Reciprocal inhibition studies

There are three basic methods for carrying out alternative substrate inhibition studies. In the first, the investigator seeks to observe numerical changes in the coefficients of the double-reciprocal form of the enzyme rate expression in the presence and absence of the alternative substrate. For some mechanisms, only certain coefficients will be altered. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes . 

In the examples above, the secondary plots utilized the slopes or intercepts of the original plot. However, replots are secondary plots for any functional dependency using data obtained from a primary graphing procedure. Secondary replots can also be used with inhibition studies. In these cases, the slope or intercept of a double-reciprocal plot is graphed as a function of the inhibitor concentration. 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

Our laboratory has done kinetic studies on the mushroom enzyme with the catechols listed in Table III, and a brief summary of results is given here. Inhibition studies with cyanide and benzoic acid show that benzoic acid is competitive with catechols but noncompetitive with the cosubstrate oxygen. In a reciprocal fashion, cyanide is competitive with O2 but non-competitive with the catechol substrate 102), The kinetic constants are given in Table IV. Since a likely hypothesis is that CN" combines with the copper site, it would appear that O2 binds near the copper and that there is an adjacent site for the catechol or phenol substrate. 

The apparent dissociation constant Aei can be calculated from the slope changes in reciprocal plots with A variable and any fixed concentration of B it will be smaller than the true dissociation constant for El if the latter can combine with B to form an inactive ternary complex. This must be borne in mind in interpreting inhibition studies with coenzyme fragments of varying size, since the larger the fragment the more likely... 

The available studies indicate that a reciprocal inhibition occurs in the desatura-... 

The kinetic mechanisms of PP-ribose-P synthetase partially purified from human erythrocytes has been studied. Double reciprocal plots of initial velocity studies with variable MgATP cencen-trations and fixed ribose-5-P are intersecting and suggest a sequential kinetic mechanism, (Figure 2). Product inhibition studies... 

Steinberg et al. (1969) have shown the existence of antibodies binding specifically with poly rl-poly rC labelled with in the sera of NZB and NZB/W mice as well as in the sera of SLE patients. This reaction can be inhibited by double-helical polyribonucleotide complexes. They showed also that these antibodies are different from anti-native DNA antibodies because the latter are not inhibited by double-helical polyribonucleotides. They later confirmed these results (Talal et al., 197I) by studies of reciprocal inhibition... 

In this scheme, EOH is the enzyme, IX is the inhibitor (either a carbamate or an organophosphate). EOH(IX) is analogous to the Michaelis Menton comploc seen with the substrate reaction. EOI is the acyl-enzyme intermediate for carbamates or a phosphoro-enzyme intermediate for the organophosphates. The equilibrium constant for this reaction (K ) is defined as k /k and the phosphorylation or carbamylation constant is defined as k2- In this study 42)y ANTX-A(S) was found to be more specific for AChE than BUChE. The double reciprocal and Dixon plot of the inhibition of electric eel AChE indicated that the toxin is a non-competitive inhibitor decreases, k remains unchanged) (Figure 2). 

In order for the dream to continue (so that it can be observed), the pontine activation would have to persist, and with it, all the outward signs of REM sleep (including the inhibition of muscle tone and the REMs themselves), as Stephen LaBerge observed in his laboratory studies of lucid dream adepts. We would also predict that the amygdala activation would decline reciprocal to the dorsolateral prefrontal increase and with that decline, negative emotionality, especially fear, might also diminish. 

Hexokinase does not yield parallel reciprocal plots, so the Ping Pong mechanism can be discarded. However, initial velocity studies alone will noi discriminate between the rapid equilibrium random and steady-state ordered mechanisms. Both yield ihe same velocity equation and families of intersecting reciprocal plots. Other diagnostic procedures must be used (e.g., product inhibition, dead-end inhibition, equilibrium substrate binding, and isotope exchange studies). These procedures are described in detail in the author s Enzyme Kinetics behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley-Interscience (1975),... 

The L-phenylalanine inhibition of rat (G5) or of (Fig. 12) human intestinal alkaline phosphatase and of human placental (G6) enzyme is of the uncompetitive type, because the double reciprocal plots of velocity and substrate concentration were all straight lines parallel to those obtained without the inhibitor. Consequently, the extent of the inhibition was greatly dependent on substrate (Fig. 11) and inhibitor concentrations (Fig. 10). Detailed studies have appeared elsewhere (G5). 

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