ISOLATION AND CHARACTERIZATION OF 2 SIALOPROTEINS PRESENT ONLY IN BONE CALCIFIED MATRIX
ISOLATION AND CHARACTERIZATION OF 2 SIALOPROTEINS PRESENT ONLY IN BONE CALCIFIED MATRIX
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DOI：
10.1042/bj2320715
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发表时间：
1985-01-01
影响因子：
4.1
通讯作者：
HEINEGARD, D
中科院分区：
文献类型：
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作者：
FRANZEN, A;HEINEGARD, D
用盐酸胍提取牛骨矿化基质中的唾液酸蛋白，分离出两种不同的唾液酸蛋白。通过在DEAE-纤维素上用乙酸钠在7 M-尿素（pH 6）中的梯度洗脱的色谱法纯化唾液蛋白。然后通过在DEAE-纤维素上用氯化钠梯度洗脱的色谱法在7 M-尿素（pH 4）中分离两种唾液蛋白（I和II）。回收的唾液蛋白I和II之间的比例为1：5。两种唾液蛋白的化学分析表明它们不同。但两者的天冬氨酸/天冬酰胺和谷氨酸/谷氨酰胺含量都很高，而亮氨酸和甘氨酸的含量差异显著。两种唾液蛋白都含有磷酸盐，唾液蛋白I比唾液蛋白II多。唾液酸的含量在更突出的唾液蛋白II（干重的13.4%）中比在唾液蛋白I（干重的4.8%）中显著更高。[125 I]碘化唾液酸蛋白I和II的胰蛋白酶消化产生的肽模式显示出结构相似性和结构差异。进一步表征了作为主要组分的唾液酸蛋白II。在6 M-氯化胍中通过沉降平衡离心测定其分子量为57，300 Da，其沉降系数（S20，w 0）为2.53 S。在旋转阴影下，唾液蛋白II表现为延长的棒，具有平均长度为40 nm的核心。两种类型的寡糖，N-糖苷和O-糖苷连接的核心蛋白，从唾液蛋白II分离。O-连接寡糖中甘露糖和唾液酸的含量高得惊人。抗唾液蛋白II的抗体在兔和酶联免疫吸附试验的发展。唾液蛋白II的抗原性不受还原和烷基化的影响，仅部分损失后，胰蛋白酶消化和完全丧失后，通过碱性硼氢化物处理的核心蛋白片段，表明所有的抗原位点都位于蛋白质部分。唾液蛋白I型与唾液蛋白II型仅表现出部分免疫交叉反应性。发现骨提取物中唾液蛋白II的量为约1.5mg/g湿重。但在许多其他牛组织（即主动脉、软骨、牙本质、肾脏、肝脏、肌肉、巩膜、皮肤和肌腱）的提取物中未检测到该蛋白。
Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialopotein II, being the major component, was characterized further. Its molecular mass was 57,300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S20,w0) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotien II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.