Sugumaran G, Silbert J E
Connective Tissue Research Laboratory, Veterans Administration Outpatient Clinic, Boston, Massachusetts.
J Biol Chem. 1990 Oct 25;265(30):18284-8.
A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.
将来自硫酸软骨素4 - 硫酸酯合成培养的小鼠肥大细胞瘤细胞的微粒体制剂，与UDP - [3H]GalNAc、UDP - GlcA和3'-磷酸腺苷磷酸硫酸酯（PAPS）在10℃下孵育30秒，以及与UDP - [14C]GlcA、UDP - GalNAc和PAPS在37℃下孵育4小时，用于合成3H和14C标记的软骨素/硫酸软骨素。后一种孵育产生的产物比在10℃下的短时间孵育多100倍以上。将分离出的标记糖胺聚糖在Sepharose CL - 6B柱上进行层析时，在37℃下孵育4小时得到的大部分[14C]糖胺聚糖被柱排除，这表明这种新生糖胺聚糖已完全聚合。相比之下，在10℃下孵育30秒得到的大部分[3H]糖胺聚糖在同一柱上共层析时大多滞后，这表明新生糖胺聚糖的大小仍在增长。通过用软骨素ABC裂解酶降解，然后对产物进行纸电泳，分析代表不同大小的软骨素/硫酸软骨素的标记组分的硫酸化程度。结果表明，在4小时孵育中形成的[14C]软骨素/硫酸软骨素的硫酸化程度为60 - 70%。在30秒孵育中形成的[3H]软骨素/硫酸软骨素的不完全链的硫酸化程度也高达20 - 25%。随着[3H]软骨素/硫酸软骨素大小的增加，硫酸化程度随之增加。这些结果表明，在这个微粒体系统中，硫酸化在新生软骨素糖胺聚糖链仍在积极延长时就发生了，尽管硫酸化在一定程度上滞后于聚合反应。这不仅表明软骨素聚合和硫酸化在膜上有共同的位置，而且还证明这些肥大细胞瘤细胞对软骨素的硫酸化可能发生在糖胺聚糖聚合过程中，而不是在糖胺聚糖链完成之后。
