A SOYBEAN SEED UREASE-NULL PRODUCES UREASE IN CELL-CULTURE
A SOYBEAN SEED UREASE-NULL PRODUCES UREASE IN CELL-CULTURE
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DOI：
10.1104/pp.69.5.1233
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发表时间：
1982-01-01
期刊：
影响因子：
7.4
通讯作者：
BLEDSOE, PJ
中科院分区：
文献类型：
--
作者：
POLACCO, JC;THOMAS, AL;BLEDSOE, PJ
黄豆（G. max [L.]）[Merr.])，种子脲酶活性正常0.2%，从美国农业部大豆种质收集的6000个条目中回收。双扩散法和火箭免疫电泳均未检测到脲酶抗原。天然凝胶染色的蛋白质或溶尿活性显示没有检测到脲酶全酶。使用抗脲酶抗体亲和柱从野生型（“Prize”）种子提取物中去除所有可检测到的脲酶活性和抗原。亲和柱流出物和未色谱的Itachi萃取物缺乏与纯化的脲酶亚基在十二烷基硫酸钠聚丙烯酰胺凝胶中相结合的物质。无法检测到脲酶抗原或脲酶蛋白，表明在“Itachi”种子发育过程中没有脲酶蛋白的合成，或者其合成为野生型的0.2%（“Prize”）。在发育或萌发的“立立”种子子叶中检测不到脲酶活性或仅检测到微量脲酶活性。从“Itachi”幼苗的子叶、茎尖、根或根尖组织诱导的愈伤组织表现出与“Prize”相当的溶尿活性。“奖品”和“立立”茎尖培养以尿素为唯一氮源。“Prize”和“Itachi”悬浮培养细胞粗提物的大部分或全部溶尿活性在热稳定性、对种子酶的抗体识别、羟基脲敏感性和合成所需的Ni等方面与种子酶相似。部分纯化的细胞培养脲酶在免疫学和电泳标准上与种子脲酶相同。显然，脲酶在种子和细胞培养中受到不同的发育控制。在“Prize”和“Itachi”培养中，尿囊素的利用与尿素不同，不依赖于Ni。尿素分解代谢可能不需要脲酶。
''Itachi'', a soybean (G. max [L.] Merr.) with 0.2% normal seed urease activity, was recovered from a screen of 6000 entries in the USA Department of Agriculture soybean germplasm collection. No urease antigen in ''Itachi'' seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from wild type (''Prize'') seed extracts. Affinity column effluent and nonchromatographed Itachi extracts lack a species which comigrates with purified urease subunits in sodium dodecyl sulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of ''Itachi'' seeds there is no synthesis of urease protein or that its synthesis is 0.2% of wild type (''Prize''). No urease activity or only traces of urease activity were detected in cotyledons of developing or germinating ''Itachi'' seeds. Callus cultures induced from cotyledon, shoot tip, root or root tip tissues of ''Itachi'' seedlings exhibited ureolytic activity equivalent to that of ''Prize'' cultures. Shoot tip cultures of ''Prize'' and ''Itachi'' grew with urea as sole N source. Most or all of the ureolytic activity in crude extracts of ''Prize'' and ''Itachi'' suspension culture cells is seed-like urease in thermal stability, recognition by antibodies to the seed enzyme, hydroxyurea sensitivity and Ni requirement for synthesis. Partially purified cell culture urease is identical to seed urease by immunological and electrophoretic criteria. Evidently, urease is under different developmental controls in the seed and in cell culture. In ''Prize'' and ''Itachi'' cultures, utilization of the ureide allantoin, unlike that of urea, is not dependent on Ni. Ureide catabolism may not require urease.