Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

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 α1ß1 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.

Kinetic determination of the GTPase activity of Ras proteins by means of a luminescent terbium complex.

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.

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin.

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [alpha-(32)P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K(M)), the v(0)(max) value of ATP turnover, and the IC(50) values for three inhibitors of EF by this newly developed fluorescent method.

Luminescent lanthanide complexes as probes for the determination of enzyme activities.

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.