Quinn G B, Trimboli A J, Prosser I M, Barber M J
Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, USA.
Arch Biochem Biophys. 1996 Mar 1;327(1):151-60. doi: 10.1006/abbi.1996.0103.
The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.
菠菜同化型NADH：硝酸还原酶氨基酸序列的C末端268个残基，对应于该酶含黄素结构域，已被选择性扩增，并作为重组蛋白在大肠杆菌中表达。以可溶和不可溶形式产生的重组蛋白，通过硫酸铵沉淀、5'-ADP-琼脂糖亲和层析和快速蛋白液相色谱凝胶过滤相结合的方法纯化至均一。通过聚丙烯酰胺凝胶电泳估计，纯化后的结构域分子量约为30 kDa，通过质谱测定，脱辅基蛋白分子量为30169，这也证实了FAD的存在。紫外/可见光谱是黄素蛋白的典型光谱，氧化形式下在272、386和461 nm处有最大值，而圆二色光谱在313和382 nm以及461和484 nm处分别产生正负最大值。纯化后的结构域与抗菠菜硝酸还原酶多克隆抗体表现出免疫交叉反应性，同时对分离肽段的N末端和内部氨基酸测序证实了该结构域一级序列的准确性。该蛋白保留了NADH-铁氰化物还原酶活性（Vmax = 84 μmol NADH消耗/分钟/nmol FAD），对NADH和铁氰化物的Km值分别为17和34 μM，最适pH约为6.5。多种NADH类似物也可作为电子供体，尽管效率有所降低，最有效的是还原型烟酰胺次黄嘌呤二核苷酸（V(max) = 35 μmol NHDH消耗/分钟/nmol FAD），Km = 22 μM）。NAD+被证明是一种竞争性抑制剂（Ki = 1.9 mM），而对多种NAD+类似物抑制作用的分析表明，最有效的抑制剂是ADP（Ki = 0.2 mM），缺乏磷酸、核糖或腺嘌呤部分的类似物被证明是效率明显较低的抑制剂。分离的结构域还能够直接还原细胞色素b5（V(max) = 1.2 μmol NADH消耗/分钟/nmol FAD，Km（细胞色素b5） = 6 μM），支持菠菜硝酸还原酶中FAD -> b557 -> Mo的电子传递序列。
