Ketchum P A, Zeeb D D, Owens M S
J Bacteriol. 1977 Sep;131(3):884-90. doi: 10.1128/jb.131.3.884-890.1977.
Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source.
粗糙脉孢菌的还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）-硝酸还原酶被纯化出来，发现它受到某些氨基酸、柠檬酸盐和乙二胺四乙酸（EDTA）的刺激。柠檬酸盐和氨基酸的刺激取决于事先从酶制剂中去除EDTA，因为少量的EDTA会导致最大程度的刺激。通过用含螯合树脂的缓冲液透析从酶制剂中去除EDTA会导致硝酸还原酶活性丧失。添加丙氨酸、精氨酸、甘氨酸、谷氨酰胺、谷氨酸、组氨酸、色氨酸和柠檬酸盐可使硝酸还原酶活性恢复并刺激29至46倍。所测试的氨基酸改变了NADPH-硝酸还原酶对NADPH的Km值，但对硝酸盐的Km值没有显著影响。硝酸还原酶对NADPH的Km值随组氨酸浓度的增加而增加，但随谷氨酰胺浓度的增加而降低。讨论了NADPH-硝酸还原酶活性的氨基酸调节与粗糙脉孢菌以硝酸盐为氮源时能量（NADPH）的保存之间的关系。
