Specific Enzyme Visualization

The most specific visualization method is the localization of particular enzymes within an acrylamide gel. Most of these methods involve coupling the enzymatically catalyzed reaction either directly or indirectly to a chemical reaction that generates an intensely colored, insoluble product. Synthesis and availability of tetrazolium salts have greatly increased the variety of enzymes that can be assayed in this way. Reference 21 provides a thorough survey of the enzyme reactions that have been successfully assayed while in an acrylamide gel. 

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The specific enzyme to be used in an EIA is deterrnined according to a number of parameters including enzyme activity and stabiUty (before, during, and after conjugation), cost and availabiUty of the enzyme substrate, and the desired end point of the EIA, such as color. Most EIAs utilize a colored end point which can be readily deterrnined both visually and spectrophotometricaHy. Table 1 Hsts a number of enzymes which have been used in immunoassays and their substrates. 

As pointed out above, the bioassay design depends on the objective(s) of the study. A bioassay to determine allelopathic interactions in the field or in an ecological setting may have a quite different design than one used to determine PGR activity of a compound or to determine its molecular mode of action. Specific bioassays can be used to follow the isolation/purification of allelochemicals, evaluate their phytotoxic (or growth simulation) effects (i.e., visual effects), determine their host range/selectivity, evaluate allelopathic action of volatile compounds, or examine physiological/biochemical effects, such as photodynamic and membrane effects, effects on photosynthesis, specific enzyme sites, and effects at the ultrastructural level to locate receptor sites or sites of injury. Several examples of useful bioassays will be presented later. 

Enzymes, as a class of highly efficient and specific catalysts, dictate myriad reactions that constitute various cascades in biological systems [81]. The gel-sol conversion stimulated by a specific enzyme allows the direct visual screening of inhibitors without the assistance of appropriate instruments [82]. 

Figure 3.4 Western blotting technique. (A) Size separation of proteins by SDS-PAGE. (B) Transfer of the separated proteins onto nitrocellulose (NC) or poly-vinylidine fluoride (PVDF) membrane by electro blotting. (C) Visualization of proteins of interest by specific enzyme-conjugated antibody using immuno-staining. (D) Visualization of whole-protein extract by Coomassie Blue staining (left side), and immunostaining (right side), respectively.

What defects in vitamin A transport or utilization are likely to occur in the diseased eye The cytosol and IPM binding proteins and their possible receptors are clearly essential components of the visual system, as are the specific enzymes that are implicated at certain stages of the visual cycle (Table II). Failure to isomerize el -trans to Il-cu-retinoid is one obvious possibility, and a clinical trial was carried out by Chatzinoff et al. (1968) in an attempt to answer this question. The compound ll-c/5- vitamin A (the retinoid used was not reported) was injected intramuscularly into retinitis pigmentosa patients over a 3-year period. This group was then compared with a parallel group of patients that had received the all-trans isomer. No beneficial effect of 11-crr-retinoid was found. However, the ease with which the 11-cis isomer isomerizes back to all-trans when dispersed in tissue preparations (see Section III,G,4,b) casts doubt on the likelihood that in this study any 11-cM-retinol would have survived to be delivered to the RPE. 

In the Chapter 19 Lagniappe, we discussed how to access enzyme structural data from the Protein Data Bank. Once the data for a specific enzyme have been located, it s then possible to visualize, manipulate, and study the structure. You can do this either by downloading the data file to your own computer and opening it with a free visualization program, such as DeepView (Swiss PDB Viewer)... 

A different application of visible microscopy was pioneered by Gomori. In 1941 he showed that alkaline phosphatase could be specifically located by its hydrolysis of soluble phosphate esters (initially glycerophosphate). If calcium ions were present in the medium in which the sections were incubated, insoluble calcium phosphate precipitated as a result of the action of the hydrolase. The site of the precipitate could be visualized if cobalt or lead salts were subsequently added to replace calcium and the sections exposed to hydrogen sulfide. In principle many hydrolases and other enzymes could be studied using the appropriate substrates and precipitants. It was important to ensure that the products of the enzyme reactions did not diffuse from the sites where the enzymes were located. It was also essential that the reagents could reach the enzyme site. 

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