Cheng S C, Ogrydziak D M
J Bacteriol. 1987 Apr;169(4):1433-40. doi: 10.1128/jb.169.4.1433-1440.1987.
Secretion of the extracellular RNase from the yeast Yarrowia lipolytica was studied in pulse-chase and immunoprecipitation experiments. A polypeptide of 45,000 daltons was immunoprecipitated from [35S]methionine-labeled cell extracts and supernatant medium by rabbit anti-RNase antiserum. The RNase was secreted rapidly; the time between synthesis and appearance in the extracellular medium was about 5 min. In pulse-chase experiments, about 50% of the RNase was still cell associated 30 min after labeling. A polypeptide of 73,000 daltons whose immunoprecipitation was blocked by an excess of purified RNase was also detected. It broke down to a polypeptide with the same mobility and same peptide map as the mature RNase. Peptide maps of the undegraded 73-kilodalton polypeptide and the intracellular mature RNase contained several peptides of identical mobility. Immunoprecipitates from cells labeled in the presence of tunicamycin contained 66- and 45-kilodalton polypeptides. Endoglycosidase H treatment of the 73-kilodalton polypeptide converted it to a 66-kilodalton form, but did not change the apparent molecular weight of the mature form of the RNase. Labeling kinetics from pulse-chase experiments did not clearly support a precursor-product relationship between the 73-kilodalton polypeptide and the intracellular 45-kilodalton form of the RNase, and other relationships between the two polypeptides are possible.
通过脉冲追踪和免疫沉淀实验研究了解脂耶氏酵母细胞外核糖核酸酶（RNase）的分泌情况。用兔抗RNase抗血清从[35S]甲硫氨酸标记的细胞提取物和上清培养基中免疫沉淀出一种45000道尔顿的多肽。RNase分泌迅速；从合成到出现在细胞外培养基中的时间约为5分钟。在脉冲追踪实验中，标记后30分钟约50%的RNase仍与细胞相关。还检测到一种73000道尔顿的多肽，其免疫沉淀被过量的纯化RNase阻断。它分解为一种与成熟RNase具有相同迁移率和相同肽图的多肽。未降解的73千道尔顿多肽和细胞内成熟RNase的肽图包含几种迁移率相同的肽。在衣霉素存在下标记的细胞的免疫沉淀物含有66千道尔顿和45千道尔顿的多肽。用内切糖苷酶H处理73千道尔顿的多肽可将其转化为66千道尔顿的形式，但不会改变RNase成熟形式的表观分子量。脉冲追踪实验的标记动力学并未明确支持73千道尔顿多肽与细胞内45千道尔顿形式的RNase之间的前体-产物关系，两者之间的其他关系也是可能的。
