Polacco J C
Department of Genetics, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, Connecticut 06504.
Plant Physiol. 1977 May;59(5):827-30. doi: 10.1104/pp.59.5.827.
Potassium citrate (10 mM, pH 6) inhibits the growth of cultured (Glycine max L.) cells when urea is the sole nitrogen source. Ureadependent citrate toxicity is overcome by three separate additions to the growth medium: (a) NH(4)Cl (20 mM); (b) high levels of MgCl(2) (10 mM) or CaCl(2) (5-10 mM); (c) low levels of NiSO(4) (10(-2) mM). Additions of 10(-2) mM NiSO(4) not only overcome citrate growth inhibition but the resultant growth is usually better than urea-supported growth in basal medium (neither added citrate nor added nickel). In the absence of added citrate, exceedingly low levels of NiSO(4) (10(-4) mM) strongly stimulate urea-supported growth in suspension cultures.Citrate does not inhibit growth when arginine is sole nitrogen source. However, cells using arginine have no net urease synthesis in the presence of 10 mM potassium citrate. When 10(-2) mM NiSO(4) is added to this medium, urease specific activity is 10 times that observed in basal medium lacking both citrate and added nickel.Citrate is a chelator of divalent cations. That additional Mg(2+) or Ca(2+) alleviates urea-dependent citrate toxicity indicates that citrate is acting by chelation, probably of another trace divalent cation; this is probably Ni(2+) since at 10(-2) mM it overcomes citrate toxicity and at 10(-4) mM it stimulates urea-supported growth in the absence of citrate. That ammonia overcomes citrate toxicity indicates that the trace Ni(2+) is essential specifically for the conversion of urea to ammonia. Ni(2+) stimulation of urease levels in arginine-grown cells supports this contention.In basal medium, soybean cells grow slowly with urea nitrogen source presumably because the trace amounts of Ni(2+) present (</=10(-6) mM) are growth-limiting.
当尿素作为唯一氮源时，柠檬酸钾（10 mM，pH 6）会抑制培养的（大豆）细胞生长。通过向生长培养基中进行三种不同添加可克服尿素依赖性柠檬酸盐毒性：（a）氯化铵（20 mM）；（b）高浓度的氯化镁（10 mM）或氯化钙（5 - 10 mM）；（c）低浓度的硫酸镍（10⁻² mM）。添加10⁻² mM硫酸镍不仅能克服柠檬酸盐对生长的抑制，而且由此产生的生长通常比基础培养基中尿素支持的生长更好（既不添加柠檬酸盐也不添加镍）。在不添加柠檬酸盐的情况下，极低浓度的硫酸镍（10⁻⁴ mM）能强烈刺激悬浮培养物中尿素支持的生长。当精氨酸作为唯一氮源时，柠檬酸盐不会抑制生长。然而，在存在10 mM柠檬酸钾的情况下，利用精氨酸的细胞没有净脲酶合成。当向该培养基中添加10⁻² mM硫酸镍时，脲酶比活性是在缺乏柠檬酸盐和添加镍的基础培养基中观察到的10倍。柠檬酸盐是二价阳离子的螯合剂。额外添加镁离子或钙离子可减轻尿素依赖性柠檬酸盐毒性，这表明柠檬酸盐是通过螯合作用起作用的，可能螯合的是另一种痕量二价阳离子；可能是镍离子，因为在10⁻² mM时它能克服柠檬酸盐毒性，而在10⁻⁴ mM时它能在不添加柠檬酸盐的情况下刺激尿素支持的生长。氨能克服柠檬酸盐毒性，这表明痕量镍离子对于尿素转化为氨的过程特别重要。镍离子对精氨酸培养的细胞中脲酶水平的刺激支持了这一观点。在基础培养基中，大豆细胞以尿素作为氮源时生长缓慢，大概是因为存在的痕量镍离子（≤10⁻⁶ mM）限制了生长。
