Paul H. Ray

[47] CTP:CMP-3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase)

Publisher Summary This chapter presents procedure for purification and assay of CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase). Escherichia coli B cells grown to mid-logarithmic phase in glucose minimal medium is used for purification. Steps of purification involve: crude extraction; acid precipitation; chromatography on DEAE-sephadex column; chromatography on Sephadex G-200; isoelecoic focusing; and chromatogrqaphy on Sephadex G-100. CMP-KDO synthetase at this step in purification is 70% pure as determined by electrophoresis on 7% non-SDS-polyacrylamide gels. The enzyme can be purified to homogeneity by preparative gel electrophoresis. The specific activity of the purified protein is 9.3–9.6. CMP-KDO synthetase activity is monitored by measuring the formation of the product, CMP-KDO, colorimetrically by a modification of the thiobarbituric acid assay.

Thermal lysis of bacterial membranes and its prevention by polyamines.

SUMMARY: Protoplasts of Sarcina lutea and Streptococcus faecalis underwent thermal lysis when heated to 60° and above. [14C]Glycine was released from the internal pool of Strep, faecalis at 50°. Spermine, spermidine, cadaverine and Mg2+ partially protected protoplasts against thermal lysis.

[46] 3-deoxy-d-manno-octulosonate-8-phosphate (KDO-8-P) phosphatase☆

Publisher Summary This chapter presents procedure for purification and assay of 3- deoxy-D-manno-octulosonate- 8-phosphate (KDO-8-P) phosphatases. Escherichia coli B cells (ATCC 11303) are grown in glucose minimal medium containing Pi to mid-logarithmic phase are used for purification. Steps of purification involve: crude extraction; acid precipitation; chromatography on DEAE-sephadex column; hydroxyapatite chromatography; isoelectric focusing; and chromatography on Sephadex G-150. Fractions containing the highest specific activity are pooled, reduced in volume by ultrafiltration to a protein concentration of 2 mg/ml, and frozen at –90 ° until used. The activity of KDO-8-P phosphatase can be monitored by either measuring the release of inorganic phosphate or, alternatively, by measuring the disappearance of [1- 14 C]KDO-8-P by liquid scintillation counting after chromatography of the reaction mixture on polyethyleneimine cellulose plates. The former method is routinely used, but the enzyme must be well dialyzed to remove Pi. The substrate, KDO-8-P is prepared enzymically using KDO-8-P synthase and purified by column chromatography.