Xxx coli

The KDO 8-P phosphatase activity was determined by either a discontinuous colorimetric assay or a continuous spectrophotometric assay.

Xxx coli-61Xxx coli-32Xxx coli-25

The p H of the supernatant was adjusted to 7.0 by 1 Tris-HCl (p H 7.4) in a final volume of 60 ml.

The cell suspension was subjected to sonication at 4 °C (ice water bath, 45-s pulses with a 2-min rest between pulses, four times), and the unbroken cells and cell debris were removed by centrifugation (40,000 × Tris-HCl (p H 7.4), sonicated, and centrifuged as above. To remove nucleic acids, a 2.2% (w/v) protamine sulfate solution (p H 7.0) was slowly added at 4 °C with gentle stirring to the supernatant to yield a final concentration of 0.267% (w/v) protamine sulfate.

After continuous stirring for 15 min at 4 °C, the precipitated material was removed by centrifugation (40,000 × Tris-HCl, p H 7.4).

The sample was dialyzed against two liters of the same buffer overnight, and the resulting protein solution was applied to a Q-Sepharose column (1.2 × 21 cm) pre-equilibrated with buffer A.

The amount of inorganic phosphate produced was quantitated by the malachite green assay (19) using KH as the standard.

The continuous spectrophotometric assay was based on the purine nucleoside phosphorylase (PNPase)-coupled phosphate assay first reported by Webb (20) and later modified by Rieger and co-workers (21).

High grade Spectra/Por® 7 dialysis tubing (10,000 molecular weight cut-off and metal-free) was obtained from VWR Scientific.

The Centriprep YM-10 Concentrators were purchased from Millipore. Mono Q (HR 5/5), phenyl Superose (HR 10/10), and Superose 12 (HR 10/30) chromatography columns were purchased from Amersham Biosciences.

The column was eluted at a flow rate of 0.5 ml/min using a linear gradient of 0–0.3 potassium chloride in buffer A over 30 min.

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