Heal R D, McGivan J D
Department of Biochemistry, School of Medical Sciences, Bristol, UK.
Biochim Biophys Acta. 1997 Jun 5;1357(1):31-40. doi: 10.1016/s0167-4889(97)00009-8.
The induction of the stress protein Grp75 in response to amino acid deprivation of Chinese Hamster Ovary cells was characterised using a specific monoclonal antibody. A 2-fold increase in the Grp75 protein content occurred over a period of 5-10 h after incubation of the cells in amino acid-free medium. A partial induction was obtained when either all non-essential amino acids or all essential amino acids were omitted from the medium indicating a broad-specificity response. Deletion of the single amino acids tryptophan, histidine or phenylalanine from otherwise complete medium also produced a partial induction of the protein. The increase in the level of Grp75 was completely blocked by cycloheximide, but only partially blocked by the inhibitors of mRNA synthesis actinomycin D and alpha-amanitin. A specific cDNA probe for Grp75 was generated by PCR and used to quantify mRNA levels. No increase in Grp75 mRNA was observed during the induction of the protein indicating that the primary regulation of Grp75 expression was not at the transcriptional level. These results contrast with the large increase in asparagine synthetase mRNA which has been shown to occur during amino acid deprivation, and indicate that cells respond to this form of stress by more than one mechanism.
利用一种特异性单克隆抗体对中国仓鼠卵巢细胞在氨基酸剥夺情况下应激蛋白Grp75的诱导过程进行了表征。将细胞置于无氨基酸培养基中培养5 - 10小时后，Grp75蛋白含量增加了2倍。当培养基中要么去除所有非必需氨基酸，要么去除所有必需氨基酸时，均可获得部分诱导效果，这表明其具有广泛特异性反应。从完全培养基中去除单个氨基酸色氨酸、组氨酸或苯丙氨酸也会部分诱导该蛋白的产生。Grp75水平的增加被环己酰亚胺完全阻断，但仅被mRNA合成抑制剂放线菌素D和α - 鹅膏蕈碱部分阻断。通过PCR生成了Grp75的特异性cDNA探针，并用于定量mRNA水平。在诱导该蛋白的过程中未观察到Grp75 mRNA的增加，这表明Grp75表达的主要调控不在转录水平。这些结果与在氨基酸剥夺期间天冬酰胺合成酶mRNA大幅增加形成对比，表明细胞通过多种机制对这种应激形式做出反应。
