Calcium-binding properties of osteopontin derived from non-osteogenic sources.
Calcium-binding properties of osteopontin derived from non-osteogenic sources.
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非成骨来源的骨桥蛋白的钙结合特性。
DOI：
10.1093/oxfordjournals.jbchem.a124240
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发表时间：
1993
影响因子：
2.7
通讯作者：
Mukherjee,BB
中科院分区：
文献类型：
--
作者：
Singh,K;Deonarine,D;Shanmugam,V;Senger,DR;Mukherjee,AB;Chang,PL;Prince,CW;Mukherjee,BB
材料与方法正常大鼠肾细胞系NRK来自美国典型培养物保藏中心(马里兰州罗克维尔)。NRK细胞在含10%新生小牛血清(GIBCO)和抗生素的DMEM中培养。由Rous肉瘤病毒温度敏感突变体转化的大鼠1细胞系tsB77是从剑桥大学RO Hynes博士那里获得的，在允许(34·Ž)和不允许(41·Ž)两种温度下，分别在含5%胎牛血清的DMEM中培养。JB6 C141。5A，取自NCL，Frederic，MD的小鼠表皮细胞系，在添加5%热灭活胎牛血清的MEM中培养。非磷酸化OP的产生-已经证明，降钙素处理JB6 141.5 a细胞只诱导非磷酸化OP的合成和分泌(19)。因此，JB6 C141。将接近融合的5A细胞用长/毫升的1ƒ，25-二羟基维生素D3(骨化三醇)处理48h，收集含有非磷酸化OP的条件培养液进行钙结合研究。为了确认OP的使用实际上是非磷酸化的，在培养的最后3小时，用32PO4或[35S]蛋氨酸标记相同处理的细胞。制备放射性标记和未标记的骨桥蛋白亚流，用[35S]蛋氨酸(130mmolCi/ml；1,200~1,400Ci/ƒ；amersham)标记生长活跃的细胞4h后，将条件培养液调至5 mM PMSF中，10,000·g离心20min。取上清液进行OP分析。为了获得未标记的OP，除细胞未被放射性标记外，采用相同的方法。
MATERIALS AND METHODSCell Cultures-NRK, a normal rat kidney cell line, was obtained from the American Type Culture Collection (Rockville, MD). NRK cells were grown in DMEM sup plemented with 10% newborn calf serum(GIBCO) and antibiotics. tsB77, a Rat-1 cell line transformed by a temperature-sensitive mutant of Rous sarcoma virus was obtained from Dr. RO Hynes(MIT, Cambridge) and was grown in DMEM containing 5% fetal calf serum at both permissive(34• Ž) and non-permissive(41• Ž) tempera tures. JB6 C141. 5a, a mouse epidermal cell line was obtained from Dr. NH Colburn(NCl, Frederic, MD) and was maintained in MEM supplemented with 5% heat inactivated fetal calf serum. Generation of Non-Phosphorylated Form of OP-It has been demonstrated that calcitrol treatment of JB6 141.5 a cells induces the synthesis and secretion of only the non-phosphorylated form of OP (19). Therefore, JB6 C141. 5a cells approaching confluence were treated for 48 h with long/ml of 1ƒ¿, 25-dihydroxyvitamin D3 (calcitriol) dissolved in DMSO, and the conditioned medium contain ing non-phosphorylated OP was collected for Ca2+-binding studies. To confirm that OP used that OP use was in fact non phosphorylated, identically treated cells were labeled with 32 PO4 or [35S] methionine during the last 3h of culture. The conditioned medium was immunoprecipitated with anti-OP antibody, and the proteins were analyzed by SDS-PAGE and autoradiography.Preparation of Radiolabeled and Unlabeled Osteopon tinSubconfluent, actively growing cells were labeled for 4 h with[35S] methionine(130ƒÊCi/ml; 1,200-1,400 Ci/mmol; Amersham), then conditioned medium was adjusted to 5mM PMSF and centrifuged at 10,000•~ g for 20min. The supernatant was used for OP analysis. For obtaining unlabeled OP, the same method was followed except the cells were not radiolabeled.