Artificial substrates

Much of what is known about the chymotrypsin mechanism is based on studies of the hydrolysis of artificial substrates—simple organic esters, such as /Miitrophenylacetate, and methyl esters of amino acid analogs, such as... 

Artificial substrates used iii studies of the mechaiiism of chymotrypsiii. 

Alkaline phosphatase catalyzes the dephosphorylation of a mmber of artificial substrates ( ) including 3-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, thymolphthalein phosphate, and phenolphthalein phosphate. In addition, as shown recently for bacterial and human enzymes, alkaline phosphatase simultaneously catalyzes the transphosphorylation of a suitable substance which accepts the phosphoryl radical, thereby preventing the accumulation of phosphate in the reaction mediim (25). 

The natural substrates for many enzymes do not show any significant spectral properties and alternative artificial substrates (analogues) may be used. Many hydrolytic enzymes can most easily be detected by using substrate analogues the glycosidases, for instance, may be measured using p-nitro-phenyl derivatives of the appropriate carbohydrates. Hydrolysis results in the production of p-nitrophenol, which can be measured at 404 nm. 

Vitro Descriptive of an event or enzyme reaction under experimental investigation occurring outside a living organism. Parts of an organism or microorganism are used together with artificial substrates and/or conditions. [NIH]... 

As AMQI was both an expensive and unstable artificial substrate, it was replaced by the native substrate of AChE, acetylcholine that is both cheap and... 

Non-isotopic assays have been developed using synthetic acetylated forms of fluorescein-labeled substrates. A number of artificial substrates containing acetylated lysines based on some proteins, such as histones, p53 and p300/CBP, are available for HDAC enzyme activities [11-14]. These include the generic peptides (Fluor-de-Lys), Ac-GA-(NH-Ac-K)-amino-4-methylcoumarin (AMC), Boc-K(Ac)-7-AMC, Ac-NH-GGK(Ac)-AMC [12] and Ac-NH-RHK(Ac)K(Ac)-AMC [14]. The generic peptide... 

The pHopt may not reflect the effect of pH on protein activity (or on biological processes) but may be a result of the effect of pH on the ionization(s) of the sub-strate(s). This may be particularly true when utilizing artificial substrates having value(s) significantly dif-... 

Acetate is known to be a good carbon source for fungi and would be expected to be the ultimate degradation product of cutin (14). The effect of acetate on the production of cutinase by the T-8 strain of F. solani was examined and compared with that of glucose. Since previous studies showed that hydrolysis of the artificial substrate p-nitrophenylbutyrate, PNB, was specifically hydrolyzed by cutinase in the T-8 strain, this activity was used to measure cutinase levels (8). Figure 1 illustrates that basal levels of cutinase activity were detected in the growth medium when T-8 was grown on... 

Figure 4. Specific activity of cutinase for the T-8 parental strain and PNB-1 mutant strain after growth on medium containing 200 mg cutin. Enzyme activity was assayed with the artificial substrate PNB (Panel A) and with the natural substrate, [14C]-labelled cutin (Panel B). (Reproduced with permission from Ref. 13. 1986, American Society for Microbiology.)...

Because of the complex structure and the insolubility of cellulose, the precise mechanism of recognition and action of cellulases have been examined by the use of inhibitors and artificial substrates. Cellulases have been found, so far, in 11 families of glycosyl hydrolases [65]. 

The abundance of L. littorea and the far more sensitive dog whelk, N. lapil-lus, can be used as indicators. TBT contamination has made the dog whelk a rare species in Dutch coastal waters and it is almost entirely absent from harbours and marinas (Harding et al., 1992 Van Moorsel, 1996). TBT potentially accumulates in sediments. However, there is no direct link between sediment concentrations and effects in gastropods, as the preferred habitats of some of the most common gastropods (e.g. N. lapillus, L. littorea) are hard substrates, including natural substrates such as rock and artificial substrates such as harbour walls, breakwaters, etc. (Oehlmann, 2002). 

Morris, P.F., Bone, E., and Tyler, B.M., Chemotropic and contact responses of Phytophthora sojae hyphae to soybean isoflavonoids and artificial substrates. Plant Physiol, 117, 1171, 1998. Zerback, R., Dressier, K., and Hess, D., Flavonoid compounds from pollen and stigma of Petunia hybrida inducers of the vir region of the Agrobacterium tumefaciens Ti plasmid. Plant Scl, 62, 83, 1989. 

The natural substrate of biotinidase is biocytin, but it also readily cleaves an amine bond between biotin and a variety of different compounds, allowing the use of artificial substrates for the quantitation of biotinidase activity. The most common method is the colorimetric assay described by Knappe and coworkers in 1963 [14, 32], which uses biotinyl-p-aminobenzoic acid (B-PABA) as substrate. The assay of plasma biotinidase activity by this method allows rapid detection of individuals with biotinidase deficiency. 

Biotinidase activity in plasma is quantitated by a simple colorimetric assay that uses the artificial substrate B-PABA. Biotinidase cleaves the amide bond of B-PABA forming free biotin and p-aminobenzoic acid (PABA). PABA is then converted to a purple azo dye and quantitated spectrophotometrically. No colour develops when biotinidase is inactive. Biotinidase with reduced affinity for the substrate (Km variant) can be detected by simultaneous assay of the activity with two substrate concentrations, a saturating standard concentration of 0.15 mM and a ten-times higher concentration of 1.5 mM [26]. Greater than 1.2 times higher activity with the higher substrate concentration indicates a Km defect. 

The natural substrate for SR (EC 1.1.1.153) is the PTP that is converted in an NADPH-dependent reaction into BH4. However, the activity of SR is assayed by measuring the conversion of the artificial substrate sepipaterin to BH2 in the presence of NADPH in the dark. BH2 is then oxidized in the presence of iodine at pH 1.0 to biopterin, which is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based, with some modifications, on the method by Ferre and Naylor [24]. 

A compelling experiment tipped the scales decidedly in favor of the SN2 pathway, as reported by Stephen Withers and colleagues in 2001. Making use of a mutant enzyme (with residue 35 changed from Glu to Gin) and artificial substrates, which combined to slow the rate of key steps in the reaction, these workers were able to stabilize the elusive covalent intermediate. This in turn allowed them to observe the intermediate directly, using both mass spectrometry and x-ray crystallography (Fig. 6-25b). 

A distinct group of synthetic depsipeptides comprises of compounds that do not originate from natural product biodiversity several artificial substrates of peptidases and esterases belong to this group, as well as polydepsipeptides that are considered as potentially biodegradable polymeric materials. A specific feature of depsipeptide synthesis is the necessity to acylate a hydroxy acid component, which requires stronger activation of the amino acid component in comparison to normal peptide synthesis. Otherwise, the main principles of depsipeptide synthesis are similar to those of peptides. Frequently, formation of the ester... 

Spectrophotometry is particularly useful with naturally occurring chro-mophores. For example, the rates of many dehydrogenases may be measured from the rate of appearance of NADH at 340 nm (e = 6.23 X 103 M-1 cm-1), because NAD+ does not absorb at this wavelength. Otherwise artificial substrates may be used, such as p-nitrophenyl esters with esterases, since the p-nitrophenolate ion absorbs at 400 nm (e = 1.8 X 104M-1 cm-1). 

Rates of supply may be controlled, in part, by the activity of hydrolytic enzymes that degrade combined molecules in dissolved or particulate organic matter to monomeric constituents prior to enzymatic transport into bacterial cells (Skoog et al., 1999 see also Chapter 13). At present, in situ rates of such exoenzymes have not been accurately measured, but enzyme potential has been estimated with the addition of artificial substrates in saturating... 

Abstract Neurotransmission in the nervous system is initiated at presynaptic terminals by fusion of synaptic vesicles with the plasma membrane and subsequent exocytic release of chemical transmitters. Currently, there are multiple methods to detect neurotransmitter release from nerve terminals, each with their own particular advantages and disadvantages. For instance, most commonly employed methods monitor actions of released chemical substances on postsynaptic receptors or artificial substrates such as carbon libers. These methods are closest to the physiological setting because they have a rapid time resolution and they measure the action of the endogenous neurotransmitters rather than the signals emitted by exogenous probes. However, postsynaptic receptors only indirectly report neurotransmitter release in a form modified by the properties of receptors themselves, which are often nonlinear detectors of released substances. Alternatively, released chemical substances... 

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