6-phosphate labeled with acetate

In common with acetate, CO, and glycerol, phosphate is expected to label lipids nonspecifically. It should be noted that the phosphate in lipids made by the CDP-base pathway and those made by the CDP-DG pathway come from ATP rather than CTP (e.g., via choline kinase or glycerol kinase). 

After administration of labeled phosphate, free phosphate and also some of the acid-soluble fractions of organs are often much more active than the phosphatide fraction. It is therefore of great importance to purify the phosphatides and free them from all other active fractions. Levene s classical method, which is based on the extraction of the mineral constituents by shaking an ether solution of phosphatides with a water solution of acetic acid, does not remove the labeled free phosphate. When, however, acetic acid is replaced by hydrochloric acid, an effective separation can be obtained (69). The effectiveness of this method of purification is seen from the figures of Table XII (9). 

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

The technique known as high speed reversed-phase HPLC was applied, and the fluorescein-labeled substrate and its product were separated within 2.5 minutes on Qg column (4.6 x 33 mm) with a 76 24 (v/v) mixture of (50 mAf sodium phosphate and 50 mM sodium acetate at pH 7) and acetonitrile. The column was run at ambient temperature at 3 mL/min, and 75 fiL... 

Procedure. Add the PA solution to [ H]acetic anhydride and allow the mixture to incubate at room temperature for 2 hr. Collect H-labeled ace-tyl-PA by chromatography on a column (1.5 x 25 cm) of Sephadex G-25 fine by elution with 10 vaM sodium phosphate, pH 7.2. Collect 0.5 ml fractions at a flow rate of 10 ml/hr. The specific activity is approximately 25 Ci/mmol. This procedure should primarily label e-amino groups of lysine residues. 

To prepare I-DOTATATE, 1 mg of DOTATATE was dissolved in 100 mL of 0.02M acetic acid in redistilled water and dispensed into 1 mL fractions in plastic coated vials. These vials were then freeze dried for 24 h and refrigerated for use in further experiments. A vial containing 10 gg of DOTATATE was added to 40 gL of O.IM phosphate buffer solution at pH7.5. A Na l solution at pH7.5 with high specific activity (>2 x 10" Bq/mg) produced in our laboratory using dry distillation of irradiated natural TeO2 powder was used for the labelling studies. 

High-performance size-exclusion chromatography (HPSEC) was used for competition studies. Solubilized HLA-DR1 (0.13 pM) was incubated for 48 h at 37 °C with the N-ter-minally 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled IM-(19-31)-peptide dissolved in 150 mM sodium phosphate, pH 5.5, containing 15 % (v/v) acetonitrile, 0.1 % (w/v) Zwittergent-12 (Calbiochem) and a cocktail of protease inhibitors (0.2 mM PMSF, 5 pM leupeptin, 10 pM pepstatin, and 1 pM chymostatin). Competition assays were performed in a 1.5 pM solution of AMCA-peptide as competitors, different peptides, the peptide library or peptide sublibraries were added in concentrations ranging from... 

The superscripts in the methods column denote the following isotopicaUy labeled nucleotide was made by this procedure nucleotide isolated as the 5 -alkyl or 5 -aryl ester only nucleotide isolated as the 2, 3 -0-isopropylidene acetal only nucleotide isolated as the 2, 3 -0-isopropylidene, 5 -alkyl or 5 -aryl ester derivative only nucleotide isolated as the 2, 3 -di-0-benzoyl, 5 -aryl ester only method involves initial formation of a pyrophosphate derivative by reaction of diphenyl phosphorochloridate with hydrobenzoin cyclic phosphate. 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Exchange between product and pack can occur in both directions, e.g. certain labelling materials such as heat sensitive and self-adhesive labels when in contact with plastic materials. Both the plastic and the adhesives may contain plasticisers or migratory constituents. Most cellulosics use phthalate, sebacate, phosphate-type plasticisers (e.g. methyl phthalate (DMP) may be used in cellulose acetate). Plasticisers may also be found in poly vinyl chloride/acetate copolymers, polyvinyl acetate and polyvinyl alcohol formulations, polymethyl methacrylate, nylon and certain thermosetting resins. 

A series of early biochemical studies have identified the primary precursors of methanofuran as phosphoe-nolpyruvate, dihydroxyacetone phosphate, tyrosine, glutamate, 2-ketoglutarate, acetate, and CO2 as illustrated in Figure 2. Isotope incorporation studies with H- and C-labeled precursors have shown that the fiiran moiety of methanofuran is formed from phosphoenolpyruvate and dihydroxyacetone phosphate. Following dehydration, the next step in the biosynthesis of the fiiran would be a reduction of the carboxylic acid to an aldehyde. The final step in the biosynthesis of the fiiran moiety of methanofuran is expected to be a transamination reaction to form the 2-aminomethyI subunit. 

Displacement by plasma of radiolabeled thrombin and radio-labeled thrombin-antithrombin III inactive complex from a heparinized surface was measured and found to be significant for example, removing 63% of the thrombin and 90% of the complex that could not be removed by phosphate-buffered saline alone. Heparin-poly(vinyl alcohol) (PVA) gel beads with a very low heparin release rate, prepared by acetal coupling of the heparin to the PVA, adsorbed thrombin and potentiated the inactivation of thrombin by antithrombin 111, as measured by both thrombin time and chromogenic substrate assays. 

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