Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

The determination of enzyme activities and the screening of enzyme regulators is a major task in clinical chemistry and the development of new drugs. A broad variety of enzymatic reactions is associated with the consumption or formation of small molecules like H(2)O(2), ATP, pyrophosphate, or phosphate. Luminescent lanthanide complexes can be applied to monitor these enzymatic conversions and therefore can serve as probes for the determination of enzyme activities. The utility of this concept will be demonstrated by means of some selected examples including europium and terbium complexes. Accordingly, this new approach could be already implemented for the determination of glucose oxidase, catalase, and peroxidase activity. In particular, enzymes that catalyze phosphorylation or dephosphorylation reactions came to the fore of interest because of their high relevance as drug targets. These include (protein) kinases, adenylyl cyclases, phosphodiesterases, phosphatases, and ATPases. The development and design of fluorescent lanthanide complexes should lead to probes with optimized selectivity and response times that can be applied for high-throughput screening of enzyme inhibitors and for real-time monitoring of enzyme kinetics. In contrast to other assays for enzyme activity determination, this method does not require the use of radioactively labelled substrates or the accomplishment of rather complex and expensive immunoassays.

Guanine nucleotide binding proteins, such as Ras proteins, play a pivotal role in maintaining the regular life cycle of cells. The involvement of Ras mutants in the progress of cancer has attracted many efforts to find detection methods for Ras activity. In this study we present a luminescent microwell plate assay for monitoring GTPase activity of Ras proteins. The luminescence intensity of the Tb-norfloxacin complex is influenced by nucleoside phosphates as well as by inorganic phosphates. Real-time kinetics of the GTPase activity of wild-type Ras and Ras mutants can be monitored online. The effect of a GTPase activating protein as well as of a downstream effector (Ras-binding domain of human Raf-1) on the GTPase activity of different Ras mutants is examined. In contrast to other methods, this assay does not require the use of radioactively labeled substrates or chromatographic separation steps. Moreover, the application of fluorescently labeled GTP substrates which often interfere with enzymatic activity can be avoided. This in vitro assay can serve as a model system for the screening of regulators affecting the GTPase activity of Ras proteins.

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Luminescent lanthanide complexes (LLCs) are versatile probes for the determination of a variety of analytes such as metal ions, anions, hydrogen peroxide, ATP, pH, or oxygen. Antenna chromophores have been introduced as ligands for sensitizing lanthanide emission. The ligand system or the lanthanide ion can act as receptors for reversible binding of analytes. Thus, the response of luminescence emission can occur via ligand or metal-centered processes, which will both be discussed. LLCs, particularly Eu 3+ and Tb 3+ complexes, have been incorporated in solid state matrices, e.g., polymer hydrogels or silica to obtain optical sensor layers. A selection of such sensitive materials will be outlined, including sensors for the analytically relevant parameters oxygen, pH, hydrogen peroxide, humidity, and temperature.