SPECIFIC CLEAVAGE OF A RECOMBINANT MURINE AMELOGENIN AT THE CARBOXY-TERMINAL REGION BY A PROTEINASE FRACTION ISOLATED FROM DEVELOPING BOVINE TOOTH ENAMEL
SPECIFIC CLEAVAGE OF A RECOMBINANT MURINE AMELOGENIN AT THE CARBOXY-TERMINAL REGION BY A PROTEINASE FRACTION ISOLATED FROM DEVELOPING BOVINE TOOTH ENAMEL
复制标题
DOI：
10.1016/0003-9969(94)90090-6
复制
发表时间：
1994-08-01
影响因子：
3
通讯作者：
FINCHAM, AG
中科院分区：
文献类型：
--
作者：
MORADIANOLDAK, J;SIMMER, JP;FINCHAM, AG
通过尺寸排阻和反相高压液相色谱（HPLC）技术从发育中的牛牙釉质中分离出48-70-kDa的蛋白酶组分。使用明胶作为底物，通过酶谱法观察HPLC级分中的蛋白水解活性。使用由179个氨基酸残基（20 kDa）组成的重组鼠釉原蛋白（M179）作为底物，以检查分离组分中酶的特异性。将M179与蛋白酶级分在37 ℃下孵育产生了主要的蛋白水解产物，其在约42%乙腈下从反相柱洗脱。该产物具有与M179亲本蛋白一致的氨基末端序列Pro-Leu-Pro-Pro-His-Pro-。这些数据表明，产物是由M179重组蛋白在羧基末端区域的切割产生的。通过反相HPLC分离的产物的质谱分析得到18.89 kDa的分子量。鉴于氨基末端序列完整，该质量图表明该产物终止于M179残基序列的pro（168）。当M179被用作底物时，EDTA在蛋白水解实验中的存在抑制了18.89-kDa产物的产生。抗痛剂、抑肽酶、亮肽素和4，（脒基苯基）甲磺酰氟（均为丝氨酸蛋白酶抑制剂）不影响蛋白水解活性。此外，用Zn ~（2+）、Mn ~（2+）或Co ~（2+）替代蛋白水解缓冲液中的Ca ~（2+）抑制酶活性。因此，在pro（168）-Ala（169）位点切割M179的“高分子量”蛋白酶是一种特异性的“钙依赖性金属蛋白酶”。
A proteinase fraction of 48-70-kDa was isolated from developing bovine tooth enamel by size exclusion and reversed-phase high-pressure liquid chromatography (HPLC) techniques. Proteolytic activity in the HPLC fraction was visualized by enzymography using gelatin as substrate. A recombinant murine amelogenin (M179) composed of 179 amino acid residues (20 kDa) was used as a substrate to examine the specificity of the enzymes in the isolated fractions. Incubation of M179 with the proteinase fraction at 37 degrees C generated a major proteolytic product eluting at about 42% acetonitrile from the reversed-phase column. This product had an amino-terminal sequence Pro-Leu-Pro-Pro-His-Pro- in conformity with that of the M179 parent protein. These data indicated that the product resulted from the cleavage of the M179 recombinant protein in the carboxy-terminal region. Mass spectroscopic analysis of the product isolated by reversed-phase HPLC gave a molecular mass of 18.89 kDa. Given an intact amino-terminal sequence, this mass figure suggests that this product terminates at pro(168) Of the M179 residue sequence. The presence of EDTA in proteolysis experiments when M179 was used as substrate inhibited production of the 18.89-kDa product. Antipain, aprotinin, leupeptin and 4,(amidino-phenyl)methanesulphonyl fluoride, which are serine proteinase inhibitors, did not affect the proteolytic activity. In addition, replacement of Ca2+ with Zn2+, Mn2+ or Co2+ in the proteolysis buffer inhibited the enzymatic activity. It is concluded that the 'high molecular-weight' proteinase cleaving M179 at pro(168)-Ala(169) is a specific 'calcium-dependent metalloproteinase'.