Takeda Takumi, Fry Stephen C
The Edinburgh Cell Wall Group, ICMB, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, EH9 3JH, UK.
Planta. 2004 Aug;219(4):722-32. doi: 10.1007/s00425-004-1267-9. Epub 2004 Apr 17.
Crude extracts of cauliflower florets had high xyloglucan endotransglucosylase (XET) activity, but this was largely lost after partial purification and de-salting. Activity was restored (promoted up to 40-fold) by any of a wide variety of inorganic and organic salts. Optimum concentrations for Na+, K+ and NH4+ salts were typically approximately 300 mM. The chlorides of Ca2+, Mg2+, Al3+ and La3+ were optimally active at lower concentrations (e.g. 0.1 mM LaCl3), but became inhibitory at higher concentrations (e.g. 5 mM LaCl3). Some anionic polysaccharides at 0.04-0.2% w/v (e.g. gum arabic, pectin and hypochlorite-oxidised xyloglucan) promoted the XET activity of de-salted enzyme, especially if a sub-optimal concentration of NaCl was also present; others (e.g. homogalacturonan, 4- O-methyl-glucuronoxylan and alginate) were inhibitory. Similar ionic effects were noted on the XET activity of the Arabidopsis protein XTH24 (heterologously expressed by insect cells); in this case carboxymethylcellulose was also stimulatory. To look for endogenous modulators of XET activity, we prepared a cold-water extract of cauliflower florets; after boiling and centrifugation, the supernatant [boiled cauliflower preparation (BCP)] promoted the XET activity of de-salted cauliflower enzyme and of XTH24. About half the activator present in BCP was an ethanol-precipitable, anionic polymer of apparent Mr <5,000. After acid hydrolysis the polymer yielded much arabinose and galactose, and small amounts of galacturonic and glucuronic acids amino acids were also present. The polymer may thus contain arabinogalactan-proteins. We suggest that acidic polymers and/or other apoplastic ions are naturally occurring regulators of XET action in vivo, and may thus control cell wall assembly, loosening, and growth.
花椰菜小花的粗提物具有较高的木葡聚糖内转糖基酶（XET）活性，但在部分纯化和脱盐后，这种活性大部分丧失。多种无机和有机盐中的任何一种都能使活性恢复（提高至40倍）。Na⁺、K⁺和NH₄⁺盐的最佳浓度通常约为300 mM。Ca²⁺、Mg²⁺、Al³⁺和La³⁺的氯化物在较低浓度（如0.1 mM LaCl₃）时具有最佳活性，但在较高浓度（如5 mM LaCl₃）时具有抑制作用。一些浓度为0.04 - 0.2% w/v的阴离子多糖（如阿拉伯树胶、果胶和次氯酸盐氧化的木葡聚糖）能促进脱盐酶的XET活性，特别是在同时存在次最佳浓度的NaCl时；其他多糖（如高半乳糖醛酸聚糖、4 - O - 甲基 - 葡糖醛酸木聚糖和藻酸盐）则具有抑制作用。在拟南芥蛋白XTH24（由昆虫细胞异源表达）的XET活性上也观察到了类似的离子效应；在这种情况下，羧甲基纤维素也具有刺激作用。为了寻找XET活性的内源性调节剂，我们制备了花椰菜小花的冷水提取物；经过煮沸和离心后，上清液[煮沸的花椰菜制剂（BCP）]促进了脱盐花椰菜酶和XTH24的XET活性。BCP中约一半的激活剂是一种乙醇可沉淀的、表观分子量<5000的阴离子聚合物。酸水解后，该聚合物产生大量阿拉伯糖和半乳糖，还存在少量半乳糖醛酸和葡糖醛酸，也有氨基酸。因此，该聚合物可能含有阿拉伯半乳聚糖蛋白。我们认为酸性聚合物和/或其他质外体离子是体内XET作用的天然调节剂，因此可能控制细胞壁的组装、松弛和生长。
