Inhibitor incubation

Preincubation time with inhibitor Incubation time with marker substrate ... 

Fig 4. Inhibition of 1-13 uptake in mouse neuroblastoma cells by antimycin and monodansylcadaverine. Confluent cells were preincubated for 30 min at 37°C in RPMI 1640 containing the inhibitor. Incubation after addition of 25pM L-T3 was for 30 min. Wells measuring 28.2 cm contained 4 ml of medium. Other conditions as in Fig 1. 

Equimolecular concentration of inhibitors incubated with 133.3 yM l-l c a-linolenic acid as described in Experimental procedures. 

Fig. 3. The effects of chloramphenicol and puromycin on protochlorophyllide regeneration by etiolated bean leaves. Panel A Absorption spectra of 7-day-old dark-grown red kidney bean (Phaseolus vulgaris) leaves taken 4 hours after illumination for 1 minute. Panel B Absorption spectra of the same leaf sets as shown in panel A, but spectra were deteimined after leaves had again been irradiated for 1 minute. Panel C Difference spectra obtained by subtracting an absorption spectrum in panel A from the corresponding one in panel B. Curves a control bi leaves incubated with 5 X 10" M chloramphenicol for 4 hours before initial illumination c leaves incubated with 10" M puromycin for 4 hours prior to initial illumination d control leaves (no inhibitors) incubated with 10 M ALA in darkness during the 4-hour period after the initial illumination e as in b, but incubated with 10 M ALA in the same manner as d. After Gassman and Bogorad (1967a). Data for actinomycin D and cycloheximide are not shown.

The activity of P-lactamase inhibitors is often expressed as an IC q value, which is defined as the concentration of inhibitor that causes 50% inhibition of en2yme activity for a given set of conditions. IC q values, which vary widely according to substrate, time of incubation, and other factors, are presented herein solely to give an indication of potency and en2yme inhibitor specificity. Values that decrease with preincubation are indicative of irreversible inhibitors. 

There are other substrates for the E. coli Met(0) peptide reductase, one of which is Met(0)-a-l-PI. The native protein is the major serum elastase inhibitor that functions by forming a binary complex with elastase which inhibits its activity. Met(0)-a-l-PI, on the other hand, which can be formed by treatment of the protein with TV-chlorosuccinimide, cannot form a complex with elastase and therefore is not able to inhibit elastase activity117,118. Table 6 shows, however, that when Met(0)-a-l-PI is incubated in the presence of Met(0)-peptide reductase and dithiothreitol the protein regains its ability to form a complex with elastase and inhibit elastase activity119. Similar to results found with Met(0)-L12 reduced thioredoxin could replace the dithiothreitol as reductant in the enzymatic reaction. 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

Effect of preincubation of PG inhibitor with PG A suitable aliquot of the inhibitor was incubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for various periods of time at 37°C. The reaction was initiated by addition of 1 ml of 1% polygalacturonic acid solution. It is apparent from Table 6 that the magnitude of inhibitory... 

Kaneko et al. (1993) have described a group of lipophilic ascorbic-acid analogues that have been studied in cultured human umbilical vein endothelial cells that were first incubated with test drug and then exposed to lipid hydroperoxides. Although ascorbate itself did not protect the endothelial cells, derivatives like CV3611 protected. Pretreatment was necessary. CV3611 was synergistic with vitamin E. The authors concluded that these lipophilic antioxidants incorporate into endothelial cell membranes where they are effective inhibitors of lipid peroxidation. In contrast, lipophobic antioxidants were not effective in their hands (Kaneko et al., 1993). 

Male Sprague-Dawl ey rats (200 -300 gm) were used in all experiments. Dopamine uptake was measured using the accumulation of tritium into a striatal synaptosomal fraction in the presence of an MAO inhibitor following a 5-minute incubation period with 10 nM 3H-DA at 37 °C. Uptake at 2 °C was used as a control for uptake by diffusion and was subtracted from uptake at 37 °C. 

E I is a kinetic chimera Kj and kt are the constants characterizing the inactivation process kt is the first-order rate constant for inactivation at infinite inhibitor concentration and K, is the counterpart of the Michaelis constant. The k,/K, ratio is an index of the inhibitory potency. The parameters K, and k, are determined by analyzing the data obtained by using the incubation method or the progress curve method. In the incubation method, the pseudo-first-order constants /cobs are determined from the slopes of the semilogarithmic plots of remaining enzyme activity... 

The gold-standard assay used for all chemokine receptor inhibitors that reach clinical-phase trials is the chemotaxis functional assay. This assay relies on the ability of chemokines to recruit cells expressing their respective receptor to areas of inflammation. In vitro, this assay was first described in detail by Taub et al. (16) for 24/48-well plates currently, this can be achieved by using 96-well plates. Cells are incubated in the upper chamber with an antagonist for a particular receptor (at different concentrations or with buffer) and challenged to migrate to the lower chamber, which has the relevant chemokine. After 2 to 4 hours of incubation at 37°C, the upper chamber inlet is removed and the cells in the lower chamber quantified by fluorescence with, for example, Calcein AM (Invitrogen, Carlsbad, CA). 

Figure 5.8 Dilution scheme for testing the reversibility of an enzyme inhibition The enzyme and inhibitor are pre-incubated at a concentration of enzyme equal to 100-fold that needed for activity assay, and at a concentration of inhibitor equal to 10-fold the IC50 value. The sample is then rapidly diluted 100-fold into an assay solution. The inhibitor concentration thus goes from 10-fold above die IC50 (corresponding to 91% inhibition) to 10-fold below the IC50 (corresponding to 9% inhibition).

Figure 5.9 Recovery of enzyme activity after rapid dilution as described in Figure 5.8. Curve a represents the expected behavior for a control sample that was pre-incubated and diluted in the absence of inhibitor. Curve b represents the expected behavior for a rapidly reversible inhibitor. Curve c represents the expected behavior for a slowly reversible inhibitor, and curve d represents the expected behavior for an irreversible or very slowly reversible inhibitor. See color insert.

The use of CL-free media required 100 times higher concentrations of the toxins for any cellular uptake. Furthermore, the entry of both toxins was denied when Vero cells were incubated in normal medium with inhibitors of CL entry, including 4-acetamido-4 -isothiocyanostilbene-2,2 -disulfonate (SITS), pyridoxal... 

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