Enzymes measurement of activity

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

Available methods provide measurements of enzyme activity rather than of enzyme concentration. In order that the measured activity be proportional to enzyme concentration, the reaction conditions which include pH, temperature, initial substrate concentration, sample and total volume and reaction time must be held constant and be carefully controlled. 

Reagents. The measurement of enzyme activities requires rigid control of the analytical conditions, including accurate measurement of reagent and sample volumes, and careful control of temperature, pH and reagent stability. 

Dihydroxybenzoic acid (DHB) is also a commonly used tool to measure the pharmacological effects of HIF-la stabilization via PHD inhibition. Recently, it was shown that mice pretreated with DHB (100 mg/kg, i.p.) showed a marked resistance to the neurotoxic effects of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) via protection of dopaminergic cell loss and striatal denervation. Importantly, this protection was seen to coincide with HIF-la stabilization, and the prevention of the MPTP-induced loss of ferroportin and striatal iron. Additionally, in these studies, DHB was also observed to block MPTP-induced reduction in mitochondrial pyruvate dehydrogenase, at both the mRNA level and through the measurement of enzyme activity in midbrain substantia nigra [26]. 

Under basal conditions, PKC is predominantly a cytoplasmic protein. Upon activation by Ca2+ or DAG, the enzyme associates with the plasma membrane, the site of many of its known physiological substrates, including receptors and ion channels. In fact, the translocation of PKC from the cytoplasm to the membrane has long been used as an experimental measure of enzyme activation. Such translocation has often been assayed by phorbol ester binding phorbol esters are tumor-promoting agents that selectively bind to and activate PKC. The molecular basis of the translocation of PKC from the cytoplasm to the plasma membrane has been solved. Subsequent to activation, PKC binds with high affinity to a series of membrane-associated proteins, termed receptors for... 

Other automated systems may be purchased for a specific purpose and are called dedicated instruments, e.g. glucose analyser. Others have fairly restricted applications, an example being the reaction rate analysers which are specifically designed for the kinetic measurements of enzyme activity. Some of the more recently developed instruments employ individual pre-prepared disposable test packs or strip devices which contain all the reagents for each particular assay in a dry form. 

End-point assays can also use coupled enzyme systems in which the product of the test reaction provides the substrate for the subsequent auxiliary and indicator reactions. The choice of pH is less critical than with the measurement of enzyme activity frequently a compromise pH is used and... 

The situation in a test tube is usually much more straightforward enzyme activity is reflected as a decrease in substrate concentration and an increase in product concentration, and measurement of the initial rate of change of concentration of either - the reaction velocity - constitutes a direct measurement of enzyme activity. 

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

The measurement of enzyme activities in tissues other than blood can also be carried out, for example, in extracts of tissue obtained by biopsy, from red or white blood cells or from cerebrospinal fluid. Such measurements are valuable in diagnosis of genetic diseases in... 

Kang, M.-J., Tholey, A., Heinzle, E. Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities. Rapid Commun. Mass Spectrom. 2001, 15, 1327-1333. 

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

Assays may range from the quick-and-easy type (e.g., instantaneous spectrophotometric measurement of enzyme activity) to long and tedious bioassays that may take days to produce an answer. The latter situation is very difficult, because by the time one knows where the protein is, it may be was, owing to degradation or inactivation. Moreover, this may not become clear until the next step has been completed and its products assayed. Any assay that is quick is therefore advantageous, even if it means a sacrifice of accuracy for speed. 

Cl.] Expression and Measurement of Enzyme Activity C1.2 Detecting Enzyme Activity A Case Study of Polygalacturonase... 

The assays presented in this section deal with the measurement of enzyme activity, which is expected to be proportional to the amount of active enzyme present in a sample, food or otherwise, unit ci.i is an overview of the important considerations in performing activity assays unitc 1.2 illustrates how these considerations are applied to the assay of a representative food-relevant glycosyl hydrolase. Chapters C2, C3, and C4 present the first units on particular types of activity assays. In Chapter C2, two units present peptidase activity assays that use either synthetic substrates (UNITC2.1) or common, commercially available protein substrates (unit C2.2). unitC3.i presents three different assays for lipase activity. unitc4.i presents assays for diphenol oxidases, and unitc4.2 for lipoxygenase. 

Fig. 5. A cell line selected for phosphate starvation resistance was con-stitutively induced for the excretion of APase into the medium. Three-day-old tomato cells selected for phosphate starvation resistance (PSR) and unselected cells (L. esculentum cv. VF36) were grown under Pi-sufficient conditions. Proteins excreted by the cells were separated by SDS-PAGE and immunoblotted with AP3 antiserum from which the Xylose-binding component had been removed via stem bromalin treatment (Goldstein, 1991). The selected cells showed constitutive excretion of high levels of APase protein based on the large signal obtained from the immunoblot. Measurement of enzyme activity gave a similar result (not shown).

With the introduction of quality assurance in the diagnostic laboratory 56 years ago [6], a kind of educational and benchmarking process started forcing laboratories, national and international organizations, and the IVD industry to improve the methods applied in clinical laboratories. Comparison of the measurements of enzyme activity demonstrate that the analytical performance of the methods applied 30 years ago were far beyond the biological variation and most probably insufficient for medical needs. Interlaboratory comparisons show that with the new routine methods based on recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) (Table 5) comparable results can be obtained irrespective of time and space and thus small individual variations can now be detected. Similar improvements in the analytical process in clinical laboratories can be reported generally for homogenous measurands. 

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