Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.
Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.
复制标题
通过正常和糖尿病皮肤成纤维细胞培养物生产[3H]己糖胺标记的蛋白多糖：用来自葡萄糖和其他来源的内源性己糖胺稀释外源性[3H]葡萄糖胺。
DOI：
10.1016/0003-9861(89)90600-0
复制
发表时间：
1989
影响因子：
3.9
通讯作者：
Silbert,JE
中科院分区：
文献类型：
--
作者：
Silbert,CK;Palmer,ME;Humphries,DE;Silbert,JE
将来自两名正常男性、三名I型糖尿病男性和一名I型糖尿病女性的人皮肤成纤维细胞单层培养物与[3 H]葡糖胺和[35 S]-硫酸盐孵育不同的时间。3 H掺入到培养基中出现的大分子中在约45分钟后呈线性，35 S掺入在约30分钟后呈线性。比较了每种培养物在5小时孵育期间形成的35 S-蛋白聚糖的量，发现六种细胞系相当相似，从0.08到0.14 nmol硫酸盐/μg DNA不等。然后用软骨素ABC裂解酶处理分离的3 H，35 S-糖胺聚糖，以表征硫酸化的位置和程度。结果表明，软骨素/皮肤素硫酸化的完整性和不同品系之间6-硫酸化至4-硫酸化的比例存在相当大的变化。分离3 H，35 S标记的二糖，测定3 H与35 S的比值，以计算孵育期间由葡萄糖或其它来源的内源性葡糖胺对[3 H]葡糖胺的稀释。在不同的细胞系中，稀释度从160倍到635倍不等，但这些变化似乎与细胞是来自正常人还是糖尿病患者无关。
Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of35S was linear after approximately 30 min. The amounts of35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/μg DNA. Isolated3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics.3H,35S-Labeled disaccharides were isolated and ratios of3H to35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.