สำนักราชบัณฑิตยสภา

«“√ “√ √“™∫— ≥±‘ µ¬ ∂“π ªï ∑’Ë Û ©∫— ∫∑’Ë Ò ¡.§.-¡’ .§. ÚıÙ¯ 61 Somsak Damronglerd, et al. 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 prepara- tion was incubated on ice for 5 min with 950 l of 50 mM Tris-HCl buffer (pH 7.5) and 50 l of a solution of Triton X-100 (20%, v/v). Finally, 200 l of this mixture was used for the azo dye reduction as described above. The remaining membrane- bound protein was stored at -20˚C until used. Enzyme assays. (i) NADH : quinone oxidoreductase. The NADH: quinone oxidore- ductase was measured spectrophoto- metrically 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, 1 ml) containing 50 mM of Tris-HCl (pH 7.5), 50 M of menaquinone dissolved in dimethyl sulfoxide, and 2.5 mM of NaCN. The reaction was started by the addition of 200 M 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, the reduction rate of NADH was measured at 348 nm (molar extinction coefficient = 5.9 mM -1 cm -1 ), where reduced and oxidized forms of menaquinone have identical absorbtivity. (ii) NAD(P)H: flavin oxidoreductase. The NAD(P)H: flavin oxido- reductase was determined by a modification of the method given by Izumi and Ohshiro (25) and Russ et al (46). The reaction mixture (final volume, 1 ml) for the measurement of the enzyme activity contained 50 mM of Tris-HCl (pH 7.5), 0.5-1 mg protein of cell extract or soluble cell membrane, and 3 M of riboflavin. The reaction was started by the addition of 250 M of NADH. The decrease in the concentration of NADHwas determined spectrophoto- metrically at 340 nm, and reaction rates were calculated by using a molar extinction coefficient of 6.2 mM -1 cm -1 . Determination of protein content. The protein content of cell extracts and soluble cell membrane were determined by the method of Bradford (6) with bovine serum albumin (Sigma, Fraction V) as the standard. Proteins in whole cells were assayed after adding NaOH to the cells to 1 N concentration, then boiling the cultures for 5 min. The supernatant fractions obtained by centrifuging the suspensions were assayed by the Bradford method, using 1 N NaOH as diluent for the standards. Reduction of AQDS by whole cells of strain A5 and subsequent chemical reduction of sulfonated azo dyes by reduced AQDS. Strain A5 was aerobically grown in MMG until reach the late- exponential growth phase. The cells were harvested by centrifugation, washed and resuspended in Na-K phosphate buffer (50 mM, pH 7.5). One millilitre of the cell suspension (protein content = 0.1 g l -1 ) was transferred to screw cap-glass tube (10 ml) containing 9 ml of 50 mM Na-K phosphate buffer (pH 7.5) plus glucose (5 mM). The anaerobic incubation was started by the addition of AQDS (final concentration = 0.25 mM) form anaerobic stock solution into reaction mixture. The concentra- tions of reduced AQDS (AH 2 QDS) were determined spectrophoto- metrically at 450 nm. After 10 h of incubation, the cells of strain A5 were removed form suspension by filtration through a 0.2- m-pore diameter filter under anaerobic atmos- phere. The resulting supernatant was then transferred into 1 cm disposable plastic cuvettes. The chemical reduc- tion of azo dyes by reduced AQDS was started by the addition of anaerobic azo dyes solutions (final concentration = 0.1 mM) into cell- free suspension. The reduction of azo dyes was determined spectrophoto- metrically at λ max of each azo dye for 30 min (using 30 sec measuring intervals). Determination of quinone com- ponent(s) in plasma membrane Paenibacillus sp. strainA5 cells were grown either aerobically or anaerobically in 100 ml volumes.