PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF CP4 (SP-D), A COLLAGENOUS SURFACTANT-ASSOCIATED PROTEIN
PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF CP4 (SP-D), A COLLAGENOUS SURFACTANT-ASSOCIATED PROTEIN
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DOI：
10.1021/bi00441a031
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发表时间：
1989-07-25
期刊：
影响因子：
2.9
通讯作者：
CROUCH, E
中科院分区：
文献类型：
--
作者：
PERSSON, A;CHANG, D;CROUCH, E
CP 4是由大鼠II型肺上皮细胞在原代培养物中合成的胶原糖蛋白（43 kDa，还原的）（Rehson等人，1988年）。为了更好地表征这种蛋白质，从大鼠支气管肺泡灌洗液和肺表面活性剂的EDTA提取物中分离出CP 4，通过吸附到硫酸钡和用柠檬酸钠洗脱，然后通过反相HPLC。纯化的CP 4的氨基酸分析表明4-羟脯氨酸（Hyp），羟赖氨酸（Hyl），和酸不稳定组分共洗脱Hyl葡糖苷。此外，两个CP 4 CNBr肽的气相氨基末端微测序证明了由9个和6个Gly-X-Y三联体组成的非重叠胶原序列，总共含有4个Hyp残基和2个Hyl残基。与大鼠SP-A胶原结构域cDNA序列同源性< 50%。二维IEF/SDS-PAGE将蛋白质解析为碱性同种型的电荷链（pI约为6-8），与新合成的CP 4和D类表面活性剂蛋白相似（Phelps和Taeusch，1985）。凝胶过滤的非变性CP 4的4%琼脂糖显示出高的表观分子量复合物组成的二硫键合的三聚体的43-kDa的亚基。纯化的灌洗液CP 4的抗体表现出特异性结合新合成的和表面活性剂相关的CP 4。我们建议根据公认的表面活性剂相关蛋白命名法（Possmayer，1988），将CP 4命名为“表面活性剂蛋白D”（SP-D）。
CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glucosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was < 50% sequence homology of these peptides with the cDNA-dreived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8) similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps and Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins (Possmayer,1988).