Assay catalytic

The LPL catalytic assay measures the hydrolysis of a [14C[- or [3H]-triolein emulsion producing the 14C- or 3H -labeled free oleic acid [6]. The 14C- or 3H-labeled oleic acid is isolated by a selective extraction procedure and its radioactivity is determined by liquid scintillation counting [40]. Lipase activity is calculated as nanomoles of oleic acid released per minute per milliliter of postheparin plasma [41]. 

Reliance on enzyme activity as the monitoring device also limits study to enzyme-type proteins, a most unfortunate situation since many interesting proteins do not have catalytically assayable functions. Some proteins are regulatory elements which modulate synthesis or activity of enzymatically active proteins others are structural elements or proteins that can perform their function only when integrated into a membrane, that is, permeases. The functions and modes of operation for many of these proteins are being elucidated with the help of immunochemical assay procedures. 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

Conditions for catalytic assay and for inactivation at pH 6.8 were as described in the text, except that pol3rmer A-2.8 and its hydrolyzate were evaluated at pH 6.2. See Rohlfing and Fox (J6) for details. 

Early studies used catalytic assays, but it was soon recognized that other proteolytic enzymes present in serum could also hydrolyze the same substrates. A major advance has been the development of immunoassays to quantify TRY in blood. In the case of TRY-1, immunoassays detect trypsinogen-1, TRY-1, and TRY-1-tti-antitrypsin complex. They do not detect the TRY-l-a2 macroglobulin complex, for which different assays are necessary. Free TRY-1 is not usually found in serum it is always complexed. 

All other panels of isolated kinases use catalytic assays and measure the conversion of a substrate (either ATP or peptide/lipid) to product. Because these are catalytic assays, they are all run in the presence of ATP plus a substrate. The ability to assay inhibitors at multiple ATP concentrations provides the scientist with the ability to obtain some information on whether the inhibitor is competitive with ATP or not and to rapidly eliminate the... 

The Millipore human kinase panel consists of 236 wt kinases (including some lipid kinases fluorescence energy transfer technology is used for lipid kinases). They also provide assays for 40 mutant kinases and for 11 kinases with special substrates or in activated states. As with most catalytic assay suppliers, whole panel screens are typically done at one ATP concentration (10 pM ATP for Millipore), but assays at the ATP Km can be requested. For more information on the Millipore panel, see http //www.millipore.com/drugdiscovery/dd3/ kinaseprofiler. 

The RBC kinase panel consists of 325 wt kinases (including some lipid kinases), 35 mutants and 6 kinases with special substrates or in activated states. A review of the technology as well as most of the other technologies discussed here was recently published by authors from RBC.23 This paper provides a nice overview of many of the standard catalytic assays used by RBC and other kinome assay providers. For more information on the RBC panel, see http // www.reactionbiology.com/pages/kinase.htm. 

Nanosyn, Caliper Life Sciences, and most Carna catalytic assays rely on a microfluidics capillary electrophoresis (CE) technology commercialized by Caliper Life Sciences. These are non-radioactive assays that measure the change in electrophoretic mobility of the substrate (usually a fluorescent-labelled peptide or lipid) upon phosphorylation. Both substrate and product are measured in these assays enabling increased assay precision. Since... 

Kinetic Assays, Catalytic Assays, and the Kinetic Advantage of FIA... 

In addition to that also the classical approach to enhancement of sensitivity of determination of traces of species, by letting them act as catalysts to promote formation of a measurable product, has been performed successfully by FIA modes [915, 1009, 1033,1041, 1121, 1134, 1135, 1159, 1186, 1203, 1216]. These ultrasensitive catalytic assays benefit from the rigorous mixing and timing of all operations performed in a FIA system, and also from the protective environment of the flow channel, where cross contamination and influence of ambient environment is minimized. 

Concise Catalytic Assays for Aldolase Antibody-catalyzed Reactions I 297... 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

The minute quantities of enzymes present in cells complicate determination of their presence and concentration. However, the abifity to rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the abifity to reveal its presence. Assays of the catalytic activity of enzymes are fre-quendy used in research and cfinical laboratories. Under appropriate conditions (see Chapter 8), the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which allows its concentration to be inferred. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Another quite general approach is to employ a coupled assay (Figure 7-10). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled. 

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

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