Shankar R A, Anderson P M
Arch Biochem Biophys. 1985 May 15;239(1):248-59. doi: 10.1016/0003-9861(85)90833-1.
Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.
海洋板鳃亚纲鱼类肝脏线粒体通过氨同化作用合成尿素，最初涉及谷氨酰胺的形成，随后谷氨酰胺被用于线粒体氨甲酰磷酸的合成[P.M.安德森和C.A.凯西（1984年）《生物化学杂志》259卷，456 - 462页]。本研究的目的是确定催化尿素合成第一步反应的谷氨酰胺合成酶是否具有与该功能独特相关的特性。谷氨酰胺合成酶已从棘鲨（一种具有代表性的板鳃亚纲鱼类）的离体肝脏线粒体中高度纯化出来。在存在Mg2 +、MgATP和L - 谷氨酸的情况下，纯化后的酶分子量约为400,000，但在不存在MgATP和L - 谷氨酸时，会可逆地解离为分子量约为200,000的物种。无法证明它与同样位于线粒体中的谷氨酰胺和乙酰谷氨酸依赖性氨甲酰磷酸合成酶存在关联。亚基分子量约为46,000。生物合成反应的最适pH值为7.1 - 7.4。纯化后的酶通过MgATP、谷氨酸和乙二醇得以稳定，并且被5 - 10%的乙二醇激活。MgATP、L - 谷氨酸和氨（NH4 + - NH3）的表观Km值分别为0.7、11.0和0.015 mM。超过与ATP络合所需量的Mg2 +以MgATP形式存在时，酶具有最大活性；Mn2 +不能替代Mg2 +。该酶被低浓度的氯离子、溴离子或碘离子激活；这种效应似乎与谷氨酸表观Km值的降低有关。该酶受到生理浓度尿素的抑制，但不受生理浓度氧化三甲胺的显著影响。除了被卤素阴离子激活以及对氨的表观Km值极低外，这种板鳃亚纲鱼类的谷氨酰胺合成酶的特性与已报道的哺乳动物和鸟类谷氨酰胺合成酶的特性相似。对氨极低的表观Km值可能与这种谷氨酰胺合成酶在尿素合成中线粒体氨同化作用中的独特作用具体相关。
