Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase.
Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase.
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21 kDa 和 25 kDa 牛釉质金属蛋白酶的纯化和测序。
DOI：
10.1111/j.1600-0722.1998.tb02196.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Li,W
中科院分区：
文献类型：
--
作者：
DenBesten,PK;Punzi,JS;Li,W
发育中的釉质基质含有金属蛋白酶，推测其在釉质基质蛋白的水解中起作用。确定这些蛋白酶的身份和功能需要进一步的信息，如它们的氨基酸组成和序列。在这项研究中，我们纯化的21 kDa和25 kDa的基质金属蛋白酶分泌期牛釉基质。提取后，这些蛋白酶通过离子交换连续分离进一步纯化。Con A亲和层析和反相HPLC。通过SDS PAGE分离蛋白酶，转移到PVDF膜上，并对N末端进行测序。两种蛋白酶的N-末端序列相同，并显示与猪釉质溶素（MMP 20）cDNA序列同源。使用PCR扩增产生的探针从牛成釉器官cDNA文库中分离牛MMP 20的cDNA。牛和猪MMP 20 cDNA的编码区高度同源，并且包含与蛋白酶N末端序列具有预测氨基酸序列同源性的相同区域。这些结果表明，21 kDa和25 kDa的釉基质金属蛋白酶是最初分泌的MMP 20的裂解产物，并且MMP 20的序列在物种间是保守的。
The developing enamel matrix contains metalloproteinases that are presumed to have a role in hydrolysis of enamel matrix proteins. Determination of the identity and function of these proteinases requires further information such as their amino acid composition and sequence. In this study, we purified the 21 kDa and 25 kDa matrix metalloproteinase from secretory stage bovine enamel matrix. After extraction, these proteinases were further purified by sequential separation by ion exchange. Con A affinity chromatography, and reversed phase HPLC. The proteinases were separated by SDS PAGE, transferred to a PVDF membrane and the N‐terminus was sequenced. The N‐terminal sequences of both proteinases were the same, and showed homology to the porcine enamelysin (MMP 20) cDNA sequence. A cDNA for bovine MMP 20 was isolated from a bovine enamel organ cDNA library using a probe generated by PCR amplification. The coding regions of bovine and porcine MMP 20 cDNAs were highly homologous and contained the same regions of predicted amino acid sequence homology with the proteinase N‐terminal sequences. These results suggest that the 21 kDa and 25 kDa enamel matrix metalloproteinases are cleavage products of the initially secreted MMP 20, and that the sequence for MMP 20 is conserved across species.