Diethylpyrocarbonate DEPC

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

Table VI. Effect of Diethylpyrocarbonate (DEPC) on Thermoanaerobe Glucose Isomerase Activity of the Wild-Type and Mutant Enzymes ...

Prohibited Additives. Diethylpyrocarbonate (DEPC) is now prohibited in the United States and other countries. Since a small but regular amount of diethyl carbonate is a constant byproduct of its use and is easily detectable with GLC, wines to which DEPC has been added are easily detected (69, 70, 71). 

Diethylpyrocarbonate (DEPC), an ester of ethyl alcohol and carbon dioxide, kills yeast and bacteria. This ester also decomposes within a few hours after addition to wine. Thus, if a small amount of diethylpyrocarbonate were added to the sweet wine blend just before bottling, the ester would kill all organisms inside the bottle and then decompose to ethyl alcohol, carbon dioxide, and the simpler ester, ethyl carbonate. 

Water treated with diethylpyrocarbonate (DEPC) as RNase-free water. 

Glassware should be baked overnight at 200°C. Nondisposable plasticware should be rinsed thoroughly with 0.1 A NaOH, 1 mill ethylenediamine-tetra-acetic acid (EDTA), and then with RNase-free (diethylpyrocarbonate [DEPC]-treated) water. Alternatively, it can be treated with RNase AWAY (Molecular BioProducts), according to the manufacturer s instructions. 

Diethylpyrocarbonate(DEPC) treated solutions are prepared by adding DEPC to 0.1% vol (in a fume-hood), mixing vigorously, and incubating either overnight at room temperature or for 2 h at 37 C. DEPC solutions are then autoclaved for 20 min to inactivate DEPC. 

Cells of Scenedesmus obliauus (wild type) were grown heterotrophically in the dark. PS II membranes were prepared as described in [7]. The following proteases were used to modify the PS II membranes carboxypeptidase A, 1 h incubation with 1 2 Chhenzyme subtilisin Carlsberg, 30 min incubation with 20 1 Chhenzyme and trypsin, 10 min incubation with 50 1 Chhenzyme. Incubation with trypsin was performed at pH 7.4, otherwise all treatments were done at pH 6.5. The reactions were stopped by 20 times dilution with ice-cold 20 mM MES-NaOH pH 6.5, 20 mM NaCl, 400 mM sucrose, and 1 mM phenylmethylsulfonyl fluoride (PMSF), except for carboxypeptidase A incubations where 4 mM 1,10-phrenanthroline was substituted for PMSF. Mn depletion of PS II membranes was accomplished by a 30 min incubation with either 0.8 M Tris-HCl pH 8.4 or 5 mM NHjOH pH 6.5. Histidine and carboxyl residues in PS II membranes were modified with 500 /xM diethylpyrocarbonate (DEPC) [8] or 10 mM l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) [9], respectively. More details regarding these procedures can be found in [5]. 

Diethylpyrocarbonate (DEPC)-treated water. DEPC (Sigma, Poole, UK) is added to double-distilled water to a concentration of 0.1% (v/v). The solutions are shaken then autoclaved. All solutions used for steps between sectioning tissue and the first posthybridization wash are prepared in DEPC water. All subsequent steps use solutions prepared in double-distilled water. 

Purified cottonseed NAPE synthase enzyme exhibited non-Michaelis-Menten biphasic kinetics with respect to the free fatty acid substrates, palmitic and linoleic acids. Kinetic parameters for the two saturable sites were calculated from various transformations e.g., double-reciprocal and Hill plots Cornish-Bowden, 1995) of initial velocity/ substrate concentration data and are summarized in TABLE 1. Preliminary experiments with several group-specific modifiers indicated that NAPE synthase was progressively inactivated by increasing concentrations of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), diisopropyl fluorophosphate (DFP), phenylmethylsulfonylfluoride (PMSF), diethylpyrocarbonate (DEPC) (TABLE 2). These results suggest that NAPE synthase may form a thioester- or ester-intermediate through a cysteine or serine residue, respectively, and a histidine residue may participate in catalysis as well. 

All stock solutions for RNA work should be made with diethylpyrocarbonate (DEPC)-treated water and, where possible, autoclaved. 

Care should be taken to prevent any contamination by ribonucleases. We prepare the m vitro transcription reagents and solutions with diethylpyrocarbonate (DEPC)-treated water. DEPC inactivates ribonucleases and is used in the following way dilute 1 mL DEPC per liter milliQ or double distilled water Mix and let stand overnight Autoclave the next day to inactivate the DEPC Note however, that some researchers simply use sterile milliQ or distilled water... 

M CsCl solution dissolve 479.85 g of CsCl (optical grade, Gibco-Brl, Life Technologies, Paisley, UK) in ddHjO Add 100 mL of 0.5 M EDTA, pH 8.0 and adjust pH to 7.0. Adjust the volume to 5(X) mL with ddH20 and filter as noted previously. Treat this solution with diethylpyrocarbonate (DEPC) (01% final concentration) overnight (0/N) and autoclave (see Note 2)... 

Diethylpyrocarbonate (DEPC) is a broad and highly sensitive histidine modifier. DEPC reacts with His residues in proteins to yield an N-carbethoxyhistidyl derivative, followed by an increase in absorbance at 240 nm [121]. The reaction is as follows ... 

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