Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8.
Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8.
复制标题
嗜热栖热菌 HB-8 中两种类型的 NADH-醌还原酶的纯化和表征。
DOI：
10.1021/bi00406a030
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Ohnishi,T
中科院分区：
文献类型：
--
作者：
Yagi,T;Hon-nami,K;Ohnishi,T
材料与方法T. Thermophilus HB-8膜。T.根据Hon-nami和Oshima（1977），嗜热菌HB-8（ATCC 27634）细胞在75 ℃需氧生长，并在指数期晚期收获。如下制备膜。使悬浮于含有50 mM Tris-S 〇 4（pH 7.5）、IOmM MgS 〇 4和ImM EDTA的缓冲液中的细胞在16000 psi（IlOMPa）的剪切力下通过弗伦奇挤压池。将悬浮液在31500 g下离心30 min。将上清液在130000 g下离心90 min。通过再离心洗涤沉淀，并用相同的缓冲液（30-40 mg/mL）悬浮。将悬浮液在液氮中冷冻并储存在-70 ℃。蛋白质含量按Lowry et al.（1951）或在1 mg脱氧胆酸钠/mL的存在下通过缩二脲（Gornall等，1949年）。根据Yagi（1962）和Faeder和Siegel（1973）确定了黄素的性质。分别根据Doeg和齐格勒（1962）以及Fogo和Popwski（1949）估算了非血红素铁和酸不稳定硫化物。酶测定基本上按照Yagi（1986，1987）进行。除非另有说明，NADH-K3 Fe（CN）6还原酶活性在420 nm下在含有1 mM NaHCO 3的50 mM磷酸钾缓冲液（pH 7.5）中用比色法测定。
Materials and Methods Isolation of T. thermophilus HB-8 Membranes. T. thermophilus HB-8 (ATCC27634) cells were grown aerobically at 75 C according to Hon-nami and Oshima (1977) and harvested at late exponential phase. Membranes were prepared as follows. The cells, suspended in a buffer containing 50 mM Tris-S04 (pH 7.5), 10 mM MgS04, and 1 mM EDTA, were passed through the French press cell at a shear force of 16000 psi (110 MPa). The suspension was centrifuged at 31500g for 30 min. The supernatant was centrifuged at 130000g for 90 min. The pellet was washed by recentrifugation andsuspended with the same buffer (30-40 mg/mL). The suspension was frozen in liquid nitrogen and stored at-70 C.Analytical Procedures. Protein was estimated by the me-thod of Lowry et al.(1951) or by biuret in the presence of 1 mg of sodium deoxycholate/mL (Gornall et al., 1949). The nature of the flavins was determined according to Yagi (1962) and Faeder and Siegel (1973). Non-hemeiron and acid-labile sulfide were estimated according to Doeg and Ziegler (1962) and Fogo and Popwski (1949), respectively. Enzymatic assays were basically carried out according to Yagi (1986, 1987). Unless otherwise indicated, NADH-K3Fe (CN) 6 reductase activity was assayed spectrophotometrically at 420 nm in 50 mM potassium phosphate buffer (pH 7.5) containing 1 mM