Functional intracellular glutaminase activity in intact astrocytes.
Functional intracellular glutaminase activity in intact astrocytes.
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完整星形胶质细胞中功能性细胞内谷氨酰胺酶活性。
DOI：
10.1007/bf01000035
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发表时间：
1989
影响因子：
4.4
通讯作者：
Landry,ME
中科院分区：
文献类型：
--
作者：
Zielke,HR;Tildon,JT;Zielke,CL;Baab,PJ;Landry,ME
许多细胞代谢物，如谷氨酰胺、谷氨酸、磷酸盐、钙、氨和乙酰衍生物，已知会影响全细胞匀浆或提取物中磷酸盐活化的谷氨酰胺酶活性。由于在非生理条件下对提取物的测量可能会模糊实际的细胞内代谢通量，因此通过ml-[2-3H]谷氨酰胺形成3h2o来测量细胞内“功能性”磷酸盐激活的谷氨酰胺酶活性。生物化学。127:134-142,1982)在完整星形胶质细胞培养中，未经处理和用二丁基c-AMP （DiBcAMP）处理，存在几种潜在的效应物。将这些值与相同细胞提取物中测定的酶水平进行比较。在未处理和DiBcAMP处理的星形胶质细胞中也测量了-[1-14C]谷氨酰胺释放14co2的速率。在含有1mM放射性谷氨酰胺的MEM培养基中，未处理细胞的谷氨酰胺酶活性为88 nmol/mg蛋白/h，而DiBcAMP处理细胞的谷氨酰胺酶活性为153 nmol/mg蛋白/h。然而，在最佳条件下，未处理和处理过的细胞提取物的酶活性要高得多，但基本相同，约为1,750 nmol/mg蛋白/h。在未处理和DiBcAMP处理的细胞中，谷氨酰胺-[1-14C]释放14co2的速率分别为74和133 nmol/mg蛋白/h。这约占细胞内谷氨酰胺酶活性的85%。此外，将培养基中谷氨酰胺的浓度从1 mM增加到6.4 mM，在未处理和处理的细胞中，谷氨酰胺酶的胞内活性均增加了约3倍。在两种处理条件下，在培养基中添加250 μM谷氨酸可抑制70%的细胞内谷氨酰胺酶活性。葡萄糖的缺失刺激谷氨酰胺酶的活性。相反，去除胎牛血清使活性降低35%。添加10 mM磷酸和α -酮酸异亮氨酸和缬氨酸略微提高细胞内谷氨酰胺酶活性。在培养基中添加0.4 mM氯化铵没有影响。培养基pH从6.8增加到7.7，细胞内谷氨酰胺酶活性增加了近2倍。这些结果提供了证据，证明磷酸盐激活的谷氨酰胺酶活性在体内受细胞代谢物的调节，其“功能”活性是使用提取物获得的速率的5-9%，这种“功能”活性足以解释谷氨酰胺氧化的速率。
Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the “functional” intracellular phosphate-activated glutaminase activity was measured by the formation of3H2O froml-[2-3H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of14CO2release froml-[1-14C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of14CO2release froml-[1-14C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 μM glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its “functional” activity is 5–9% of the rate obtained using extracts, and this “functional” activity is sufficient to account for the rate of glutamine oxidation.