Assay of Other Enzymes

The tested acetolactate synthase (ALS) was from pea (pisum sativum). The ALS extraction, assay protocols, and the procedure of in vitro ALS assay followed the literature [41]. 

The tested peroxidase (POD) was from rice (Oryza Sativa). The POD assay in vitro was conducted according to the procedure reported in the literature [42, 43]. 

3 Assay of Superoxide Dismutase from Rice (in Vitro) 

The biochemical mechanism of herbicidal action of the alkylphosphonates was explored on the basis of biochemical experiments. Representative herbicidal active alkylphosphonates were selected to examine their inhibition against plant PDHc through enzyme activity assays, kinetic experiment, and enzyme-selective inhibition experiment. Based on the above study we can give the following conclusions  

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LDH is used for diagnosis of heart infarction and hepatitis, since its level in serum is considerably elevated in both these diseases. Purified LDH is used in coupled optical tests for the assay of other enzymes, e.g. pyruvate kinase, enolase, transaminases, and for the enzymatic determination of many metabolites, such as ADP, ATP, L-lactate and pyruvate. 

GO often is used in solution phase chemical reactions as well as being immobilized on dip-sticks and electrodes. Although its overall clinical usage is widespread, its use as conjugated to antibodies in enzyme-linked assay systems is minor compared to the popularity of other enzymes like horseradish peroxidase and alkaline phosphatase. 

For the assay of enzymes with products and reagents that have no absorption, fluorescence or luminescence in the ultraviolet or visible region, developments in analytical infrared spectroscopy can be used. In particular, mid-Fourier transform infrared (mFTIR) spectroscopy has been successfully applied to the determination of enzyme activities and kinetics, e.g. of /i-fructosidasc, phosphoglucose isomerase and polyphenol oxidase [90]. The method could very well be a tool that may also be applied to a variety of other enzyme classes. The potential of high-throughput applications, however, has yet to be demonstrated. 

The first is the presence of proteolytic activities, which must be inhibited early in the procedure to prevent the degradation of other enzyme proteins. Second, the amount of protein present in these fluids is usually in excess of what an HPLC analytical column can handle without becoming clogged. And finally, these fluids often contain many low molecular weight compounds, either those added as nutrients or those present as a result of cellular metabolism. Since such compounds may resemble either the substrate or product, or both, of the enzymatic reaction under study, their presence in the reaction mixture could interfere with the assay. At the very least, such compounds will pass through the analytical column and appear on a chromatogram, confusing the experimental results. 

Analytical Determinations Involving Total Change. The concentration of a compound which serves as a substrate for an enzyme may be determined by a rate assay or by a total change method. The advantages of the rate assay method are its speed, even at low enzyme concentrations, and its selectivity. With the usual spectrophotometric equipment a rate assay may be completed in one to five minutes. In the total change method the enzyme concentration is usually increased so that the reaction is complete in 10-30 minutes. Contamination by traces of other enzymes which convert the primary product to compounds not detectable by the assay procedure is more serious in the total change method than in the rate assay method. On the other hand, small variations in pH, tem-... 

The HPLC/MS/MS assays of other CYP enzymes are very similar in principle and use the identical instrumentation but employ different internal standards. As a consequence of the high degree of specificity of MS/MS selected reaction monitoring, batteries of CYP assays can be robotically programmed for high throughput with little additional manpower. 

The other cholinesterase is acylcholine acylhydrolase (EC 3.1.1.8, acylcholine acylhydrolase, CHE) it is also called pseudocholinesterase, serum cholinesterase, butyryl-choHnesterase, or choline esterase II. Although it is found in the liver, pancreas, heart, white matter of the brain, and serum, its biological role is unknown. The assay of this enzyme is clinically useful. 

Two situations must be distinguished, (i) assays of pure enzyme and (ii) assays of cell extracts. When purified enzyme preparations are available, no labeled substrate is required. Natural or synthetic sphingomyelin is prepared, pure or mixed with other lipids, in the form of extruded large unilamellar vesicles (LUV) ca. 100 nm in diameter. When pure sphingomyelin vesicles are used extrusion must take place at a temperature close to or above the gel-fluid transition temperature of the lipid, i.e. often 45-50 °C. LUV and enzyme are mixed in the appropriate assay buffer and aliquots are removed at fixed time intervals. The aliquots are mixed with chloroform-methanol and, after phase separation, phosphorous (from phosphorylcholine) is assayed in the aqueous phase. The procedure has been described in detail by Ruiz-Arguello et al. [91]. 

In strictly diffusion-controlled reactions, i.e. at low efficiencies, simply spoken, only a small fraction of the total immobilized enzyme quantity is working. If in the course of operation actively working enzyme is destroyed, some other previously resting fraction may substitute to some extent. This is one reason why immobilized enzymes may erroneously appear to be more stable than free enzymes. To avoid such misinterpretation requires the assay of the enzyme activity in the absence of diffusional limitations. For industrial applications it is more useful to consider the enzyme s performance as indicated in Fig. 1, instead of just assessing its half-life. 

These enzymes, abundant in heart and liver, are released as part of the cell injury that occurs in myocardial infarction, infectious hepatitis, or other damage to either organ. Assays of these enzyme activities in blood serum can be used both in diagnosis and in monitoring the progress of a patient during treatment. 

Too much P-amyloid is the result of the action of other enzymes with other roles, so that blocking them may cause side effects. Using cultured human and mouse cells, as well as test tube assays. University of Texas Southwestern researchers singled out how just one portion of the enzyme, a protein called nicastrin, is involved in the pathway that produces P-amyloid, thereby leading to AD. They hope next to work on ways to specifically block nicastrin (August 12 issue of Cell). 

Activity of salivary a-amylase was measured by incubation of the enzyme with 0.5% soluble starch in 0.05 M phosphate buffer (0.2 ml), pH 6.8, containing 0.005 M sodium chloride at 37° for 20 min, and the reducing sugars were measured by the method of Nelson-Somogyi. One unit was defined as the amount of enzyme which produces one ymole of reducing group (as glucose) per min. Activities of other enzymes were assayed by similar methods. 

Assay of adenosine deaminase and of other enzymes of purine and pyrimidine metabolism were performed by previously described radioisotopic assays (3). Electrophoresis of RBC ADA was performed on hemolysates after Spencer et al. (4). The enzymes of qlu-cose and glutathione metabolism were assayed after Beutler (5). Erythrocyte glucose consumption was assayed according to Cartier et al. (6). Adenosine deaminase was partially purified from human red blood cells the specific activity of the preparation was 1.2 pmol.min l.mg protein l. Rabbit antiserum to human adenosine deaminase was raised by immunization of white male rabbits with this preparation. 

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