Pyrophosphatase elution

Figure 11.4 Analysis of in vitro synthesized RNAs. 32P-Radiolabeled RNAs (48 nucleotides) capped with m7Gp3G (A and C) or m27,3 °Gp3G (B and D) were digested with either RNase T2 (A and C) or RNase T2 plus tobacco acid pyrophosphatase (TAP) (B and D) followed by anion-exchange HPLC on a Partisil 10SAX/25 column as described in the text. Fractions of 1 ml were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by ultraviolet (UV) absorption, are indicated with arrows 3,-CMP (Cp), S UMP (Up), 37-AMP (Ap), 3 -GMP (Gp), 3, 5 -m7GDP (pm7Gp), 3, 5 -GDP (pGp), 5 -GDP (p2G), 5 -GTP (p3G), and guanosine-SCtetraphosphate (P4G).

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

Figure 9.141 Time course of formation of adenosine 5 -phosphosulfate (APS) by activity in rat liver. The reaction mixture contained, in a final volume of 350 /xL, 30 ju.mol of Tris-HCl (pH 8.0), 0.9 /xmol of ATP, 3 /unol of magnesium sulfate, 6 /xmol of sodium fluoride, and 50 fiL of inorganic pyrophosphatase (2.5 U). A50/xL supernatant sample (19 mg of protein) from rat liver was added to start the reaction. Then samples were removed at intervals, and the reaction was terminated and analyzed. Chromatograms were obtained after incubation for (A) 0 minutes, (B) 15 minutes, and (C) 45 minutes. Arrow indicates elution time for the reaction product APS. (From Mina and Rossomando, 1988.)...

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