Assay curvature

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

If the enzyme under consideration is assayed via a coupling enzyme(s) system, downward curvature in the plot will be observed when the primary enzyme fails to limit the observed velocity. In such instances, it would be necessary to increase the amount of coupling enzymes such that they remain in excess under aU conditions. This problem is often encountered in manometric assays, where the rate of diffusion of gas into the liquid begins to limit the enzyme reaction rate. 

Fig. 5. Example of dysmorphology induced by assay treatment. A range of body shape defects are shown, (a) Normal body shape, (b) Abnormal body shape moderate lordosis (ventral curvature), (c) Abnormal body shape severe lordosis. (Reprinted from Panzica-Kelly JM et al. (23), with permission from John Wiley and Sons.).

In practice it is desirable to be able to detect substrate-related porous diffusion reliably by simple means. A popular method is to assay the enzyme activity at varying substrate concentrations. At low substrate concentrations diffusional restrictions are likely to be predominant. They can be detected by graphical evaluation. Instead of straight lines somewhat curved Hues are obtained in the case of v/S vs. v-plots according to Eadie-Hofstee [89]. However, the extent of the curvature is not necessarily as great as would be required to detect diffusional control beyond all doubt. Thus one caimot exclude diffusional effects if nonlinearity is not observed. 

Bioassay Studies of Brassins - BARC As indicated by Mandava (3), bioassay systems have been rather extensively used to study brassins physiology. In addition to the bean 2nd internode bioassay, the bean first internode curvature bioassay (28) and the mung bean epicotyl bioassay (29) were found to be useful by Meudt and Gregory. In many cases, details of early brassins studies using these and other assay systems were not published because the chemical nature of the active component(s) of brassins was unknown at the time. However, types of responses obtained with the synthetic brassinosteroids were essentially the same as those obtained with brassins (29, 30, 31, 32, 33). ... 

Cu(II) bleomycin was also analyzed using the FID assay (Figure 4). In stark contrast to the others, this metalloWeomycin failed to produce an appreciable decrease in overall F and did not exhibit FID plot curvature. Notably also, Cu(II) bleomycin did not discriminate against binding to the A/T-only cassettes the A/T-only cassettes were distributed throughout the oligonucleotide rank order in contrast to the other metallobleomycins studied. These results indicated that Cu(II) bleomycin did not bind to these hairpin substrates appreciably under... 

The assembly-disassembly cycle of CO PI and COPII coats is controlled by the GTPase cycle of the small G proteins Arfl and Sar. We describe here two spectroscopic assays that enable real-time studies of some elementary steps of coat assembly and disassembly on artificial liposomes of defined composition and curvature. A flotation assay to assess the effect of membrane curvature on protein adsorption to liposomes is also presented. 

Despite the overall complexity of coat assembly and vesicle formation, major advances have been made in the understanding of COP machineries. One breakthrough was the reconstitution of COPI and COPII assembly using purified components and artificial liposomes of defined composition (Bremser et al, 1999 Matsuoka et ah, 1998 Spang et al, 1998). In this chapter we describe two spectroscopic assays that complement the biochemical reconstitution and that enable the study of some dynamics aspects of protein coats, notably their assembly-disassembly cycle under the control of the small G-proteins Arf and Sar. In addition a biochemical flotation assay is detailed that permits fair determination of protein binding to liposomes of increasing curvature. 

The fluorescence assay is useful to dissect physical and chemical factors of the hpid membrane that influence the Arfl activation-inactivation cycle. Some GEFs or GAPs harbor PH domains and introducing phosphoinosi-tides at few % in the liposome formulation can have a dramatic effect on the rate of Arf activation or inactivation. As shown in Fig. 2, the tryptophan fluorescence assay can also be used to study the effect of membrane curvature (Bigay et al, 2003). In these experiments, Arfl has been activated by artificial nucleotide exchange (low Mg concentration) on small or large liposomes obtained by extrusion. 

On first view, the Hill coefficient n reports on the cooperativity of the transport process.Measnred below ECso to avoid artifacts from assay satnration, Hill plots with n > 1 show an upward curvature (Figure 11a), Hill plots with n < 1 show a downward curvature (Figure 11b), and Hill plots with n = 1 are linear. With n > 1, Hill coefficients correspond to the number of monomers (or stable dimers, trimers, etc.) in the active supermolecule, and ECsos can represent its /fo- With < 1, the situation is more complex. 

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