Cell-free assays

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.

Oldfield s work confirmed the complete pathway and identified all the intermediates via an exhaustive experimental scheme, which included whole cell assays, cell-free extracts of IGTS8 as well as extracts from clones containing individual genes expressed in Escherichia coli. The need for NADH was clearly demonstrated in cell-free assays by amendment of NADH. The experimental evidence for involvement of FMN in the pathway was demonstrated [53,66,67],... 

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). 

B. Fubini, Use of physico-chemical and cell free assays to evaluate the potential carcinogenicity of fibres, in Mechanisms of Fibre Carcinogenesis, edited by A. B. Kane, P. Boffetta, R. Saracci, and J. D. Wilboum (IARC Scientific Publication no 140, International Agency for Research on Cancer, Lyon 1996), pp. 35-54. 

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. 

Paris, A., Strukelj, B., Renko, M. and Turk, V. 1993. Inhibitory effect of cornosolic acid on HIV-1 protease in cell-free assays. J. Nat. Prod. 56 1426-1430. 

Analyzing the minimal inhibition concentration (MIC) data for nine different strains, resulted in two significant PCs being obtained, accounting for 77.1% and 16.1%, respectively, of the data variance. Thus, the intrinsic dimensionality of the data matrix of nine assays was only two. The loading plot, that is, a plot of the calculated PCs with respect to the descriptors, shows that the first component is mainly related to the seven cell-free test systems, while the second component represents the two whole-cell test results. Thus, there was much correlation between the results of the seven cell-free assays and the two whole-cell assays were correlated with each other but not with the cell-free assays. In other words, much redundant information was obtained... 

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. 

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