Diethylpyrocarbonate

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... 

Abbreviations DEPC, diethylpyrocarbonate DCCD, Af.iV -dicyclohexylcarbodiimide EEDQ, N-ethyoxycarbonyl-2-ethoxy-1,2-dihydroquinoline NEM, A-ethylmaleimide PAO, phenylarsine oxide NPM, IV-phenylmaleimide PLP, pyridoxal phosphate DIDS, diisothiocyanostilbene disulfonate PITC, phenylisothiocyanate. 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Nuclease-free water (not DEPC (diethylpyrocarbonate) treated) (Ambion). 

Caution All specimen handling and subsequent procedures must be carried out using ultra pure, diethylpyrocarbonate-treated deionized distilled auto-... 

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

Kim, Y.S. Park, C. Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate. Biochim. Biophys. Acta, 956, 103-109 (1988)... 

The binding of one Zn ", presumably at the Zn site, organizes the active site, as shown with BESOD by H NMR spectroscopy and by chemical modification studies with diethylpyrocarbonate The ligands of the Zn rite are close together in the sequence (Scheme 2). 

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. 

However, diethylpyrocarbonate also will react with the traces of ammonia in wines under high pH conditions to produce a few parts per billion urethane. Therefore, the compound is no longer used in any foods produced commercially, and its use by the home winemaker cannot be recommended. 

Imidazole, lysine amino groups, and tyrosine hydroxyl groups react with diethylpyrocarbonate... 

Dicyanocobalamin 870 3,6-Dideoxyhexoses, formation of 747 Dielectric constant 47 Diesenhofer, Johan 84 Diethanolamine, pKa value of 99 Diethylmalonic acid, pKa value of 99 Diethylpyrocarbonate reaction with histidine 126... 

To obtain a template for the RACE, RNA isolation is necessary to rewrite it into cDNA. Precautions have to be taken, as RNA is a considerably more unstable molecule than DNA and, to add to the situation, its degrading enzymes are among the most stable known. Nowadays, most suppliers provide RNAse-free buffers and water, so DEPC (diethylpyrocarbonate) treatment to destroy RNAse activity is necessary only for home-made buffers. Kits for RNA preparation, either for total RNA or for mRNA isolation, do not require any DEPC treatment... 

Biophysical studies of the urease metal centre in the presence and absence of inhibitors, in conjunction with kinetic data provide the model of the bi-Ni site shown in 1. Certain inhibitors are thought to bridge the two nickel atoms consistent with a bridged transition state during urea hydrolysis. The ligands for nickel are believed not to contain sulphur, however, an essential cysteine is proximal to the active site. Comparisons of diethylpyrocarbonate reactivity for apo- and halo-enzyme are consistent with His as a ligand to nickel (Lee et al., 1990). 

Figure 6. Modifications of imidazole groups (a) ethoxyformic anhydride (diethylpyrocarbonate) (b) iodoacetic acid (c) iodination.

Modified histidine residues of HRP elucidated by the specific chemical modification of histidine residues of CPO with diethylpyrocarbonate The catalytic activity of HRP was not [60]... 

The structure and enzyme kinetics of bovine erythrocyte superoxide dismutase are reviewed. The protein has a novel imidazolate-bridged copper(II)-zinc(II) catalytic center in each of two identical subunits. Since a C /Cu1 redox couple is responsible for the dismutase activity of the enzyme, the role of zinc is of interest. Both 220-MHz NMR measurements of the exchangeable histidine protons and chemical modifications using diethylpyrocarbonate demonstrate that zinc alone can fold the protein chain in the region of the active site into a conformation resembling that of the native enzyme. Other possible roles for zinc are discussed. Synthetic, magnetic, and structural studies of soluble, imidazolate-bridged copper complexes of relevance to the 4 Cu(II) form of the enzyme have been made. 

The extreme sensitivity of RNA to the ubiquitous inter- and intracellular nudeases (for example on the skin of the investigator) makes special precautions necessary for effective RNA preparations. The use of disposable containers is recommended, or glassware that has been soaked in dilute hydrochloric acid and rinsed with autoclaved distilled water. Disposable gloves must be worn in all procedures where RNA is handled, or is likely to come into contact with RNA, such as solutions, chemicals, glassware, spatulas etc. Buffers for RNA work should be prepared from reagents reserved for this purpose, and stored separately. Buffers can be treated with 0.2 % (v/v) diethylpyrocarbonate (care - this is carcinogenic) and autodaved to inactivate RNases, or at least those with adive site histidines. Since most nucleases require Mg2 for activity, the addition of EDTA in mM concentrations to solutions is also recommended. 

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