Molecular probing enzymes

Bonfante et al. (73) used monoclonal antibodies and enzyme-gold complexes to reveal pectins and cellulose at the interface between the fungal wall and the host plasma membrane in AM roots (Fig. 6), and additional wall components have been investigated with other molecular probes (74-76). These studies indicate that the interface is an apoplastic space of high molecular complexity where the boundaries of the partners are defined. The examination of other endomycorrhizal systems has demonstrated that their interface is morphologically similar but different in composition. Cellulose and pectins are present at the interface... 

By keeping the same fluorophores, but reducing the cavity size of the ion-ophore, we obtain PCT-13 (Mag-Indol) and PCT-14 (Mag-Fura2) (Figure 10.22), which are selective for magnesium. PCT-8, PCT-9 and PCT-11 to PCT-14 are commercially available in the non-fluorescent acetoxymethylester form so that they are cell permeant and they recover their fluorescence upon hydrolysis by enzymes (Molecular Probes, Inc., Eugene, OR). 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

Figure 6.24 Enzyme-labeled fluorescence. ELF-97 is a soluble phosphorylated substrate cleaved by alkaline phosphatase into a highly fluorescent, insoluble product. (Molecular Probes, Inc., Eugene, OR.)...

Among the items that have been measured are vitality, intracellular pH, DNA and RNA content, and specific plasmids [77,408]. Besides nucleic acids [204], other intracellular components can also be analyzed, e.g. storage materials [2, 82,294], enzymes and protein content [6,338], or the cell size [60,61]. The physiological state can also be rapidly assessed [331]. Furthermore, this technique allows the separation of certain cells using a cell sorter, e.g. for strain improvement [28]. The flow cytometry technique has also been used in connection with molecular probes for identification and viability determination of microbial communities [98]. This application of viability estimation is becoming increasingly important [63, 136, 188, 454]. Unfortunately, the equipment is expensive and most of the measurements are tricky and laborious and not well designed for on-line application. 

A/PI4Ka 1 (Figure 3) is probably the major enzyme contributing to the previously reported F-actin associated Ptdlns 4-kinase activity (Tan and Boss, 1992 Xu et al., 1992). While it was also reported that PIP5K activity was associated with the actin-enriched fraction from plants, the specific isoform of the enzyme has not been identified (Tan and Boss, 1992). As more specific antibodies and molecular probes become available, it will be important to monitor lipid protein interaction in vivo and in vitro. 

One key aspecf of applicafions of combinatorial libraries in chemical biology is fhe mapping of subsfrafe specificities for enzymes. Alfhough the early studies that use peptide libraries were focused on studying antibody epitopes (12), the substrate mapping for enzymes that transform peptides and the development of molecular probes found widespread interest. 

Because of the various problems, the search for better methods of differentiating the cholinesterase genotypes will undoubtedly continue. Ideally, what is required is a molecular probe that is capable of interacting in a distinctly different manner with each genotypic variant of the enzyme, and which will produce an unequivocal signal characteristic of each genotype. At the present time, such a universal probe is not in sight. 

The role of amide bonds in molecular recognition, enzyme inhibition and protein stability has been probed by a variety of amide-bond surrogates with a variety of chemical modification to the peptide bond. Early work in this area on pseudopeptides, or amide-bond surrogates, has been reviewed by Spatola (95, 96). 

Kozikowski AP et al (2008) Use of the nitrile oxide cycloaddition (NOC) reaction for molecular probe generation a new class of enzyme selective histone deacetylase inhibitors (HDACIs) showing picomolar activity at HDAC6. J Med Chem 51(15) 4370-4373... 

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