Smith E M, Watford M
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
Arch Biochem Biophys. 1988 Feb 1;260(2):740-51. doi: 10.1016/0003-9861(88)90504-8.
A method for the purification of phosphate-activated glutaminase from the liver of streptozotocin-diabetic rats is described. The procedure involves solubilization of glutaminase activity from isolated mitochondria by sonication, followed by ammonium sulfate precipitation, polyethylene glycol precipitation, and sequential chromatography on DEAE, hydroxylapatite, and zinc-chelated resins. The enzyme was purified 600-fold to a specific activity of 31-57 U/mg protein. The purified enzyme has an apparent subunit molecular mass of 58,000-Da and is greater than 80% pure by scanning densitometry of sodium dodecyl sulfate-polyacrylamide gels. The purified enzyme has an apparent Km for glutamine of 17 mM and a pH optimum between 7.8 and 8.2. The physical and kinetic properties of this enzyme are similar to those of the enzyme from normal rat liver. Polyclonal antibodies raised against the enzyme specifically inhibit hepatic glutaminase activity and react primarily with a 58,000-Da peptide in liver fractions on immunoblots. These antibodies were used in equivalence point titrations and immunoblots to provide evidence for increased concentration of glutaminase protein in the liver of diabetic rats with no change in specific activity of the enzyme. In addition, the antibodies cross-react, at low affinity, with kidney-type glutaminases. On immunoblots, the antibodies did not react with fetal liver, mammary gland, or lung. Antibodies to rat hepatic glutaminase should prove useful as tools to study the long-term regulation of the enzyme.
本文描述了一种从链脲佐菌素诱导的糖尿病大鼠肝脏中纯化磷酸激活型谷氨酰胺酶的方法。该方法包括通过超声处理从分离的线粒体中溶解谷氨酰胺酶活性，随后进行硫酸铵沉淀、聚乙二醇沉淀，以及在DEAE、羟基磷灰石和锌螯合树脂上进行连续层析。该酶被纯化了600倍，比活性达到31 - 57 U/mg蛋白质。纯化后的酶表观亚基分子量为58,000道尔顿，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶扫描密度法测定其纯度大于80%。纯化后的酶对谷氨酰胺的表观Km为17 mM，最适pH在7.8至8.2之间。该酶的物理和动力学性质与正常大鼠肝脏中的酶相似。针对该酶产生的多克隆抗体可特异性抑制肝脏谷氨酰胺酶活性，并且在免疫印迹中主要与肝脏组分中的一种58,000道尔顿的肽发生反应。这些抗体用于等效点滴定和免疫印迹，以证明糖尿病大鼠肝脏中谷氨酰胺酶蛋白浓度增加，而酶的比活性没有变化。此外，这些抗体与肾型谷氨酰胺酶有低亲和力的交叉反应。在免疫印迹中，这些抗体不与胎儿肝脏、乳腺或肺发生反应。大鼠肝脏谷氨酰胺酶抗体应可作为研究该酶长期调节的有用工具。
