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- Cebula, Marcus, et al.
(författare)
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Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.
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| 4. |
- Zhang, Jie, et al.
(författare)
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Synthesis and Characterization of a Series of Highly Fluorogenic Substrates for Glutathione Transferases, a General Strategy
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 133:35, s. 14109-14119
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.
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