Potassium phosphate, modifications

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

LO was purified from two weeks-old bovine calf aorta by a modification of published procedures (14, 15). The aortas (ca. 600 g) were cleaned and finely ground prior to protein extraction. Extractions and all subsequent procedures were carried out at 4 °C. The ground aortas were extracted with 0.15 M NaCl-16 mM potassium phosphate (KPi) (saline buffer), 16 mM KPi, and 4 M urea-16 mM KPi buffer (pH 7.8), sequentially, in the presence of protease inhibitors PMSF (1 mM) and iodoacetamide (0.04% w/v). The saline buffer and KPi extracts contained negligible activity and were discarded. The urea buffer extracts were pooled (total of 4 to 5 L) and mixed with hydroxyapatite gel (100 g, preequilibrated in 4 M urea-16 mM KPi). After batch elution from hydroxyapatite, the urea-soluble enzyme solution was concentrated (ca. 700 ml) and dialyzed against 16 mM KPi (pH 7.8) buffer. 

All tubes contained the following components (in /nmoles unless stated otherwise) potassium phosphate, pH 6.5, 100 ascorbate, 6.0 fumarate, 50 tyramine-/3/3 -3H, 2.0, specific activity 1.15 X 10 CPM//nmole catalase, 300 units, dopamine /3-hydroxylase (4), Final volume, 0.68 ml. at 25°C. Octopamine determined by a minor modification of a published procedure (17). A 0.05 ml. sample of water was obtained by lyophilization and dissolved in 10 ml. of Bray s scintillation mixture. Radioactivity determined in a Packard liquid scintillation spectrometer total counts collected were sufficient to yield a 5% coefficient of variation. The expected tritium release was calculated from the octopamine formed, assuming that the amount of tritium was the same in both /3 positions of the tyramine. The results for the amount of tritium released have been corrected for the amount of exchangeable tritium initially present in the tyramine. 

Chromophore modifications 1) Reduction with borohydride (modified from ref. 10). A solution of PC or isolated subunits (chromophore concentration 7-21/iM, 0.9M potassium phosphate, pH 7, 8M urea) was treated with NaBH (170 mM). After complete reduction (spectrum, 45 min) excess reductant was destroyed by glucose. For reoxidation experiments, the samples (lOOmM potassitim phosphate, pH 7, containing 70% ammonium sulfate to prevent protein degradation) were allowed to stand at room temperature in the dark for up to nine days. This was followed by recombination (if not yet done), denaturation (8M urea in lOOmM potassium phosphate, pH7) and renaturation as described below. 2) Photobleaching of the o-subunit (8/xM protein, 100 mM phosphate, 8M urea, pH 7.5) was... 

Recombination Isolated subunits were modified and then hybridized with the respective "partners by dialysis of combined samples containing 5 mM mercaptoethanol, against lOOmM potassium phosphate, pH 7, 25 C over night, and then with new buffer at 4 0 for 5hrs. In control experiments, PC was modified in toto. Linker-peptide preparations were also added in some experiments. The modification and recombination products were tested by UV-vis-absorption, SDS-PAGE, sedimentation behaviour (13) and (in some cases) gel-filtration on sephadex-G-75 (Serva, Heidelberg). The chromoprotein bands (which were distinct by their color) were marked and quantitated by absorption spectroscopy. 

With the following modifications the losses of label could be kept below 5% Reduction of disulfide bonds with dithiothreitol is performed at pH 6.7 in 0.2 M potassium phosphate in 6 M guanidinium chloride (4 hr at room temperature, 100-fold excess with respect to disulfide bonds). Cyanoethylation is carried out at pH 8 for 15 min with a 5-fold excess of acrylonitrile. After readjustment to pH 4, the protein solution is dialyzed and lyophilized. 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

Research on the various biochemical systems affected by tumor promoters, in particular TPA, has been recently reviewed by Diamond et al. (54) and Werner and coworkers (55) have reviewed the early effects of phorbol esters on the membranes of cultured cells. The latter group reports that TPA causes permeability changes in 3T3 cell membranes and experimental evidence is cited that phorbol esters interact specifically with a membrane-specific macromolecule rather than passive adsorption by the membrane lipid matrix. One of the earliest observed effects of TPA is a significant modification in the transport of potassium, sodium and phosphate. Lee and Weinstein (56) have found that the addition of phorbol esters immediately stimulated the uptake of 2-deoxyglucose in... 

The sensor developed by Gawley and co-workers is based on de Silva s modular approach comprises an azacrown ether linked to a fluorophore by a short link. The molecule combines the aza[18]crown-6 recognition element with a fluorescent coumaryl group attached by a methylene spacer. In tests it was shown to bind to saxitoxin with a binding constant on the order of 105 M 1, even in a phosphate buffer at physiological pH and concentrations of sodium and potassium, and could detect levels of the toxin down to 1(T7 M. Subsequent modification of the sensor allowed it to be linked to the surface of a quartz slide to allow the fluorescent response to concentrations of saxitoxin between 10-4 and 10-6 M to be detected via a fibre optic system. This level of sensitivity is comparable to the current mouse bioassay that requires the inoculation of a large number of animals to determine the concentration of saxitoxin present in the test sample [25],... 

In other modifications of the process, some sulfuric acid is substituted for part of the phosphoric acid. A number of patents describe the use of sodium, potassium, and calcium phosphate as other substitutes for phosphoric acid. 

The 2-(acetoxymethyl)benzoyl-protective group (4,5,6) has initially been used in the synthesis of particularly base-labile backbone modifications, such as phosphate methyl esters or methylphosphonates [51, 52], again thymine remains unprotected. Cleavage is achieved by elegant intramolecular reaction within 90 min at room temperature by use of potassium carbonate in dry methanol (Fig. 11). 

As has been described in Section V-A, crystals and glasses of sodium meta-arsenate-phosphates are composed of anions of long-chain structures, and there is no indication of the presence of ring structures. However, it has been found by Thilo and co-workers (24,61,62) that some modifications of crystals of potassium meta-arsenate-phosphates have long chain structures, while others have ring structures. The structures... 

The Relations among the Modifications of Potassium Metaphosphate, Potassium Metaarsenate, and Potassium Meta-Arsenate-Phosphate... 

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