Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.
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PMID:Behavior of a fluorescent analogue of calmodulin in living 3T3 cells. 404 38

The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC50 of 4.2 nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine.
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PMID:Fluorescence polarisation for high-throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay. 3295 83