Protection experiment

The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

ANSWER No, we haven t done the Fink-Heimer work at the longer periods with the blocking agents. We have done the AMT protection experiment 2 weeks later. That seems to work on the 5-HT neurons. 

Application of irreversible antagonists (receptor protection experiments, attempted... 

Aluminum can be cathodically protected, but caution must be employed. If there is too much alkali developed as a result of too high a cathodic current, the aluminum will be attacked. Figure 4 shows a cathodic-protection experiment being conducted at Battelle s marine station. 

In a separate line of research, the majority of 129 strain mice were shown to be resistant to a primary infection with 5. japonicum (Mitchell et al., 1984). Moreover, these mice had a dominant antibody response to a 26 kDa antigen from adult worms that was also strongly reactive with sera from rabbits multiply immunized with adult worm extract (Beall and Mitchell, 1986). This allowed the cDNA to be cloned from an expression library and identified as a GST (Smith et al., 1 986). The native protein was easily purified on a glutathione affinity column and used for protection experiments, with mostly negative results except for >50% on one occasion in C57BL/6 mice (Mitchell et al., 1988). The natural resistance of 129 strain mice for S. mansoni was also demonstrated (Tiu et al., 1 986) but unfortunately proved to be the result of a defective portal vasculature rather than acquired immunity (Cox, 1990). This finding undermined the whole concept of immunity induced by GST in 129 strain mice and as a consequence vaccine work with the Philippines strain of S. japonicum largely petered out. 

If the photoaffinity reagent has been designed well and there are a measurable number of tight binding sites for it, the protection experiment will usually succeed. Occasionally it will not for example, four polypeptides of sarcoma cells were labeled with low concentrations of 8-N3-CAMP but only three of the sites could be protected by cAMP. Presumably, the fourth site is not part of a cAMP binding protein but a different nucleotide binding site that just happens to have affinity for the reagent but not for cAMP. 

Another test for specific labeling is to determine whether the ligand binding site is blocked. But, as 1 pointed out in the discussion of photoinactivation experiments, there are several other possible causes of apparent binding site occupation besides the covalent attachment of a ligand. It has also been noted that specific labeling as defined by a protection experiment may not always yield a blocked receptor when the photoaffinity label is a macromolecule. When sodium channels in tissue culture cells were labeled... 

A final possibility is to label in the presence of a molecule that bears a strong structural resemblance to the reagent but is not photoactivatable. This is analogous to a protection experiment in photoaffinity labeling. 

Pingoud, A., Urbanke, C. (1979) The Determination of Binding Parameters from Protection Experiments, Anal. Biochem. 92,123-127. 

In vivo protection experiments with DMS that is able to transfer a methyl group to the N7 position of guanines enabled us to demonstrate that telomestatin engages the telomeric overhang in vivo in a DNA structure resistant to action of DMS. These experiments represent the first proof of the existence of G-quadruplexes at the telomeric G-overhang under the effect of the treatment by a G-quadruplex ligand. 

Intact mitochondria were isolated by the method of Johnson and Lardy with the modifications previously published. Mitochondrial outer membranes were isolated by the method of Parsons et and their purity assessed as described previously. Protease treatment of intact mitochondria and protection experiments were carried out as described previously. " Briefly, this method consisted of incubating the mitochondria (5mg/ml), and outer membranes (1 mg/mL) with Nagarse (5 t.g/mL) at 37°C for lOmin after which the proteolytic activity was stopped by addition of 200 pi of 20% (w/v) BSA/mL of incubation volume plus 40mL of ice-cold isolation medium. After centrifugation (5,600 xg for lOmin), the mitochondria were resuspended (4mg/mL) in isolation medium and used as indicated. Protein determination was by a biuret method. In some experiments intaet mitoehondria or the isolated outer membranes were first ineubated... 

We have employed nuclease protection experiments to directly probe the nature of the contacts between poly(ADP-ribose) polymerase and defined DNA sequences. The data suggest that the polymerase exhibits a broad preference for A + T rich regions and may recognize certain unusual conformation(s) on the DNA molecule. 

A protection experiment is performed and the results are compared with those of the normal labeling experiment. This technique has been used so often in conventional labeling experiments that it need not be expanded upon here. The protecting agent does not prevent nonspecific labeling but indicates what proportion of the observed labeling is relevant. 

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