Department of Genetics, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504.
Plant Physiol. 1976 Sep;58(3):350-7. doi: 10.1104/pp.58.3.350.
Cultured soybean (Glycine max, Kanrich variety) cells grow with 25 mm urea as the sole nitrogen source but at a slower rate than with the Murashige and Skoog (MS) (Physiol. Plant. 15: 473-497, 1962) nitrogen source of 18.8 mm KNO(3) and 20.6 mm NH(4)NO(3). Growth with urea is restricted by 18.8 mm NO(3) (-), 50 mm methylammonia, 10 mm citrate or 100 mum hydroxyurea, substances which are much less restrictive or nonrestrictive in the presence of ammonia nitrogen source. The restrictive conditions of urea assimilation were examined as possible bases for selection schemes to recover urease-overproducing mutants. Since urease has higher methionine levels than the soybean seed proteins among which it is found, such selections may be a model for improving seed protein quality by plant cell culture techniques.Callus will not grow with 1 mm urea plus 18.8 mm KNO(3). Urease levels decrease 80% within two divisions after transfer from MS nitrogen source to 1 mm urea plus 18.8 mm KNO(3). Hydroxyurea is a potent inhibitor of soybean urease and this appears to be the basis for its inhibition of urea utilization by callus cells.Stationary phase suspension cultures grown with MS nitrogen source exhibit trace or zero urease levels. Soon after transfer to fresh medium (24 hours after escape from lag), urease levels increase in the presence of both MS or urea nitrogen source. However, the increase is 10 to 20 times greater in the presence of urea. NH(4)Cl (50 mm) lowers urease induction by 50% whereas 50 mm methylammonium chloride results in more drastic reductions in urea-stimulated urease levels. Citrate (10 mm) completely blocks urease synthesis in the presence of urea.Ammonia and methylammonia do not inhibit soybean urease nor do they appreciably inhibit urea uptake by suspension cultures. It appears likely that methylammonia inhibits urea utilization in cultured soybean cells primarily due to its "repressive" effect on urease synthesis.Citrate does not inhibit urease activity in vitro and exhibits only a partial inhibition (0-50% in several experiments) of urea uptake. It appears likely that the citrate elimination of urease production by cultured soybean cells is due to its chelation of trace Ni(2+) in the growth medium. Dixon et al. (J. Am. Chem. Soc. 97: 4131-4133, 1975) have reported that jack bean (Canavalia ensiformis) urease contains nickel at the active site.
培养的大豆（Glycine max，Kanrich 品种）细胞以 25mm 尿素作为唯一氮源生长，但生长速度比以 Murashige 和 Skoog（MS）（Physiol. Plant. 15: 473-497, 1962）氮源 18.8mm KNO3 和 20.6mm NH4NO3 为氮源时慢。尿素同化的限制性条件被认为是恢复脲酶过度产生突变体的选择方案的可能基础。由于脲酶的蛋氨酸水平高于其所在的大豆种子蛋白，因此在存在氨氮源的情况下，这些选择可能是通过植物细胞培养技术提高种子蛋白质量的模型。
愈伤组织不能在 1mm 尿素加 18.8mm KNO3 中生长。从 MS 氮源转移到 1mm 尿素加 18.8mm KNO3 后，在两次分裂内，脲酶水平下降 80%。羟基脲是大豆脲酶的有效抑制剂，这似乎是其抑制愈伤组织细胞利用尿素的基础。
以 MS 氮源生长的静止期悬浮培养物表现出痕量或零脲酶水平。在从滞后期逃脱后 24 小时转移到新鲜培养基时，MS 或尿素氮源的存在都会增加脲酶水平。然而，在存在尿素的情况下，增加了 10 到 20 倍。50mmNH4Cl 将脲酶诱导降低 50%，而 50mm 甲基氯化铵导致尿素刺激的脲酶水平大幅降低。柠檬酸（10mm）完全阻断尿素合成。
氨和甲基氨不会抑制大豆脲酶，也不会显著抑制悬浮培养物对尿素的吸收。似乎很可能，甲基氨主要通过对脲酶合成的“抑制”作用来抑制培养的大豆细胞中尿素的利用。
柠檬酸在体外不抑制脲酶活性，仅对尿素摄取有部分抑制（在几次实验中为 0-50%）。似乎很可能，柠檬酸通过螯合生长培养基中的痕量 Ni（2+）来消除培养的大豆细胞中脲酶的产生。Dixon 等人（J. Am. Chem. Soc. 97: 4131-4133, 1975）已经报道，刀豆（Canavalia ensiformis）脲酶在活性部位含有镍。
