Shapiro R A, Haser W G, Curthoys N P
Biochem J. 1987 Mar 15;242(3):743-7. doi: 10.1042/bj2420743.
Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.
制备了针对分离的大鼠肾谷氨酰胺酶的抗体，并针对纯化的大鼠脑谷氨酰胺酶中所含的65 kDa肽进行了亲和纯化。然后使用亲和纯化的IgG来比较经过SDS/聚丙烯酰胺凝胶电泳并转移至硝酸纤维素膜上的样品中所含的谷氨酰胺酶免疫反应性肽。纯化的脑谷氨酰胺酶和分离的脑线粒体含有68 kDa和65 kDa的肽，它们表现出几乎相同的免疫染色。用金黄色葡萄球菌V8蛋白酶对分离的68 kDa和65 kDa肽进行部分蛋白水解产生了相同模式的免疫反应性蛋白水解片段。然而，用胰凝乳蛋白酶消化这两种肽产生了相似但略有不同的模式。即使脑线粒体用Triton X-100溶解并在4℃下保存2天，免疫染色模式也未改变。当完整的肾线粒体进行免疫印迹分析时观察到非常相似的模式。然而，当肾线粒体溶解时，68 kDa肽迅速降解为65 kDa形式。在4℃下，该反应以明显的一级动力学发生，t1/2为35分钟。肾谷氨酰胺酶65 kDa形式的降解动力学要慢得多，但24小时后几乎完全降解。从肾脏不同区域分离的线粒体的溶解表明，负责的内源性蛋白酶主要定位于皮质。从肠道或肾乳头组织分离的线粒体含有四种谷氨酰胺酶免疫反应性肽（Mr 68,000、65,000、61,000和58,000）。这些肽中最小的与在肝组织中观察到的单一免疫反应性肽大小相同。
