosphorylating particle have also been elucidated. The particle consists mainly of two basic components, protein (65%) and phospholipids (20%). In addition to the various components of the electron transport chain, the phosphorylating particle also contains ATPase, the activity of which is so high that it may well explain the particle s low phosphorylating ability. Like that associated with mitochondria, the ATPase found in the particle is a highly specific enzyme that splits ATP to yield ADP, and is without effect on inosine, guano-sine, or cytidine triphosphate. There is more bound copper and iron in these particles than can be accounted for by the ferroproteins and cuproproteins of the electron transport chain. 

The chemical structure of the hormone that is active under physiological conditions is still under investigation. Although thyroxine administration undoubtedly corrects the metabolic and clinical alterations that occur after thyroidectomy, and although thyroxine acts on mitochondria in vitro, these observations constitute no proof that thyroxine is the compound that is active in normal physiological conditions. The belief that thyroxine is the active hormone was shattered when 3,5, 3 -triiodothyronine was isolated from blood and thyroid and when it was established that triiodothyronine is more potent than thyroxine on a molecular basis. The discovery of active compounds different from thyroxine has stimulated more research on the physiological effects of thyroxine analogs. 

In contrast to the regulation of many other enzymes, there is no evidence for any chemical factors which directly influence the activity of the H,K ATPase other than the availability of the necessary substrates Mg ATP, H and K. Since within the parietal cell the availability of protons and Mg ATP are not likely to be rate-limiting given the large number of mitochondria, it follows that the major, if not the only, factor which controls proton transport is the availability of at the extracytosolic surface of the H,K... 

In addition to effects on biochemical reactions, the inhibitors may influence the permeability of the various cellular membranes and through physical and chemical effects may alter the structure of other subcellular structures such as proteins, nucleic acid, and spindle fibers. Unfortunately, few definite examples can be listed. The action of colchicine and podophyllin in interfering with cell division is well known. The effect of various lactones (coumarin, parasorbic acid, and protoanemonin) on mitotic activity was discussed above. Disturbances to cytoplasmic and vacuolar structure, and the morphology of mitochondria imposed by protoanemonin, were also mentioned. Interference with protein configuration and loss of biological activity was attributed to incorporation of azetidine-2-carboxylic acid into mung bean protein in place of proline. 

A further option to investigate the phototoxic potential of substances is the use of reconstructed human skin models. The evaluation of the cell viability is based on the MTT-assay that is sensitive for the mitochondria activity in cells. Currently, these in vivo substitutes are still under validation and are not approved as full standard test methods for the investigation of the phototoxicity potency of a test chemical. However, several existing models are in use for prevalidation studies and are described elsewhere in more detail [92],... 

The synthesis of ATP is catalyzed by the enzyme ATP synthase (or FiFq-ATP synthase) the Fj portion of this enzyme was first isolated by Racker and coworkers in 1960 [4]. ATP synthase is present in abundance in the membranes of animal mitochondria, plant chloroplasts, bacteria and other organisms. ATP synthesized by our ATP synthase is transported out of mitochondria and used for the function of muscle, brain, nerve, liver and other tissues, for active transport, and for synthesizing myriad compounds needed by the cell. Since the pool of adenosine phosphates in the body is limited, the use of ATP must be continually compensated by its synthesis, and an active person synthesizes his own body weight of ATP every day. The synthesis of ATP is the most prevalent chemical reaction in the body [5]. This is indeed a very important reaction. How exactly does it occur ... 

Hexachlorobutadiene is a nephrotoxic industrial chemical, damaging the pars recta of the proximal tubule. Initial conjugation with GSH is necessary, followed by biliary secretion and catabolism resulting in a cysteine conjugate. The conjugate is reabsorbed and transported to the kidney where it can be concentrated and becomes a substrate for the enzyme p-lyase. This metabolizes it into a reactive thiol, which may react with proteins and other critical macro molecules with mitochondria as the ultimate target. The kidney is sensitive because the metabolite is concentrated by active uptake processes (e.g., OAT 1), which reabsorb the metabolite into the tubular cells. 

The discussion to this point has focused on the isolation of intact mitochondria. By various chemical and physical treatments, mitochondria may be separated into their four components. This allows biochemists to study the biological functions of each component. For example, by measuring enzyme activities in each fraction, one can assign the presence of a particular enzyme to a specific region of the mitochondria. Studies of mitochondrial subfractions have resulted in a distribution analysis of enzyme activities in the four locations (Table E10.1). This type of study is often referred to as an enzyme profile or enzyme activity pattern and the enzyme may be considered a marker enzyme. For example, cytochrome oxidase, which is involved in electron transport, is a marker enzyme for the inner membrane. 

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