2 indicator enzyme

Figure 10.3-23. Metabolic model of glycolysis and tbe pentose phosphate pathway in E. coli. Squares Indicate enzyme activities circles indicate regulatory effects,...

An indicator enzyme is the enzyme that catalyses the final measurable reaction in a coupled enzyme assay. 

In this example, malate dehydrogenase is known as the indicator enzyme. 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

The only rate-limiting factor in a coupled assay should be the concentration of the initial and linking products and all other reagents should be in excess. The role of the auxiliary and indicator enzymes is essentially that of a substrate assay system and under optimum assay conditions the rate of the indicator reaction should be equal to the rate of formation of the initial product. The indicator reaction must be capable of matching the different test reaction rates and its velocity can be defined by the Michaelis-Menten equation in the usual way ... 

In order for the indicator reaction to parallel the test reaction the ratio, Vmax/ATm, for the indicator enzyme should be significantly greater than the same ratio for the test enzyme. The value for Vmax is the only variable in the equation and is determined by the amount of enzyme present, usually quoted in units per assay volume. An increase by a factor of 100 is usually recommended for each stage and will permit concentrations of approximately 1/100 of the Km value to give a rate comparable to the test reaction rate. It may be necessary, if pH or inhibition effects reduce the activity of the enzyme by a known proportion, to increase further the amount of enzyme added to the assay. Table 8.3 lists some examples of coupled and direct assays for a range of enzymes. 

In coupled assays, it is essential that the concentration of the indicator enzyme be kept low... 

Glucose-6-phosphate dehydrogenase is used as an indicator enzyme in the hexokinase assay of glucose... 

GPRT- defective cells appear in population. Using the method of the selective pressure by 5-BDU and 8-Ag, the mutant cultures of cells, defective by the indicated enzymes, are obtained. The intraspecific and interspecific hybrid cultures of cells are firstly obtained in the laboratory of the cellular biotechnology, A4H (SPEV TK- x lymphocytes of horse) and others. Hybrid cultures possess the unique properties. They are the ability to grow in the monolayer and in suspension, the high sensitiveness to the viruses. Hybrid cultures PO-TK-KHLO, PO-TK-KHSO are sensible to the agent scrappy, what allows using them for the study of diseases, caused by prions. 

Figure 6-8. Conversion of phosphoenolpyruvate to glucose during gluconeogenesis. Except for the indicated enzymes that are needed to overcome irreversible steps of glycolysis, all other steps occur by the reverse reactions catalyzed by the same enzymes as those used in glycolysis.

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

Indications Enzyme replacement therapy for adenosine deaminase (ADA) deficiency in patients with severe combined immunodeficiency disease (SCID) who are not suitable candidates for—or who have failed— bone marrow transplantation... 

Sample quality is generally assessed by the determination of /J-galactosidase activity. For MPS type IVB, the a-mannosidase activity is chosen as an indicator enzyme. Leukocyte homogenates that are sufficient for at least 20 separate runs are prepared from one source and aliquots are kept frozen. These samples serve as quality controls for each run. Heat-inactivated leukocyte homogenates may serve as a positive control (patient-mimics). 

Illustrated is a generalized metabolic pathway in which capital letters indicate major metabolites in the pathway, small letters indicate cofactors and numbers indicate enzymes catalyzing the reactions. 

HeLa cells were cultured for 24 hr with (open bars) and without (closed bars) 5 mM sodium butyrate, harvested, and assayed for indicated enzyme activity. A GM3-sialidase activity assayed with GM3 specifically labeled with [,iC]- N-acetylneuraminic acid as substrate ("14JJ. B Sialyltrasferase activity assayed with CMP-[, acetylneuraminic acid and lactosylceramide as substrates, and synthesis of labeled GM3 determined (17). Data... 

Scheme 1 Classification of elementary variational motifs describing evolution at the level of biochemical phenotypes. Indicates enzyme catalysed chemical reactions, and indicates evolutionary progression. The fundamental operations are shown in bold...

Figure 7. Activity measurements of low molecular weight urokinase refolded by continuous flow from 9.3 M urea to 20 mM Bis-Tris, pH 7.3 buffer and collected during run (Fig. 1). Circles indicate urea concentrations in each fraction as measured by refractive index. Squares indicate enzyme activity measured with urokinase substrate S-2444 (12). Inset shows a plot of activity versus urea concentration.

Classification The classification of the hepatobiliary enzymes essential for enzyme diagnostics is based on their characteristic nature - i. e. excretory, secretory and indicator enzymes, (s. tab. 5.5) They are located predominantly within the liver cells and the biliary ducts as well as within the hepatic lobules. The speed of enzyme elimination does not depend on the blood enzyme levels, but follows an exponential curve. This allows the computation of the half-life of enzymes within the plasma, which is not influenced either by gender or age and is a typical enzyme characteristic. The velocity of enzyme elimination is largely constant, (s. tab. 5.5) However, in chronic diseases of the liver, it is known, for example, that GPT is usually eliminated faster than GOT despite its longer half-life. 

The relation between cholinesterase and indicator enzymes gives insight into the stage of a chronic liver disease. 

The term enzymatic markers of cholestasis (or cholestasis-indicating enzymes) comprises alkaline phosphatase, leucine aminopeptidase (or leucine arylamidase) as well as 5 -nucleotidase and y-glutamyl transferase, (see chapter 13)... 

Determination of total bilirubin with differentiation between direct (conjugated) and indirect (unconjugated) bilirubin Determination of cholestasis-indicating enzymes (AP, LAP, y-GT) bile acids and bile pigments in the urine (s. tab. 12.6) Test for signs of haemolysis (s. tab. 12.3)... 

The spread of the damaging processes to adjacent hepatocytes is signalled by an increase in transaminase activity the liver cell membranes are also affected. The result is an enhanced release of cytoplasmic enzymes (GPT, to a lesser extent also GOT) into the serum. Ultimately, cell necrosis must be expected with a rise in mitochondrial enzymes in the serum (GDH, mGOT). From the point of view of biochemistry, the term hepatitis presupposes an increase in indicator enzymes once when the hepatocytes have been damaged or destroyed, (s. pp 93-95) (s. tabs. 5.3-5.5)... 

Boswellinic acids Leukotrienes appear to play a special role as inflammation mediators in the pathogenesis of PBC they also correlate closely with the increase in cholestasis-indicating enzymes. Boswellinic acids are selective non-redox inhibitors of 5-lipoxygen-ase and therefore inhibit leukotriene biosynthesis. So far, they have not been used in PBC treatment. Based on existing pharmacological data, their application should now be considered. 

Figure 4-S9 The activation energy, for a reaction can be determined by measuring the reaction rate constant at different temperatures and plotting log k versus 1 /T. For enzyme-catalyzed reactions, log Vms /[E]( or just log can be plotted. Curve A The usual plot. Curve B Sometimes the plot will show a definite change in slope if at some temperature a different step becomes rate-limiting. Curve C A sudden drop in the plot indicates enzyme inactivation.

The most commonly used indicator enzymes are dehydrogenases and peroxidases. The reactions catalyzed are shown in Eq. 3.5. 

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