Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts.
Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts.
复制标题
单价阳离子转运对培养的人成纤维细胞中鞘糖脂合成代谢的影响。
DOI：
10.1021/bi00333a038
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Rosenberg,A
中科院分区：
文献类型：
--
作者：
Saito,M;Saito,M;Rosenberg,A
Materials and Methods Fibroblast Culture. Human diploid skin fibroblasts (GM3440), obtained from the Institute for Medical Research (Camden, NJ), were cultured in 10 mL of growthmedium consisting of 83% Dulbecco’s modified Eagle’s medium (Gibco), 15% fetal calf serum(Gibco), and 2% penicillin-streptomycin (5000 IU and 5 mg/mL, respectively, Flow Laboratories) in 75-cm1 2 plastic tissue culture bottles (Falcon) at 37 C in a 5% C02 atmosphere in air. To observe the effects of monensin (Calbiochem), confluent fibroblasts (10-13 days after seeding) were incubated wih 10-9—10-6 M monensin in the culture medium for 1-43 h. The effects of other com-pounds were studied by incubating confluent fibroblasts with 10" 6 M ouabain (Sigma), 6 A23187 (Sigma), 10"* M valinomycin (Sigma), or 1CT3 Mnh4c1 (Sigma) in the culture medium for 18 h. For labeling the cells with [3H] Gal,‘con-fluent fibroblasts were incubated with [3* H] Gal (11.5 Ci/mmol, New England Nuclear) in the culture medium at a concen-tration of 1.5 X 1 O'6 Ci/mL. Isolation and Identification of Glycosphingolipids. Fi-broblasts in 75-cm2 plastic tissue culture bottles were washed 3 times with 2 mL of 0.15 M NaCl-0.01 M sodium phosphate buffer, pH 7.4 (PBS), scraped with a rubber policeman, and centrifuged at 600g for 5 min. Total lipids were extracted from the pellets 3 times with 1 mL of chloroform-methanol (2: 1 v/v). The isolation of glycosphingolipids was done according to a method (Irwin & Irwin, 1979) modified as described previously (Saito & Rosenberg, 1982). The gangliosides were separated by silica gel G high-performance thin-layer chromatography (HPTLC) plates (E. Merck) with chloroform-methanol-0.25% CaCl2 in water (65: 35: 8 v/v/v) and visualized by resorcinol reagent. Neutral glycosphingolipids were developed in chloroform-methanol-water (65: 25: 4 v/v/v) and visualized by anthrone reagent. Glucosylceramide and galactosylceramide were separated on HPTLC plates presprayed with 1.5% sodium borate and developed in chloroformmethanol-water-15 M NH4OH (280: 70: 6: 1 v/v/v/v). For the determination of each ganglioside and neutral glyco-sphingolipid, direct densitometric measurement was performed in a Kratos (Schoeffel) densitometer after TLC development. The isolation of glycosphingolipids from cells labeled by [3H] Gal was done as described above. The patterns of ra-dioactivity were obtained by scraping each 2 mm wide strip from TLC plates, transferring the samples into scintillation vials, adding 5 mL of Aquasol (New England Nuclear), so-nicating, and counting in a Beckman scintillation spectrometer. Glucosylceramide was purified from a total lipid extract of splenic tissue from a subject with Gaucher’s disease (Kuske & Rosenberg, 1972). Galactosylceramide, lactosylceramide, trihexosylceramide, and globoside were purchased from Supelco, Inc., Bellefonte, PA. GM1 and GDlawere prepared from
