Cell-free assays activity

AMPK can also be activated by a Ca2+-mediated pathway involving phosphorylation at Thr-172 by the Ca2+/calmodulin-dependent protein kinase, CaMKK 3. CaMKKa and CaMKK 3 were discovered as the upstream kinase for the calmodulin-dependent protein kinases-1 and -IV they both activate AMPK in a Ca2+/ calmodulin-dependent manner in cell-free assays, although CaMKK 3 appears to much more active against AMPK in intact cells. Expression of CaMKKa and CaMKK(3 primarily occurs in neural tissues, but CaMKKp is also expressed in some other cell types. Thus, the Ca2+-mediated pathway for AMPK activation has now been shown to occur in response to depolarization in rat neuronal tissue, in response to thrombin (acting via a Gq-coupled receptor) in endothelial cells, and in response to activation of the T cell receptor in T cells. 

Figure 5.12 Diagramatic illustration of the possible correlation between compound potency in cellular and enzymatic activity assays when the cellular phenotype is a direct result of inhibition of the target enzyme. Compounds that fall into the lower left and upper right quadrants demonstrate a correlation of rank-order potency between the cellular and cell-free assays. Compounds in the upper left quadrant may represent potent enzyme inhibitors that for some reason do not achieve adequate intracellular concentrations, as described in the text. Note the absence of any compound points in the lower right quadrant. Population of this quadrant would usually be inconsistent with enzyme inhibition being the direct cause of the observed cellular phenotype.

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

I. Screening for GSK3 Inhibitory Activity Using a Cell-Free Assay (Note 1)... 

Several cell-free assay systems have been used to dissect the mechanisms of DNA damage sensing in vitro. The most extensively applied system is the one using Xenopus extracts. As DNA replication can occur efficiently in Xenopus extracts, this system has been used to analyze how replication interference is sensed by checkpoint sensors (60, 61) and how activated checkpoint regulates DNA replication. The checkpoint can also be elicited in a replication-independent manner by various synthetic DNA structures in Xenopus extracts (9, 62). The checkpoint response in Xenopus extracts is often monitored by the phosphorylation of various checkpoint proteins. The association of checkpoint proteins with damaged chromatin or DNA is used to monitor the recognition of DNA damage in Xenopus extracts. 

The )8-lactamase inhibitory activity of a particular compound may be determined against isolated enzymes in a cell-free assay as well as against whole bacterial cells in combination with an appropriate y5-lactamase-sensitive yS-lactam antibiotic. 

For each BoNT serotype, the dichain form constimtes the active configuration of the neurotoxin the isolated LC and HC are devoid of systemic toxicity. The absence of toxicity is consistent with findings that the LC cannot gain access to the cytosol unless it is coupled to the HC and that the HC lacks the ability to inhibit neurotransmitter release (Stecher et al., 1989 Goodnough et al., 2002). The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit exocytosis from permeabilized chromaffin cells (Stecher et al., 1989), by its ability to cleave SNARE proteins in cell-free assays (Adler et al., 1998), and by its capacity to inhibit ACh release in skeletal muscle when delivered by liposomes (de Paiva and Dolly, 1990). It is not clear whether any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in enhancing the catalytic activity or stability of the LC. 

Based mainly on cell-free assays, 10 enzymatic activities that degrade phospholipids, intermediates in the phospholipid biosynthetic pathway, or triacylglycerol have been reported (Table 2). The detergent-resistant phospholipase A, (encoded by pldA) of the outer membrane, characterized by Nojima and colleagues (Y. Nakagawa, 1991), is the most studied of these enzymes. This enzyme is unusually resistant to inactivation by heat and ionic detergents... 

From the compounds with the highest similarity to the 43 queries, 18 were screened in cellular and cell-free assays for inhibition of the synthesis of 5-LO products. Two were found to be sufficiently active. These two hits were in turn used as queries for a second round of virtual screening on the same in-house compound collection. None of the further 55 analogues screened showed higher potency, but nevertheless the 2 original hits represented novel chemotypes of inhibitors of the 5-LO pathway. 

A cell-free system from photosynthetically active species has to be developed that is able to produce substantial amounts of intermediates and desaturated carotenes. Noteworthy a recent report using isolated chromoplasts from daffodil flowers. Narcissus pseudonarcissus > and isopentenyl pyrophosphate as substrate noted increased levels of phytoene and geranylgeraniol vs. control in the presence of high concentration (50 j,M) of SAN 6706 (27). These cell-free assays should be investigated further with regard to possible different sensitivity against inhibitors because of the species used. 

In cell-free assays the native 4OS subunits carrying the viral proteins appear to be as active, if not even slightly more active, than those from uninfected cells in terms of met-tENA binding (51) It is possible that the viral proteins exert tneir effect at some stage other than met-tENA binding, but this is not yet known. 

FELCs were cultured for 36 hr at these increasing NAm concentrations, DNA and crade nuclear extracts were isolated, and DNA methylase was assayed as previously described (17). DNA from these cells exhibited a dose-dependent hypomethylation, measured by increased methyl accepting ability, which peaked with 20 mM NAm treatment (Fig. 1). DNA isolated from cells treated with 40 mM NAm was not hypomethylated and was presumed to be due to lack of DNA synthesis and cell division, caused by cytotoxic effects of this compound. DNA methylase activity (Fig. 1) was found to be normal at non-cytotoxic doses of NAm. NAm added to the cell-free assay system was unable to significantly inhibit the DNA methylase activity. These results suggest that the DNA from FELCs grown in NAm is hypomethylated and that flie activity of the DNA methylase enzyme is not directly altered by NAm. 

Niacin analogs were compared at 10 mM for ejects on induction of differentiation, Hb accumulation, poly(ADP-ribose) synthetase in vitro, and on DNA methylase activity in cell-free assays and in growing cells (Table 1). None of these compounds were active as inhibitors of DNA methylase activity in cell-free assays. In contrast, these compounds were found to have differential effects on poly(ADP-ribose) synthetase activity in isolated nuclei, as measured by a modified procedure of Hayaishi (23). Inhibition of poly(ADP-ribose) synthetase by these compounds did not, as previously hypothesized, correlate with their ability to induce Hb synthesis and differentiation. N -MNA and 6-MNA were not able to inhibit poly(ADP-ribose) synthetase activity in isolated nuclei when present at 5 mM, whereas NAm and 5-MNA both possess inhibitory activity in vitro. When these compounds were incubated at 10 mM in cell culture for 24 hr, all those able to induce differentiation were also able to cause DNA hypomethylation, as judged by their effect on methyl-accepting abilities of DNA isolated from these cells. 

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