EFFECTS OF TEMPERATURE ON THE DEGRADATION OF PROTEINS IN RABBIT RETICULOCYTE LYSATES AND AFTER INJECTION INTO HELA-CELLS
EFFECTS OF TEMPERATURE ON THE DEGRADATION OF PROTEINS IN RABBIT RETICULOCYTE LYSATES AND AFTER INJECTION INTO HELA-CELLS
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DOI：
10.1073/pnas.81.1.90
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发表时间：
1984-01-01
期刊：
影响因子：
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通讯作者：
RECHSTEINER, M
中科院分区：
文献类型：
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作者：
HOUGH, R;RECHSTEINER, M
标记牛血清白蛋白、丙酮酸激酶、血红蛋白和IgG的Fc片段，并通过红细胞介导的显微注射将其导入[人宫颈癌] HeLa细胞。然后在6 ℃至10 ℃的温度下培养的细胞中测量注射蛋白质的降解。C和37度。C.阿耳忒弥斯曲线显示常数Ea [活化能]为27 ± 1。5千卡/摩尔。类似地，降解长期内源性标记的HeLa蛋白的表观Ea为22-26 kcal/mol。局部蛋白质解折叠和蛋白质水解由确定的酶，如胰蛋白酶或木瓜蛋白酶，进行与Eas之间的5和15千卡/摩尔。在本研究中获得的2倍更高的值表明，蛋白质解折叠或简单的蛋白质水解在注射或长寿命的内源性HeLa蛋白质的降解中不是速率限制。此外，相对均匀的Eas表明，类似的生物化学事件是特定蛋白质降解的速率限制，与其半衰期无关。该事件可能涉及来自兔网织红细胞裂解物的ATP依赖性蛋白水解途径中的反应，因为该系统中ATP依赖性蛋白水解的Eas也为27 ± 1。5千卡/摩尔。
Bovine serum albumin, pyruvate kinase, Hb and the Fc fragment of IgG were labeled and introduced into [human cervical carcinoma] HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6.degree. C and 37.degree. C. Arrhenius plots revealed a constant Ea [activation energy] of 27 .+-. 5 kcal/mol over this temperature interval. Similarly, the apparent Ea for the degradation of long-term endogenously labeled HeLa proteins was 22-26 kcal/mol. Both local protein unfolding and proteolysis by defined enzymes, such as trypsin or papain, proceed with Eas between 5 and 15 kcal/mol. The 2-fold higher values obtained in this study indicate that protein unfolding or simple proteolysis is not rate limiting in the degradation of injected or long-lived endogenous HeLa proteins. Moreover, the relatively uniform Eas suggest that a similar biochemical event is rate limiting in the degradation of a specific protein independent of its half-life. This event may involve a reaction in the ATP-dependent proteolytic pathway from rabbit reticulocyte lysates because Eas for ATP-dependent proteolysis in this system were also 27 .+-. 5 kcal/mol.