ICI Plant Protection Division, Jealott's Hill Research Station, RG12 6EY, Bracknell, Berkshire, UK.
Planta. 1984 Jul;161(5):470-4. doi: 10.1007/BF00394580.
Kinetics for the inhibition of glutamine synthetase (EC 6.3.1.2) in situ by the herbicidal glutamate analogue, phosphinothricin, have been generated, and produce an inhibitor dissociation constant (Ki) of 6.5 μM. This has been achieved through the development of a rapid technique for the isolation of mesophyll cells from the cladophylls of young asparagus (Asparagus sprengeri) plants to provide starting material for the direct measurement of enzyme activities in situ. A modification of the technique developed by Rhodes and Stewart (Planta 118, 133-144 (1974) for the direct determination of enzyme activities in higher-plant tissues has been applied to these asparagus cells. Treatment of the cells by a single freezing in liquid nitrogen for a very short period (10 s), followed by thawing, alters the permeability of cell and organelle membranes allowing enzymes to become accessible to many small molecules, and yet remain concentrated and active within the cell. The activities of enzymes known to be located specifically in the organelles as well as the cytoplasm can be measured in asparagus cells treated in this way. Comparisons have been made between the activity and inhibition of glutamine synthetase in situ, and the enzyme isolated and partially purified from asparagus cells by fast protein liquid chromatography. Similarities in Km and Ki values obtained between these two emphasize the efficacy of the freeze-thaw technique. There is only a single glutamine-synthetase isoenzyme in asparagus mesophyll cells, which copurifies with the one normally associated with the chloroplast (GS2).
已生成草丁膦（谷氨酸类似物）原位抑制谷氨酰胺合成酶（EC 6.3.1.2）的动力学数据，并得出抑制剂解离常数（Ki）为 6.5 μM。这是通过开发一种从年轻芦笋（Asparagus sprengeri）植物的营养叶中分离叶肉细胞的快速技术来实现的，该技术为直接原位测量酶活性提供了起始材料。罗兹和斯图尔特（Rhodes and Stewart）（Planta 118，133-144（1974））开发的用于直接测定高等植物组织中酶活性的技术得到了改进，已应用于这些芦笋细胞。通过将细胞在液氮中进行一次短暂的冷冻（10 秒），然后解冻，可改变细胞膜和细胞器的通透性，使许多小分子能够进入细胞，同时使细胞内的酶保持浓缩和活性。用这种方法处理的细胞可以测量已知位于细胞器和细胞质中的酶的活性和抑制作用。对用这种方法处理的芦笋细胞中谷氨酰胺合成酶的原位活性和抑制作用以及从芦笋细胞中通过快速蛋白质液相色谱法分离和部分纯化的酶进行了比较。这两种方法获得的 Km 和 Ki 值相似，强调了冻融技术的有效性。在芦笋叶肉细胞中只有一种单一的谷氨酰胺合成酶同工酶，它与通常与叶绿体（GS2）相关的同工酶一起共纯化。
