Enzymatic activity, visual

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

In general, these distributions all share the intuitive property that the vast majority of ligands have low to moderate affinity, and fewer and fewer ligands have higher affinity. Though the Sips, log-normal and RAD distributions can be made visually quite similar, they will always differ in their moments and other mathematical properties. It should be stressed that these distributions are for affinities. For other properties, such as enzymatic activity, these distributions may not be relevant, though at least one model for catalytic activity is based on these distributions [19]. 

The visual transduction system of retinal membranes contains a plethora of prenylated proteins. One of the prenylated components is a protein called transducin. This protein is a member of a family of G proteins that contain three distinct subunits (a, P, and y) and function in mediating signal transduction from cell membrane receptors to downstream effector proteins. In the case of visual transduction, photoactivation of rhodopsin leads to a conformational change that is sensed by transducin. In the absence of photoactivation of rhodopsin, transducin exists in a resting state in which Gt)P is bound to its a-subunit. Photoactivated rhodopsin catalyzes the exchange of GDP bound to transducin with GTP. This GTP-bound activated transducin then stimulates the enzymatic activity of another membrane-bound protein termed cyclic GMP phosphodiesterase. The latter... 

Activity-based protein profiling (ABPP) is a powerful technique to identify enzymatic activities and to study their functioning - and the effect of inhibitors on this - in vitro, in situ, and in vivo. In this chapter some case studies on activity-based profiling of mammalian proteasomes are discussed. Both direct and two-step bioorthogonal ABPP strategies and their merits are discussed, and the value of ABPP in the establishment of previously uncharted enzymatic activities and the direct visualization of inhibitor specificity are presented. 

Feedback mode SECM imaging was successfully employed to map metabolic activities with high spatial resolution. Heterogeneous distributions of active NADH cytochrome reductase within individual mitochondria membrane surfaces were visualized [43]. However, as suggested by Bard s group, this mode will not generally be accessible for surfaces with relatively lower enzymatic activities. On the other hand, the G/C mode may offer wider applicability for imaging biocatalytic reactions... 

All ee screening systems using UV/Vis spectroscopy as a detection system have a number of advantages, including the possibility of visual pre-screening for activity on microtiter plates in some cases. Moreover, if a reliable UV/Vis signal arises as a consequence of an enzymatic reaction, commercially available UV/Vis plate readers can be used to screen thousands of mutant enzymes catalyzing the reactions of interest in the wells of microtiter plates. 

Impressed by the specificity of enzymatic action, biochemists early adopted a "lock-and-key" theory which stated that for a reaction to occur the substrate must fit into an active site precisely. Modem experiments have amply verified the idea. A vast amount of kinetic data on families of substrates and related competitive inhibitors support the idea and numerous X-ray structures of enzymes with bound inhibitors or with very slow substrates have given visual evidence of the reality of the lock-and-key concept. Directed mutation of genes of many enzymes of known three-dimensional structure has provided additional proof. 

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