Microsomal oxidases, measurement

Superoxide-dismuting activity of copper rutin complex was confirmed by comparison of the inhibitory effects of this complex and rutin on superoxide production by xanthine oxidase and microsomes (measured via cytochrome c reduction and by lucigenin-amplified CL, respectively) with their effects on microsomal lipid peroxidation [166]. An excellent correlation between the inhibitory effects of both compounds on superoxide production and the formation of TBAR products was found, but at the same time the effect of copper rutin complex was five to nine times higher due to its additional superoxide dismuting capacity. 

Spin trapping has been widely used for superoxide detection in various in vitro systems [16] this method was applied for the study of microsomal reduction of nitro compounds [17], microsomal lipid peroxidation [18], xanthine-xanthine oxidase system [19], etc. As DMPO-OOH adduct quickly decomposes yielding DMPO-OH, the latter is frequently used for the measurement of superoxide formation. (Discrimination between spin trapping of superoxide and hydroxyl radicals by DMPO can be performed by the application of hydroxyl radical scavengers, see below.) For example, Mansbach et al. [20] showed that the incubation of cultured enterocytes with menadione or nitrazepam in the presence of DMPO resulted in the formation of DMPO OH signal, which supposedly originated from the reduction of DMPO OOH adduct by glutathione peroxidase. 

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. 

Aryl hydrocarbon hydroxylase (AHH) is part of the microsomal mixed-function oxidase system involved in the detoxification of polycyclic aromatic hydrocarbons. In the HPLC assay developed for the AHH activity, benzo[a]pyrene (BaP) is used as the substrate, and the activity is determined by measuring the unreacted BaP during the reaction. 

The predicted stoichiometry in microsomal mixed function oxidations for NADPH substrate oxygen is 1 1 1 325), In the absence of substrate NADPH oxidase is measured and in the presence of substrate this background oxidase activity is present though oxygen consumption increases 326, 326). As the level of substrate increases the expected stoichiometry is approached 326, 328). The addition of potential substrates which cannot be hydroxylated, such as perfluoro-n-hexane, leads to increased oxygen consumption and this has been termed uncoupling 327). It has been demonstrated that some of the electrons lost from the system are... 

In this equation, tt is the hydrophobic parameter, sc the Hancock steric parameter, and a the electronic constant for the bridgehead substituent. Median lethal dose is expressed as mol/fly. The bicy-clic phosphates have been shown to be mainly degraded by microsomal mixed-function oxidases in the housefly (12). Thus, the toxicity determined by injection after pb pretreatment is regarded as a good measure of the intrinsic toxicity of BPs, and it is probable that this equation reflects significant interactions between the bridgehead substituent of BPs and its site of action, i.e., a type of receptor. Little is known about the hypothetical BP receptor in the insect while a considerable amount of information has been obtained on a mammalian site. Three-dimensional receptor models have been proposed based on structure-activity relationships for BPs and related compounds (13,14). It is now important to substantiate the existence of the putative receptor in insects for the elucidation of the detailed mode of insecticidal action of BPEs. 

Most of the microsomal reactions can be classified as oxidations by what are referred to as mixed-function oxidases utilizing molecular oxygen and cofactors. The key enzyme is an iron-hemecytochrome P-450, a flavoprotein dependent in its reduction and reoxidation on the NADPH to NADP reaction. The 450 notation is based on the 450 nm absorption peak the enzyme exhibits on reaction with carbon monoxide. Thus, drug interactions with this enzyme system can be evaluated by measuring absorption spectra changes. 

The experimental results given above clearly indicate that cytochrome P-450 is the oxygen-activating catalyst of a wide variety of mixed-function oxidations in tissues of vertebrate animals. Spectrophoto-metric measurements and determinations of the photochemical action spectra did not reveal any significant differences between the pigments from different sources. Yet the substrate specificity of these mixed-function oxidase systems is pronounced, as shown by studies on the induction of microsomal hydroxylase activity toward different xenobiotics (27) and on the affinity of different C-21 methyl corticosteroids toward... 

When measuring mt DNA content it only has to be shown that no contamination with nuclei or nuclear fragments is present. This is conveniently achieved with appropriate gradient centrifugation or floatation techniques (Schatz et al., 1964 Neubert et al, 1968 Nass, 1969). However, because of the similar densities of mitochondria and microsomal or other cell particles a clean separation of mitochondria is seldom achieved with small amounts of material. The interpretation of the data, therefore, is greatly facilitated when the activity of a typical mitochondrial enzyme like cytochrome oxidase is given in addition to the amount of mitochondrial protein. 

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