Derda D F, Miles M F, Schweppe J S, Jungmann R A
J Biol Chem. 1980 Dec 10;255(23):11112-21.
The mechanism of isoproterenol and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) induction of lactate dehydrogenase (EC 1.1.1.27) was investigated in the C6 rat glioma cell line. [3H]Leucine-labeled lactate dehydrogenase in noninduced and induced cells was quantitatively immunoprecipitated with rabbit anti-rat lactate dehydrogenase-5 antiserum. The immunoprecipitates were analyzed for 3H-labeled lactate dehydrogenase by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and isoelectrofocusing. Using this technique, it was shown that isoproterenol + 3-isobutyl-1-methylxanthine and dibutyryl cAMP cause an increase of the [3H]leucine incorporation into glioma cell lactate dehydrogenase. Analysis of the kinetics of induction and deinduction revealed no change in the rate of degradation of lactate dehydrogenase in the presence and absence of inducing agent, indicating that the induction was due to an increase in the rate of synthesis of the enzyme. The increased rate of synthesis was prevented by actinomycin D. Isoproterenol + 3-isobutyl-1-methylxanthine increased only the specific rate of synthesis of lactate dehydrogenase-5 isozyme and of the M subunit. The mechanism was further studied by assaying the level of functional mRNA coding for lactate dehydrogenase in a reticulocyte cell-free protein-synthesizing system using glioma cell poly(A)-containing RNA isolated from either isoproterenol or dibutyryl cAMP-induced cells. Analysis of the immunoprecipitated translation product by isoelectrofocusing revealed that isoproterenol or dibutyryl cAMP produced an approximately 8-fold stimulation of the poly(A) + RNA-directed synthesis of the lactate dehydrogenase M subunit. These data demonstrate that isoproterenol and dibutyryl cAMP control the level of functionally active lactate dehydrogenase mRNA in glioma cells which, in turn, determines the extent of synthesis of the lactate dehydrogenase M subunit.
在C6大鼠胶质瘤细胞系中研究了异丙肾上腺素和N6,O2'-二丁酰腺苷3':5'-单磷酸（二丁酰环磷酸腺苷）诱导乳酸脱氢酶（EC 1.1.1.27）的机制。用兔抗大鼠乳酸脱氢酶-5抗血清对未诱导和诱导细胞中[3H]亮氨酸标记的乳酸脱氢酶进行定量免疫沉淀。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和等电聚焦分析免疫沉淀物中的3H标记乳酸脱氢酶。使用该技术表明，异丙肾上腺素+ 3-异丁基-1-甲基黄嘌呤和二丁酰环磷酸腺苷导致胶质瘤细胞乳酸脱氢酶中[3H]亮氨酸掺入增加。诱导和去诱导动力学分析表明，在存在和不存在诱导剂的情况下，乳酸脱氢酶的降解速率没有变化，表明诱导是由于酶合成速率增加。放线菌素D可阻止合成速率的增加。异丙肾上腺素+ 3-异丁基-1-甲基黄嘌呤仅增加乳酸脱氢酶-5同工酶和M亚基的合成比速率。通过在无细胞蛋白质合成系统中使用从异丙肾上腺素或二丁酰环磷酸腺苷诱导的细胞中分离的胶质瘤细胞含聚（A）RNA来测定编码乳酸脱氢酶的功能性mRNA水平，进一步研究了该机制。通过等电聚焦分析免疫沉淀的翻译产物表明，异丙肾上腺素或二丁酰环磷酸腺苷对聚（A）+ RNA指导的乳酸脱氢酶M亚基合成产生约8倍的刺激。这些数据表明，异丙肾上腺素和二丁酰环磷酸腺苷控制胶质瘤细胞中功能性活性乳酸脱氢酶mRNA的水平，这反过来又决定了乳酸脱氢酶M亚基的合成程度。
