สำนักราชบัณฑิตยสภา

สารฮิ วมิ กช่วยเพิ่ มความสามารถการลดสี ... 200 The Journal of the Royal Institute of Thailand Vol. 37 No. 1 Jan.-Mar. 2012 Preparation of cell membranes Strain A5 was grown aerobically under the conditions described above until they reached the late exponential growth phase. Cells were harvested by centrifugation at 10,000 x g for 10 min, washed twice, and resuspended in Na-K phosphate buffer (50 mM, pH 7.5) to an OD 600 of about 5. Cell-free extracts and cell membranes were prepared by disruption of a suspension of whole cells (protein content, about 1 g l -1 ) by ultrasonication using an ultrasonic processor (Sonicator ® W-385, Heat systems- Ultrasonics, Inc.). After removing the cell debris by centrifugation at 10,000 x g for 15 min at 4 0 C, the supernatant was ultracentrifuged at 100,000 x g for 30 min at 4 0 C (29, 43). The supernatant of ultracentrifugation was used in certain experiment as the “cytoplasmic fraction”. In the same time, the transparent pellet formed after ultracentrifugation was resuspended in a volume of about 10 ml in 50 mM Tris-HCl buffer (pH 7.5) and used in enzyme assay as the “membrane fraction” (29, 43). For the isolation of the membrane-bound azo reductase, 1 ml of this preparation was incubated on ice for 5 min with 950 µl of 50 mM Tris-HCl buffer (pH 7.5) and 50 µl of a solution of Triton X-100 (20%, v/v). Finally, 200 µl of this mixture was used for the azo dye reduction as described above. The remaining membrane-bound protein was stored at -20 0 C until used. Enzyme assays (i) NADH:quinone oxidoreductase The NADH:quinone oxidoreductase was measured spectrophotometrically by a modification of the method given by Matsushita et al. (32). In this anaerobic assay, this enzyme activity is determined by the reduction of menaquinone (MK-2) or NADH. In standard assay, cell extract and soluble cell membrane were added to a solution (final volume, 1ml) containing 50 mM of Tris-HCl (pH 7.5), 50 µM of menaquinone dissolved in dimethyl sulfoxide, and 2.5 mM of NaCN. The reaction was started by the addition of 200 µM of NADH. Due to the behavior of menaquinone, which has an intense peak at 336 nm in the oxidized form and 326 nm in reduced form (16), thus,