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首页> 外文期刊>The protein journal >Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the sauromatum guttatum inflorescence
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Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the sauromatum guttatum inflorescence

机译：从金刚尾花序的生热阑尾纯化NAD（P）还原酶样蛋白

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摘要

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.
机译：Nau（P）还原酶样蛋白的分子质量为34.146±34 Da，被从齿形金花序的花序的附件中纯化至同质。在线液相色谱/电喷雾电离质谱法用于分离和定量蛋白质。为了鉴定蛋白质，对蛋白质的胰蛋白酶消化物进行了液相色谱/电喷雾串联电离质谱分析。所获得的质谱用于数据库搜索，从而鉴定了单个胰蛋白酶肽。发现该12个氨基酸的胰蛋白酶肽（FLPSEFGNDVDR）与拟南芥基因组中异黄酮还原酶的108-120位的氨基酸残基相同。 BLAST搜索将该序列区域确定为独特的，并且对参与苯丙烷生物合成的一类NAD（P）依赖性还原酶具有特异性。埃德曼降解显示该蛋白被N端封闭。蛋白质（称为RL，NAD（P）还原酶样蛋白质）的数量从D-4（开花前4天，指定为D天）到D天增加了60倍，而在第二天下降，当发热停止时。当水杨酸（在Sauromatum阑尾中产生热量的内源性触发因素）应用于过早的阑尾时，相对于未处理的部分，在处理的部分中检测到的RL量减少了五倍。在细胞质中发现约40％的RL。在Percoll纯化的线粒体中检测到另外30％，其余的约30％与由于核和淀粉状体定位而导致的低速离心沉淀物相关。 RL也在其他产热植物中发现，并在拟南芥叶片中检测到。 RL在热源和非热源植物中的功能需要进一步研究。

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