A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase
A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase
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DOI：
10.1007/bf00201824
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发表时间：
2004
期刊：
影响因子：
4.3
通讯作者：
M. Weil;S. Krausgrill;A. Schuster;T. Rausch
中科院分区：
文献类型：
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作者：
M. Weil;S. Krausgrill;A. Schuster;T. Rausch
烟草细胞壁转录酶(CWI)的表达。未转化或转化根癌农杆菌的细胞悬浮培养物，从完整细胞中进行盐洗脱，经pH梯度洗脱后在磺丙基葡聚糖(SPS)上进行纯化，在pH为4.8的条件下培养1h，该酶的活性损失约50%。然而，Western-印迹分析表明没有明显的酶降解。在SPS上用氯化钠梯度洗脱对CWI峰组分进行重新层析，发现在CWI活性低但免疫信号强的组分中存在一个17 kDa的多肽(P17)(Weil和Rausch 1994，Planta193,430-437)。当用刀豆蛋白A(ConA)-琼脂糖柱层析分离CwI和p17时，可通过将含抑制物组分与无抑制CwI组分孵育来恢复抑制作用。超过90%的CW I可被抑制，提示所有CW I都对p17结合敏感。二价金属离子(Ca~(2+)、Mg~(2+)、锌~(2+))与p17预孵育时，可显著降低CWI的抑制作用。此外，蔗糖还可保护CWI免受p17的抑制(1.3 mM处的半最大保护)。在预孵育1h期间，p17与CWI的结合是pH依赖的，pH 4.5时抑制作用最大，而在pH 6.5时未观察到抑制作用。凝胶渗透层析表明，天然缓蚀剂是以单体形式存在的。免疫共沉淀证实了p17与CWI的直接结合。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)和随后的Western blotting分析含有CWI和p17的组份时，观察到86-90 kDa的弥漫免疫信号(除了69 kDa的显著CWI信号外)。在第二次SDS-PAGE之前，将该区带与含有尿素的样品缓冲液进行平衡，在87(±2)kDa处产生了强烈的免疫信号，这表明在p17-CWI复合体形成的一步中，两个多肽形成了牢固的聚集。CWI和葡萄糖-6-磷酸脱氢酶活性在细胞壁蛋白组分和盐洗脱细胞之间的分布表明，细胞保持了结构完整性，从而表明p17和CWI在原位共定位(Weil和Rausch 1994)。我们对p17进行了纯化，并对其N-端进行了测序，与其他已知的蛋白质序列没有相似性。讨论了p17可能的生理作用。
When cell-wall invertase (CWI) fromNicotiana tabacumL. cell-suspension cultures, either non-transformed or transformed withAgrobacterium tumefaciens, was salt-eluted from intact cells and purified on Sulfopropyl-Sephadex (SPS) by pH-gradient elution, the enzyme lost about 50% of its activity during a 1-h incubation at pH 4.8. However, Western-blot analysis indicated no appreciable enzyme degradation. Re-chromatography of CWI peak fractions on SPS using NaCl-gradient elution showed the presence of a 17-kDa peptide (p17) in fractions with low CWI activity but strong CWI immunosignal (Weil and Rausch 1994, Planta193, 430–437). When separating CWI from p17 by Concanavalin A (Con A)-Sepharose chromatography, inhibition could be restored by incubating the inhibitor-containing fraction with inhibitor-free CWI. More than 90% of CWI could be inhibited, suggesting that all CWI was susceptible to p17 binding. The presence of divalent metal ions (Ca2+, Mg2+, Zn2+) during pre-incubation of CWI with p17 reduced CWI inhibition substantially. Also, sucrose protected CWI against inhibition by p17 (half-maximum protection at 1.3 mM). Binding of p17 to CWI during a 1-h pre-incubation was pH-dependent, pH 4.5 causing maximum inhibition, whereas at pH 6.5 no inhibition was observed. Gel-permeation chromatography revealed that the native inhibitor acts as a monomer. Immunoprecipitation of CWI co-precipitated p17, confirming direct binding of p17 to CWI. When fractions containing CWI and p17 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting a diffuse immunosignal of 86–90 kDa was observed (in addition to the prominent CWI signal at 69 kDa). Equilibration of this zone with urea-containing sample buffer prior to a second SDS-PAGE run resulted in a strong immunosignal at 87 (± 2) kDa, suggesting that during one step in the formation of the p17-CWI complex the two polypeptides became firmly aggregated. The distribution of CWI and glucose-6-phosphate dehydrogenase activities between the cell-wall protein fraction and salt-eluted cells shows that cells retained their structural integrity, thus indicating co-localization of p17 and CWI in situ (Weil and Rausch 1994). We have purified p17 to homogeneity and its N-terminus has been sequenced, revealing no similarity to other known protein sequences. Possible physiological roles of p17 are discussed.