Luskey K L, Chin D J, MacDonald R J, Liscum L, Goldstein J L, Brown M S
Proc Natl Acad Sci U S A. 1982 Oct;79(20):6210-4. doi: 10.1073/pnas.79.20.6210.
UT-1 cells, a clone of Chinese hamster ovary (CHO) cells, have a 100- to 1,000-fold elevation in the amount of 3-hydroxy-3-methylglutaryl CoA reductase and therefore grow in the presence of compactin, an inhibitor of reductase. In this paper, we report that UT-1 cells also have a markedly increased amount of another protein with a Mr of 53,000 and an isoelectric point of approximately equal to 6. Whereas the reductase is an enzyme of the endoplasmic reticulum, the 53,000-dalton protein (termed the "53k" protein) is in the cytosol. It is not precipitated by an antireductase antibody. Synthesis of the 53k protein, like that of the reductase, is suppressed when UT-1 cells are incubated with plasma low density lipoprotein (LDL). We prepared a library of recombinant plasmids containing double-stranded cDNAs from UT-1 cells. Using differential colony hybridization, we identified recombinant plasmids containing double-stranded cDNA inserts encoding mRNAs expressed at high levels in UT-1 cells as compared with CHO cells. One of the plasmids, designated p53k-3, contained a 0.97-kilobase double-stranded cDNA that hybridized to a 3.8-kilobase mRNA. When translated in vitro, this 3.8-kilobase mRNA directed the synthesis of a protein identical to the cellular 53k protein as determined by two-dimensional gel electrophoresis. Hybridization studies showed that the mRNA for the 53k protein was present in much larger amounts in UT-1 cells than in parental CHO cells. In both cell types, the content of this mRNA decreased markedly when the cells were incubated with LDL. Although the function of the 53k protein is not known, circumstantial evidence suggests that it may represent cytosolic 3-hydroxy-3-methylglutaryl CoA synthase, the enzyme preceeding the reductase in the cholesterol biosynthetic pathway. The current data indicate that the synthesis of at least two proteins, the reductase and the 53k protein, are induced to high levels in compactin-resistant UT-1 cells and that the synthesis of both is suppressed coordinately by LDL.
UT-1细胞是中国仓鼠卵巢（CHO）细胞的一个克隆，其3-羟基-3-甲基戊二酰辅酶A还原酶的量升高了100至1000倍，因此能在还原酶抑制剂美伐他汀的存在下生长。在本文中，我们报道UT-1细胞中另一种蛋白质的量也显著增加，该蛋白质的相对分子质量为53,000，等电点约为6。还原酶是内质网的一种酶，而这种53,000道尔顿的蛋白质（称为“53k”蛋白质）存在于胞质溶胶中。它不会被抗还原酶抗体沉淀。与还原酶一样，当UT-1细胞与血浆低密度脂蛋白（LDL）一起孵育时，53k蛋白质的合成会受到抑制。我们构建了一个包含UT-1细胞双链cDNA的重组质粒文库。通过差异菌落杂交，我们鉴定出了与CHO细胞相比，含有在UT-1细胞中高水平表达的mRNA的双链cDNA插入片段的重组质粒。其中一个质粒，命名为p53k-3，包含一个0.97千碱基的双链cDNA，它与一个3.8千碱基的mRNA杂交。当在体外进行翻译时，通过二维凝胶电泳确定，这个3.8千碱基的mRNA指导合成一种与细胞53k蛋白质相同的蛋白质。杂交研究表明，53k蛋白质的mRNA在UT-1细胞中的含量比亲本CHO细胞中多得多。在两种细胞类型中，当细胞与LDL一起孵育时，这种mRNA的含量都会显著下降。尽管53k蛋白质的功能尚不清楚，但间接证据表明它可能代表胞质溶胶中的3-羟基-3-甲基戊二酰辅酶A合酶，即胆固醇生物合成途径中位于还原酶之前的酶。目前的数据表明，在对美伐他汀耐药的UT-1细胞中，至少两种蛋白质，即还原酶和53k蛋白质的合成被诱导到高水平，并且两者的合成都受到LDL的协同抑制。
