Evans D P, Tomasovic S P
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Radiat Res. 1990 Oct;124(1):50-6.
The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.
在大鼠腺癌细胞中研究了钙调蛋白（CaM）与热休克蛋白及其他结合蛋白的相互作用。细胞在43℃加热1小时前进行48小时的平衡标记，或在加热后于37℃脉冲标记2小时，以监测热对在这些条件下合成的CaM结合蛋白亲和力的影响。一种已证明能使细胞对热杀伤敏感的CaM拮抗剂W-7 [N-(6-氨基己基)-5-氯-1-萘磺酰胺]用于竞争分析，以帮助监测亲和力的任何变化。我们发现，相对于其37℃对照，加热往往会降低加热前合成的蛋白质与CaM的结合，而加热后合成的蛋白质相对于其各自对照往往具有增加的结合。热休克蛋白（hsp）90 kDa、70 kDa和26 kDa家族的成员是与CaM结合并被W-7洗脱的蛋白质之一。hsp和其他细胞蛋白的洗脱峰馏分各不相同，但hsp-70在早期馏分中洗脱。hsp-70家族也被发现是众多W-7结合蛋白之一。我们得出结论，认为CaM拮抗剂仅通过非特异性竞争CaM上的CaM结合蛋白位点来增强加热细胞的杀伤作用这一假设并不能完全解释这一过程。这些拮抗剂还可能通过与特定蛋白质竞争CaM结合位点来发挥作用，这些蛋白质与CaM的相互作用对加热后的存活很重要，或者通过直接结合到对存活很重要且抑制其活性的其他蛋白质上来发挥作用。我们没有足够的数据来辨别这些可能性中哪种是主要机制，但我们认为所有这些情况都可能在加热的细胞中发生，并推测抑制hsp-70家族的功能在这些拮抗剂的几种作用中很重要。
