Substrate mixtures

When phenyl phosphate is the substrate, the released phenol is conveniently measured using the reaction described by Powell and Rind and Ring based on 4-amino antipyrine (4-AAP) and ferri-cyanide. It is possible to incorporate the 4-AAP directly into the buffered substrate mixture, so that color can be developed... 

The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaCl2 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma medicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM Ca [25]. The lyase from Fusarium solani f sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent the pectate lyase from Rhizoctonia solani [34] and the endopectate lyase fi om Fusarium solani f sp. pisi [31]. 

McGeehan, and J. Wiseman, Rapid optimization of enzyme substrates using defined substrate mixtures, J. Biol. Chem. 267 1434 (1992). 

Stock Solution of H+-ATPase Substrate Mixture for Vanadate- or Nitrate-Sensitive H+-ATPase ... 

When nitrate-sensitive H+-ATPase (tonoplast) activity is measured, 250 mM (final concentration, 200 mM) KN03 is added to the stock solution of the substrate mixture. When vanadate-sensitive H+-ATPase (plasma membrane) activity is measured, 125 pM Na3V04 (final concentration, 100 pM) is added to the stock solution of the substrate mixture. 

Stock Solution of H+-Pyrophosphatase (PPase) Substrate Mixture ... 

To prevent the precipitation of Mg2+ salts, MgS04 should be added to the PPase substrate mixture just before use. 

I. H+-ATPase, H+-PPase, and UDPase. Enzyme activities of vanadate- or nitrate-sensitive H+-ATPase, H+-PPase, and UDPase are measured by the quantitation of released inorganic phosphate from the substrate by enzymatic hydrolyzation. When H+-ATPase activities are assayed, at least three substrate mixtures should be prepared (1) a substrate mixture containing nitrate, (2) a substrate mixture containing vanadate, and... 

We conclude the discussion of formal kinetics with a practical consideration. When two isotopomers simultaneously present in an enzyme substrate mixture compete for the same active site on the free enzyme E, one can write ... 

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

Further studies, with the substrate ratio altered from 1 3 to 1 100, were performed in order to suppress the formation of benzoin 12 and increase the yield of DMA-HPP 10. Although this strategy was successful, the results showed that even a 100-fold excess of dimethoxyacetaldehyde 8 (500 mM) relative to benzaldehyde 4 (5 mM) could not completely supress benzoin 12 formation catalyzed by variant 55E4. Furthermore, the formation of the mixed product DMA-HPP 10 was decreased by a factor of 2 relative to the 1 3 substrate mixture. Variant 55 E4 showed a 55-fold higher productivity with respect to the formation of DMA-HPP 10 under these conditions as compared to variant L476Q. The overall carboligation... 

Eutectic melting (and also similar systems with added adjuvants/solvents) has been used to prepare homogeneous substrate mixtures with extremely high concentration levels as media for enzymatic reactions [37, 68, 69]. 

Table 4.6.18 Substrate mixture for the phosphorylase reaction. Instructions for the preparation of these solutions are given in section 4.6.21.3...

On the other hand, sensitized oxidation of highly concentrated sensitizer-substrate mixtures have been successfully developed in falling film reactors (for example, the synthesis of 2-hydroxy-5H-furanone (Eq. 48 [82, 83]) [12]. 

The treatment of iodosylbenzene (2) with two equivalents of trimethylsilyl azide in dichloromethane results in the formation of [azido(trimethylsilyloxy)iodo]-benzene (3) and [b/s(azido)iodo]benzene (4), (Scheme 2) [11 -14]. Although the azidoiodanes are unstable and decompose in solution at temperatures above -30°C, they can be employed in situ for azidonations of organic compounds. The addition of trimethylsilyl azide to iodosylbenzene/substrate mixtures enables such azidonations to be effected at temperatures higher than - 30 °C. 

Fig. 8. Heteronuclear single-quantum coherenc (HSQC) spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free system. The FT protein was synthesized in the same way as in Fig. 6 except that Ala, Leu, Gly, and Gin in both translation and substrate mixture were replaced with their -labeled forms (Isotec, Inc ). After incubation for 48 h, the reaction mixture (1 mL) was dialyzed against 10 mMphosphate buffer (pH 6.5) overnight, and then centrifuged at 30,000g for 10 min. The supernatant containing 30 xMof the protein was directly subjected to nuclear magnetic resonance spectroscopy. The spectrum was recorded on a Broker DMX-500 spectrometer at 25°C, and 2048 scans were averaged for the final H- WHSQC spectrum.

The bi-layer-translation method can synthesize protein without any membrane (see Chapter 10 [17]). To initiate the translation, the substrate mixture is simply overlaid onto the translation mixture. The system works for more than 10 times longer than batch-mode reactions, yielding sub-milligram quantities of proteins sufficient for functional analysis and determining solubility of gene products from cDNA libraries. Work to further improve the bi-layer system is in progress in our laboratory, where yields similar to those of the CFCF method have been obtained. 

The purpose of every crossover experiment is to determine whether reactions take place intra- or intermolecularly. In a crossover experiment two substrates differing from each other by a double substituent variation are reacted as a mixture. This substrate mixture is subjected to the same reaction conditions in the crossover experiment that the two individual substrates had been exposed to in separate experiments. This double substituent variation allows one to determine the origin of the reaction products from their structures, i.e., from which parts of which starting materials they were formed (see below for details). 

The product mixture is then analyzed. There are two possible outcomes. It can contain nothing other than the two products that were already obtained in the individual experiments. In this case, each substrate would have reacted only with itself. With the substrate mixture of the crossover experiment, this is possible only for an intramolecular reaction. The product mixture of a crossover experiment could alternatively consist of four compounds. Two of them could not have arisen from the individual experiments. They could have been produced only by crossover reactions between the two components of the mixture. A crossover reaction of this type can only be intermolecular. 

Sonication should be performed on ice with a probe sonicator (usually during the immunoprecipitation step). Typically, three 10 s bursts interspersed by periods of 30 s is sufficient. However, the precise sonication time will depend on the power of the particular sonicator and whether it is in tune. The Ptdlns appears cloudy at first and should eventually clear. Sonicating too long results in oxidized, precipitated lipid. Do not store the substrate mixture, but rather make up sufficient amounts of Ptdlns/PtdSer mixture for the assay and discard any excess. The sonicated mixture of Ptdlns and PtdSer may be further diluted as required, to obtain optimal substrate concentration. 

To study the durability of immobilized lipase, the two- and three-step methanolyses were repeated by transferring the enzyme to each fresh substrate mixture every 36 and 48 h, respectively. Consequently, more than 95% conversion was maintained during 70 cycles (105 days) in the two-step reaction, and during 52 cycles (104 days) in three-step reaction (Shimada et al, 1999 Watanabe et al., 2000). These results showed that the lipase can be used at least for 100 days without significant loss of the activity. 

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