Rescue experiments

The crystal structure of the cobalt-substituted enzyme was obtained with bicarbonate bound to the metal (Iverson et al. 2000). The structure shows Asn 202 and Gln75 hydrogen bonded to the metal-bound bicarbonate, suggestzing potential roles for these residues in either transition-state stabilization or orientation and polarization of CO2 for attack from the zinc-hydroxyl (Fig. 11.5). The crystal structure also shows three discrete conformations for Glu 84, suggesting a role for this residue in the transfer of protons out of the active site indeed, kinetic analyses of Glu 84 variants combined with chemical rescue experiments establish this residue as critical for proton transfer (Tripp and Ferry 2000). The location of Glu 62 adjacent to Glu 84 suggests a potential role in proton transfer as well. Although kinetic analyses of site-specific variants establish an essential role for Glu 62 in the CO2 hydration steps (Eqs. 11.3 and 11.4), the results were inconclusive regarding an additional role in proton transfer (Eqs. 11.5 and 11.6). 

Figure 6.16 Bases used in substitution and rescue experiments.

It is clear that the analysis of thio effects, rescue experiments and other experiments with derivatives have contributed significantly to our understanding of the mechanism of the action of the large group I intron ribozyme of Tetmhymena. All the available data appear to support the Lewis acid catalysis for activation of the attacking nucleophile and enhancement of the leaving group that is shown in Fig. IIB. 

Rescue experiments Target noncoding sequence 3 imtranslated regions (UTRs), alternatively 5 UTRs, can be targeted by siRNA rescue can be performed by ectopically expressed genes lacking UTRs... 

Mechanistic studies based mainly on metal ion rescue experiments have identified six oxygen atoms involved in metal ion coordination in the active site (the oxygens in bold font in Figure 19) . Metal ion rescue experiments substitute a potential oxygen ligand with a soft atom, usually sulfur, that is much less inclined to coordinate a hard Mg + ion. If the addition of a soft cation such as Cd + restores activity, the oxygen... 

FIGURE 19. The two- and three-metal-ion mechanisms for phosphoryl transfer in the group I introns. Oxygens in bold have been identified as metal ligands by ion rescue experiments. From Reference 177. Reprinted with permission from AAAS... 

Another dwarf mutant of Arabidopsis, sax], defines a step upstream of DWF1 in the brassinosteroid biosynthesis pathway [27]. Rescue experiments with intermediates showed that saxl is involved in the oxidation and isomerization of 3P-hydroxyl,A5 6 precursors to 3-oxo-A4 5 steroids (Fig. (4)). 

Although remarkable progress has been made in matching the secondary structure and the function in the various members of the mitochondrial carrier family (del Arco and Satrustegui 2005), satisfying information about the function of potential ADP/ATP transporters can only come from a detailed functional analysis (see later). Several methods are currently available reconstitution in liposomes, in vivo expression, rescue experiments, and functional tests in bacteria (Escherichia coli, Lactococcus lactis). Such tests involve competition experiments, effector and inhibitor studies (Voncken 2001 Voncken et al. 2002a Haferkamp et al. 2002 van der Giezen et al. 2002 Tjaden et al. 2004 Chan et al. 2005 Leroch 2006). 

Dementin, S., Bouhss, A., Auger, G., Parquet, C., Mengin-Lecreulx, D., Dideberg, O., van Heijenoort, J., and Blanot, D. (2001) Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. Eur. J. Biochem., 268, 5800-5807. 

Generally, it is thought that these compounds are only effective in vivo against insects in the orders of Hemiptera, Homoptera, Dictyoptera and Orthoptera, with exceptions [42, 137]. Holometamolous insects are believed to be insensitive high doses of precocenes may affect development in these and other insect species but it is thought that this action does not occur through inhibition of JH biosynthesis. Rescue experiments with reversal of precocene-induced effects counteracted with JH or JH analogues will allow the differentiation between endocrine-specific and non-specific effects. 

Fujioka et al. [37] found through rescue experiments of the det2 mutant using brassinolide precursors that 6-deoxo intermediates of brassinolide show stronger activity than 6-oxo... 

Fig. 6. Messenger rescue" experiment. The HTC cell suspensions (800,000 cell/ml) were incubated in induction medium with 1 x M cortisol for 17 hours. At that time an 8-ml sample of cell suspension was added to fresh warmed induction medium free of steroid (37 C) and to warmed induction medium containing cortisol (1 x Both suspensions were further incubated at 37°C,...

Fig. 1. Schematic diagram of pCR with the sphaeroides operon as a donor. Three of the classes of recombinants that arose from the chimera rescue experiments are also shown. The hatched boxes represent sphaeroides sequences the open boxes r resent capsulatus sequences. Only the Pstl sites that define the su lone of pJWl (3) used in this experiment are shown. Restriction sites B=BamHI E = coRI H = //i/iDIII P = Pstl S = Sad.

Fig. 8. Messenger rescue experiment. HTC cells in suspension culture were incubated with 10" M cortisol for 17 hours after which the culture was diluted into prewarmed medium free of inducer (zero time). Samples were removed from the deinduced suspension at 0, 15, 45, 90, and 180 minutes and further incubated in the presence of AMD (5 /xg/ml). From each of these subcultures aliquots were removed as indicated, and the cytosols of the cells were assayed for specific TAT activities. The activities are expressed as milliunits of enzyme per milligram of cell protein. From Tomkins et al. [46].

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