Enzymes activity assay techniques

Proteolytic enzymes in the respiratory mucosa play important role(s) in the regulation of lung inflammation and remodelling [123, 124], Pulmonary proteolytic enzymes, however, also comprise one of the barriers which pulmonary-administered protein/peptide drugs have to overcome in order to achieve adequate bioavailability [125]. Intriguingly, the pulmonary enzymatic barrier is an aspect that has been little investigated and is poorly understood. Inconsistencies in the data available to date are most likely a result of the use of different techniques (e.g., PCR, immunotechniques and enzyme activity assays), different species and different cell (pheno)types, for example primary cells vs. cell lines. 

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

The PMC-technique permits miniaturization of most pre-existing macro enzyme activity assays. It allows incubations in exceedingly small volumes (lowering of the blanc, improving the signal/noise ratio) for as many hours as the enzyme is stable (increased sensitivity) This is a reliable way to assay enzymes routinely 1. at the single level, 2. with very low costs. 

The relation between microbial diversity and soil functions is poorly understood because we cannot measure easily the microbial diversity, even if we can detect unculturable microorganisms by molecular techniques (Nannipieri et al. 2003). In addition, the present assays for measuring microbial functions determine the overall rate of entire metabolic processes, such as respiration, or specific enzyme activities, without identifying the active microbial species involved. The recent advances in RNA extraction from soil might permit us to determine active species in soil (Griffiths et al. 2000 Hurt et al. 2001). Further advances in understanding require us to determine the composition of microbial communities and microbial functions in microhabitats. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Several things may be done if the researcher has difficulty in detecting an enzyme activity of interest in a homogenate, or elsewhere. A more sensitive assay technique may be used, if one is available. The concentration of enzyme may be increased, as the rate of product formation is directly proportional to [E]. The incubation time for enzyme with substrate can be increased, although the caveats discussed in O Section 3.3.2 must be borne in mind. The reaction volume may also be increased, while maintaining concentrations of reactants constant this approach is particularly useful if product is separated and detected by chromatography, or if a column is used to separate radiolabeled substrate from product, because the increased amount of product formed in unit time will result in enhanced signal size. 

Enzyme activity measured, in vitro, in extracts of the muscle (for details of assay technique, see Chapter 3). 

Addition of ethyl acetate to a specimen having a transaminase activity of 47 units was responsible for the following increases in enzyme activity 10 mg/100 ml, 60 units 20 mg/100 ml, 77 units 40 mg/100 ml, 107 units and 80 mg/100 ml, 150 units. Transaminase activity in these specimens determined by another method ranged from 32 to 34 units (C7). Thus, when serum from patients with ketosis is assayed for aspartate aminotransferase activity by the diazo method, false elevations of activity may be recorded due to reaction of acetoacetic acid. In Table 11 are shown some values obtained by the diazo method and by an ultraviolet NADH NAD aspartate aminotransferase technique (B12). Examination of the medical records of these patients indicated that they were either diabetics who were in ketosis or individuals who were eating very poorly and had some degree of starvation ketosis. Similar elevations have been observed in patients receiving p-aminosalicylic acid (G6). 

WlO. Winsnes, A., Variable effect of phenobarbital treatment of mice on hepatic UDP-glucuronyltransferase activity when judged by slightly different enzyme-assay techniques. Biochem. Pharmacol. 20, 1853-1857 (1971). 

The simplest, but least accurate, method of assaying DPO activity is to record the final color yield when the enzyme is incubated with a suitable chromogenic substrate such as catechol, DOPA, or 4-methylcatechol. DOPA is the most frequently used substrate in colorimetric assays because it yields a dark brown/black end-product. In this reaction, catecholase catalyzes the conversion of DOPA to dopaquinone and then to the red dopachrome, which subsequently polymerizes to yield dark brown melanin-type pigments. Unfortunately, this simple procedure has serious limitations, as it measures the end-product of a sequence of reactions rather than the true initial reaction rate. Furthermore, because different substrates yield different final colors, valid kinetic comparisons between substrates are not possible. Nevertheless, this simple assay technique has proved adequate for useful comparative studies of the levels of enzymic browning in different fruit varieties and similar problems (Vamos-Vigyazo, 1981 Machiex et al., 1990). 

The significance of all these observations is at present far from clear. Any rationalization is difficult because many variables are involved. As Stadtman has pointed out (38) the nature of the buffering ions, the presence of activating cations, the different assay techniques, substrates, and enzyme preparations can each influence the overall activity. Attempts have been made to isolate particular factors and these will be briefly discussed. 

It is essential for a successful assay that the vitamins be quantitatively extracted from the food matrix in a form that can be accurately measured by the particular HPLC technique to be used. An effective extraction procedure serves to homogenize and concentrate the sample, isolate the vitamin analyte from its association with protein, eliminate as far as possible known interfering substances, and destroy any indigenous enzyme activity. The vitamin-rich fraction thus obtained may require some form of cleanup before the vitamins can be measured, particularly when measuring the trace amounts of naturally occurring vitamins D and K. 

High-molecular-weight solid substrates such as hide powder azure, and cellulose, chitin, and agar stained with remazol brilliant blue R have been used in enzyme assays to investigate activities in extracts from sediments (Reichardt, 1986). This technique is one of the few means of examining the particle — dissolved transition in relationship to sedimentary enzyme activity, but the necessity of extracting enzymes from sediments complicates interpretation of the results, because extraction efficiency, as well as the... 

Poly-C-SpecificRibonuclease (P-RNase) (EC3.1.27.5). Warshawand Fournier (W3) showed that an increase in plasma enzyme activity of pancreatic P-RNase in patients with AP may indicate necrotic lesions, and is one of the few direct markers of pancreatic tissue injury (Nl, W4). Due to the time-consuming and cumbersome nature of the P-RNase assay procedure and the development of effective visualization techniques providing direct information on the structure of the inflamed pancreas, the diagnostic utility of the P-RNase assay has not been extensively studied (Table 3). 

Besides enzyme activity, enzyme induction assays can also utilize mRNA and enzyme protein level as endpoints. Gene expression studies now can be performed using branch-chained DNA and microarray techniques. Protein level quantification in general is performed using isoform-specific antibodies and Western blotting. Enzyme activity represents the most relevant endpoint for drug-drug interaction evalua-... 

Cofactors. Many enzymes require relatively loosely bound organic cofactors (coenzymes) or metal ions for activity. A biochemist must be alert for indications of such a requirement during purification of an enzyme, because it may be necessary to add such cofactors to the assay solution to obtain enzyme activity. The interactions of coenzyme with enzyme can be analyzed by kinetic and spectral techniques. Similar techniques are used to evaluate interactions with metal ions. 

While most of the problems in the assay of an activity purified by HPLC are expected and typical of chromatographic work with enzymes, the introduction of this technique into the purification scheme may lead to problems if the fractions obtained from the HPLC purification step are to be measured for enzymatic activity. For example, the salt in each fraction may inhibit any enzymatic activities it contains. Moreover, when ion-exchange HPLC is used the salt concentration will vary in the fractions. Thus it is prudent to study the effects of salt, at the concentration used for elution, on enzyme activity before the chromatography. If the salt is found to be detrimental, it will have to be eliminated or at least reduced in concentration before the chromatography. Removing the salt by dialysis may not be the appropriate way to proceed, however, since the inactivation of enzyme activities is not always reversible. 

In vitro translation is the method of choice for preparing small quantities of protein for analytical purposes and, with the recent availability of commercial reactors and reagents, also for preparative purposes on a modest scale (several mg). Proteins prepared in this way can be analysed by imunoprecipitation or SDS-PAGE they can also be tested for specific biochemical activity, such as enzyme activity or specific DNA binding activity by techniques such as gel electrophoretic mobility shift assays. 

In 1983, Falkowski observed, there is little information on the N enzymology of marine organisms and also noted that there was scanty information about transporters (Falkowski, 1983). At that time, most of the information available for enzymes was based on activity assays with relatively few attempts to purify enzymes and characterize their structural and functional diversity, and very few attempts to use molecular or other techniques to probe the full range of enzymes mediating... 

Assay techniques GS is an excellent example of a reversible enzyme that is highly regulated and modified at multiple levels. GS is assayed in both the forward and reverse directions. In the forward direction, GS assays the biosynthesis of gin in the presence of the divalent cation Mg (biosynthetic activity). Because Mg appears to inhibit the activity of modified enzyme in the forward reaction, biosynthetic assay is thought to represent in vivo potential for gin synthesis via GS (Lee et al, 1988). In the reverse direction, GS assays measure total potential GS activity of both active and inactive enzyme in the presence of Mn as the divalent cation (transferase activity) (Lee et al, 1988 Stadtman et al, 1979). In both of these... 

Assay techniques Assays of GOGAT rely on measuring fluorometricaUy or spectrophotometricaHy the oxidation of reductant (Ahmed et al., 1971 Avila et al, 1987 Clayton and Ahmed, 1986 Inokuchi et al, 1999), or measuring products of enzyme activity (e.g., C-glutamate production CuUimore and Sims, 1981 Prusiner and Milner, 1970). As for other enzyme assays, these assays are... 

Assay techniques GDH utilizes both nicotinamide nucleotide cofactors NAD+ in the direction of N liberation (catabolic) and NADP+ for N incorporation (assimilatory). In the forward reaction, GDH catalyzes the synthesis of amino acids from free ammonium and Qt-kg. The reverse reaction links amino acid metabolism with TCA cycle activity. In the reverse reaction, GDH provides an oxidizable carbon source used for the production of energy as weU as a reduced electron carrier, NADH, and production of NH4+. As for other enzymes, spectrophotmetric methods have been developed for measuring oxoglutarate and aminotransferase activities by assaying substrates and products of the GDH catalyzed reaction (Ahmad and Hellebust, 1989). 

Enzyme kinetics is an important tool for assaying enzyme activities and for determining enzyme mechanisms. Although other techniques can provide useful information on enzyme mechanisms, the kinetics has to be the ultimate arbiter because it looks at the reaction while it is taking place. Initial velocity patterns, inhibition patterns, patterns of isotopic exchange, pH profiles, and isotope effects are all kinetic tools that allow one to determine kinetic mechanisms, chemical mechanisms, and transition state structures. 

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