Martin-De Las Heras S, Valenzuela A, Overall C M
Department of Forensic Medicine and Forensic Odontology, Faculty of Medicine, University of Granada, 18071 Granada, Spain.
Arch Oral Biol. 2000 Sep;45(9):757-65. doi: 10.1016/s0003-9969(00)00052-2.
A dentine protein extraction protocol was modified in order to identify matrix metalloproteinase gelatinolytic activities in the non-mineralized and mineralized phases of human dentine. Dentine proteins from 24 individual permanent molars from patients aged 15-73 years were sequentially extracted, first with guanidinium chloride (G1 extract), then EDTA (E extract), and after this demineralization step, again by guanidinium chloride (G2 extract) to dissociate collagen-associated proteins. Extracts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the gels were processed by Western blotting and zymography to detect gelatinolytic activities. Active and latent forms of gelatinase A were identified in the non-mineralized dentine fraction (G1 extract) of 58% of the teeth. Other gelatinolytic species were also detected by zymography with apparent M(r) of 92, 54 and 30 kDa. Although gelatinase A was detected in the G1 extracts of teeth from all ages, indicating more recent synthesis and remodelling of the predentine, gelatinase A was never detected in any E extract or in the G2 extracts of patients older than 41 years. The presence of the active form of gelatinase A in mineralized human dentine implicates this enzyme in dentine mineralization.
为了鉴定人牙本质非矿化和矿化阶段的基质金属蛋白酶明胶分解活性，对牙本质蛋白提取方案进行了修改。从15至73岁患者的24颗单个恒磨牙中依次提取牙本质蛋白，首先用氯化胍提取（G1提取物），然后用乙二胺四乙酸提取（E提取物），在这个脱矿步骤之后，再次用氯化胍提取（G2提取物）以解离与胶原相关的蛋白。提取物通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析，凝胶通过蛋白质印迹法和酶谱法处理以检测明胶分解活性。在58%的牙齿的非矿化牙本质部分（G1提取物）中鉴定出了活性和潜伏形式的明胶酶A。通过酶谱法还检测到其他明胶分解物种，其表观分子量为92、54和30 kDa。尽管在所有年龄段牙齿的G1提取物中都检测到了明胶酶A，这表明前期牙本质有更新的合成和重塑，但在任何E提取物或41岁以上患者的G2提取物中从未检测到明胶酶A。矿化人牙本质中活性形式的明胶酶A的存在表明该酶参与牙本质矿化。
