Fellini S A, Hascall V C, Kimura J H
J Biol Chem. 1984 Apr 10;259(7):4634-41.
Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.
用[3H]丝氨酸对来自Swarm大鼠软骨肉瘤的软骨细胞进行30分钟的脉冲标记，并在存在环己酰亚胺的情况下追踪长达300分钟。研究了蛋白聚糖新合成的核心蛋白前体在内质网和高尔基体元件中的移动情况。通过在不连续蔗糖梯度上离心细胞匀浆的线粒体后上清液获得粗面和滑面微粒体部分。通过以下方法在亚细胞部分鉴定核心蛋白前体：(a) 用针对核心蛋白透明质酸结合区域和连接蛋白的抗血清进行免疫沉淀，以及(b) 在聚丙烯酰胺凝胶上测定其大小。标记的核心蛋白前体从微粒体中减少，半衰期为60±8分钟，与完整蛋白聚糖单体中标记出现的半衰期（t1/2 = 58±13分钟）几乎相同，这与前体-产物关系一致。在校正了微粒体部分核心蛋白前体的不完全回收率以及粗面内质网元件对滑面微粒体的交叉污染后，核心蛋白前体和完整蛋白聚糖在细胞内区室中的重新分布以及标记的细胞外蛋白聚糖符合三室模型。粗面微粒体中核心蛋白前体损失的半衰期为98±7分钟，细胞内区室（高尔基体和分泌小泡）中完整蛋白聚糖的半衰期为10±4分钟。数据表明，核心蛋白前体在细胞内转运时间的至少70%花费在粗面内质网中。糖胺聚糖链的添加随后从细胞分泌相对较快，占总细胞内停留时间的不到30%。
