Glutamine solution preparation

Unlabeled amino acid solution (10 x stock) See Table 3.2.6. These amino acids, which are included in the natural L-amino acid kit (ICN Biomedical, 100586), except for the two alkaline amino acids (cystine and tyrosine), are mixed into a 100-ml volumetric flask. Then, 10 ml of L-glutamine (200 mM, Sigma G7513 must be in solution before use) and 50 ml water are added and the solution stirred until all components are dissolved. For L-cystine and L-tyrosine, the indicated amount is added to a 10-ml volumetric flask and the pH adjusted to > 8.0 by adding 2N NaOH drop by drop until the amino acids are dissolved. Then water is added to the 10-ml mark and the solution transferred to a 100-ml flask to complete the solution. Aliquots of 10 ml are prepared using 15-ml conical screw-top tubes and stored at -80°C. 

The kinetic approach that has been most extensively applied to glutamine synthetase is that of equilibrium isotope exchange (85-88). Experimental procedures involve preparing a series of reaction solutions at chemical equilibrium and monitoring the following exchanges ... 

Candida albicans cells were prepared and used immediately to determine the minimum inhibitory concentration (MIC). RPMI-1640 was used as medium with L-glutamine buffered to pH 7 with 0.15 M 3-(Ar-morpholino)propane sulphonic acid solution. Candida albicans cells, 1.5 x 103 cells/ml, were added to the wells of a 96-well plate containing RPMI-1640 and treated with selected experimental... 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

Tissue Preparation 1. P2 Sprague-Dawley Rat pups. 2. Dulbecco Modified Eagle s Medium (DMEM), high glucose, no glutamine, no pyruvate (Cambrex). 3. Ca +-Mg free Hanks Balanced Salt Solution (Cambrex). 4. 2.5% trypsin solution (Cambrex) diluted 1 10 in HBSS for 0.25% final, aliquot and store at -20°C, stable for 1 year. 5. DNAse 1 (Boehringer Mannheim) prepare 1 mg/mL stock in PBS aliquot store at -20°C stable for 1 year. 6. Heat-inactivated FBS (Cambrex) aliquot and store at -20°C. 

An alternative solution for the problem is the preparation of active esters of (N -blocked) asparagine and glutamine and to separate the desired carboxamide derivative from the nitrile. The purified reactive intermediates... 

Asparagine and glutamine were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium bicarbonate and sodium phosphate monohydrate were obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). All aqueous solutions were prepared in deionized water purified with a Milli-Q Reagent Water System (Millipore, Bedford, MA). 

Sixty-six different binary mixtures were prepared by randomly mixing different milligram quantities of asparagine and glutamine in 10 ml of buffers. The buffer was conq)osed of 4.17 mM bicarbonate and 8.44 mM phosphate, and the pH was set at 6.35. All measurements for a particular solution were conq)leted in less than one hour after the solution was prepared. Fresh sanq)les were used to avoid conq)Hcations caused by hydrolysis of these amino acids. 

Before DMEM is used for culture, add 400pL each of glutamine and penicillin-streptomycin solntion to 40 mL of DMEM. Eresh glntamine and penidUin-streptomycin shonld be added to the DMEM working solntion after 2wk. The working solution is good for 4 wk after preparation when kept at 4°C. 

Rotations at the sodium o-Iine for the majority of the y-glutamyl derivatives in either water or hydrochloric acid have been reported. Although the y-glutamyl linkage is unstable towards acid (see Section IV. 9), it is possible to obtain reliable rotations for hydrochloric acid solutions if the rotation is measured within 5 min of the preparation of the solution furthermore, one can check that no measurable change in rotation takes place in the first 15 min. The rotation of glutamine itself has previously been determined under these conditions 218). 

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