Enzymes tissue preparation

Superoxide dismutase has been approved by the FDA for preventing reperfusion injury or damage to donor organ tissue (178). This enzyme is prepared by recombinant DNA technology and marketed by Bristol-Myers and Pharmacia-Chiron. 

Principle Biosensors consist of paper matrixes and tissue enzyme preparations, often the pure enzyme AChE or AChE-containing cells. As seen in Fig.l, main scheme of the preparation technology and procedure includes (i) the preparation of same kinds of biotests-biosensors, which are paper matrixes impregnated with tissue preparation of AChE and covered by polymer film. (ii) biochemical reactions of the AChE activity with and without inhibitors tested and (iii) the photometric analysis of the samples for quantitative estimation of the biochemical reactions. 

In essence, an enzyme-catalyzed equivalent exists for almost every type of chemically catalyzed reaction, and thousands of these have been documented in comprehensive monographs and reviews (9-26). Many reactions have been observed in relatively specialized areas, particularly with groups of organic compounds such as the steroids, other terpenoids, antibiotics, aromatics, and alkaloids. Specific chemical reactions have been accomplished with intact and growing microbial cells, with plant and mammalian tissue preparations, and with... 

To obtain tissue preparations whose constituents were maintained as closely as possible to their state in vivo, the material had to be fixed, i.e. the enzymes inactivated so that cell structures were instantaneously preserved, an almost unattainable ideal. Formalin was the favored fixative, but others (e.g. picric acid), were also employed. Different methods of fixation caused sections to have different appearances. Further artifacts were introduced because of the need to dehydrate the preparations so that they could be stained by dyes, many of which were lipid-soluble organic molecules. Paraffin wax was used to impregnate the fixed, dehydrated material. The block of tissue was then sectioned, originally by hand with a cut-throat razor, and later by a mechanical microtome. The sections were stained and mounted in balsam for examination. Hematoxylin (basophilic) and eosin (acidophilic) (H and E staining) were the commonest stains, giving blue nuclei and pink cytoplasm. Eosinophils in the blood were recognized in this way. 

Monooxygenase Assays. Incubation media contained the following (final concentrations) 0.05M phosphate buffer, pH 7.A, glucose-6-phosphate (G-6-P, 2.3 mM), G-6-P dehydrogenase (3 units), NADP (0.23 mM), and KC1 (2.8 mM), and various tissue preparations. Substrates were added in small volumes (25 yl or less) of MeOH. Samples (1.1 ml) were shaken in a thermostated (usually at 22°C) water bath and reactions terminated by enzyme denaturation. Specific analytical procedures for aldrin epoxi-dation (13), 1 CH30-p-nitroanisole 0-demethylation (1A), and 3H-benzo(a)pyrene oxidation (15) have been described. 

Tissue lysate (or homogenates), post-mitochondrial supernatants and microsomes offer several practical advantages for the study of xenobiotic metabolism. The principal advantages are that the human tissues provide a complete system containing all the enzymes in ratios found in vivo, and tissue fractions are stable in relatively long-term storage. Within the different types of tissue fractions, microsomes provide an enrichment of the membrane-bound enzymes, and post-mitochondrial supernatants provide a means to study both membrane-bound and soluble enzymes. Tissue fractions are easily prepared from a variety of tissues including human liver and can be cryopreserved for several years. This allows detailed characterization of the tissue prior to use with xenobiotics of unknown routes of metabolism... 

Identification of the P450 form(s) responsible for the metabolism of a xenobiotic can be achieved by correlation of the rates of metabolism of the new entity with the rates of metabolism for marker substrates for specific enzymes in different liver specimens with the levels of the different P450 forms (for review see Wrighton et al., 1993b). A typical approach involves the selection of multiple human tissue preparations with high , medium and low levels of the enzymes of interest. The rate of metabolism of the new entity is assessed in these samples and correlation analyses are performed versus the different marker activities. The enzyme with the highest correlation with the metabolism of the new entity is likely to be the principal enzyme involved in its metabolism. Alternatively, or in addition to correlation analyses, the roles of specific enzymes can be analysed by selective immunoinhibition or chemical inhibition of different P450 forms. The use of chemical inhibitors has been reviewed recently (Halpert et al.,... 

Since a principal P450 is defined by its catalytic activity, and activity per unit enzyme is variable for the same enzyme/substrate when the enzyme is expressed in different heterologous systems, it seems more appropriate to establish relative contributions based on enzyme activities in the incubations of cDNA-expressed enzymes and human tissue preparations. 

This approach makes the assumption that any effects on V ax are independent of substrate, i.e. the rank order of rates of metabolism is the same for a particular cDNA-expressed enzyme and the same enzyme present in human tissue preparations, and any factor which affects V ax for one substrate also does so equally for other substrates. The validity of this assumption has not been rigorously tested but for most enzymes an appropriate set of test compounds is available. 

Scientists working with cell and tissue preparations recognized that caffeine and the other methylxanthines can block an enzyme called phosphodiesterase. It seems now, however, that this action is carried out at caffeine doses that are much higher than what people normally... 

In recent studies protoplasts prepared from apple tissue did not produce ethylene. The loss of ethylene-producing ability by tissue slices incubated in cell wall-digesting enzymes during preparation of protoplasts is shown in Fig. 9. Methionine, the precursor of ethylene, delayed the loss of ethylene production somewhat during preparation of the protoplasts. 

Oxidation-reduction reactions similar to the Cannizzaro process are brought about in the living cell by certain enzyme systems. Numerous exanfples 7-8- 10 of these have been studied in vitro by the aid of tissue preparations, and certain of them 6 suggest possible application in preparative methods. The dismutation. of aldehydes in basic or neutral solution also has been effected by catalytic metals, such as nickel and platinum.11 12 It seems likely that there is a closer analogy between... 

Repeat the above procedure, but use antibodies that bind to enzyme B instead of enzyme A. This portion of the experiment will reveal the relative contribution of enzyme B to the biotransformation of the carcinogen in the tissue preparation. Taken together, results will establish the relative contribution of each enzyme to the biotransformation of the chemical carcinogen in the tissue sample (Figure 7.3). 

Assay of Cu/Zn-SOD and Mn-SOD or Fe-SOD separately may be achieved as follows total SOD activity is measured as described above and the assay is then repeated following the addition of 2 mM cyanide which completely blocks the activity of the Cu/Zn-SOD. The activity of the Cu/Zn enzyme and the Mn enzyme (in mammalian tissue preparations) may then be obtained by difference. 

Metabolism studies. GC-MS is a powerful technique for following and identifying the metabolic products from the in vitro incubation of tissue preparations with steroid substrates. Examples of such studies include the 16a-hydroxylation of 18-hydroxydeoxycorticosterone by human adrenal gland [254], the eiromatization of 3jS,15, 16 -trihydroxy-5-androsten-17-one by placental homogenates [255], and the demonstration of 1/3, 12/3, 6a and 6/3 hydroxylase enzyme activities in microsomal preparations of human foetal hepatic tissue [256]. In the latter study, testosterone was used as substrate and in addition to the hydroxylated metabolites isolated, several other testosterone derivatives indicated the presence of 3a, 3/3 and 17/3-hydroxysteroid oxidoreductase in the adrenal gland preparation. 

Composite carbon paste electrodes prepared by mixing carbon paste with biocomponents (enzymes, tissues, etc.) is a recent interesting development in the field of electrochemical biosensors. The biological entities show a strong decrease in activity when incorporated in the carbon paste matrix due to their confinement in a hydrophobic environment. A new approach to the preparation of electrochemical biosensors based on a Sepharose-carbon paste electrode has been reported by Gasparini et al. [115]. As another example of a modified carbon electrode, Chen et al. [U6]. developed a sensitive and selective detection scheme for dipeptides based on carbon electrodes. The modified electrode was found to be more selective for a-dipeptides over 3- and ydipeptides as well as amino... 

Unfortunately, no useful therapeutic drug has been designed by this approach so far. Inhibiting the transaminase enzyme has no medicinal use since the enzyme is crucial to mammalian biochemistry and inhibiting it would be toxic to the host. The main use for suicide substrates has been in labelling specific enzymes. The substrates can be labelled with radioactivity and reacted with their target enzyme in order to locate the enzyme in tissue preparations. 

Electronic factors and the relative lipophilicity of the molecule probably help to determine the affinity of the substrate for the enzyme as well as turnover properties. It is likely that the fundamental instability of the enzyme has hampered progress in the characterization of human liver aldehyde oxidase. At least in animals, the specific activity of the enzyme is quite dependent on the way the tissue is procured, processed, and stored this may lead to considerable intersample variability. Enzyme instability may at least in part explain why aldehyde oxidase activity from different species is so variable (Duley et al. 1985). However, it is likely that in addition to intrinsic differences in stability, the determination of aldehyde oxidase activity for a given substrate in various tissue preparations is dependent on the analytical methodology employed to assay the enzyme and the likelihood of the presence of different forms of the enzyme that possess distinct substrate specificity and kinetic properties (Johns 1967 Beedham 1985). For example, in the... 

The pink azo dye formed is completely soluble in alcoholic borate solution the color was remarkably stable even up to 24 hours at room temperature. The method is so sensitive that a fraction of a microgram of phenol can be estimated with ease. It is very economical of enzyme, requiring only 0.1 ml of properly diluted serum or tissue preparations, and the tedious deproteinization step becomes unnecessary. 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

Mason and Sammons (1979) introduced an elegant method to lower background staining, caused by non-specific adsorption of IgG to tissue sections, by coupling the enzyme to purified antigen. An excess of the primary antibody is incubated with the tissue preparation, but among the IgG adsorbed only the antibodies reacting... 

Cell or tissue preparations are first incubated with the primary antibody (1-25 pg/ml) in a humid chamber for at least 1 h at room temperature. The preparations are washed three times with PBS for 5 min and enzyme-labeled anti-IgG antibodies (5-100 Xg/ml) are subsequently reacted with the primary antibody for 90 min at room temperature. Tween 20 at 0.05% in PBS is often beneficial. 

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