Hirschman A, Deutsch D, Hirschman M, Bab I A, Sela J, Muhlrad A
Calcif Tissue Int. 1983 Sep;35(6):791-7. doi: 10.1007/BF02405125.
Extracellular matrix vesicles from bovine fetal alveolar bone and from a dog osteosarcoma were isolated by differential centrifugation and then fractionated on a discontinuous sucrose density gradient. The fractions were examined by electron microscopy and were analyzed for protein, alkaline phosphatase, aminotripeptidase, and four different beta-naphthylamidase activities. The low-density peak of enzyme activities was shown by electron microscopy to be much more homogeneous than the crude matrix vesicle fraction. Two major peaks of protein and enzyme activities were present, one in the high and one in the low density layers. There was good correlation between the activities of alkaline phosphatase and the various peptidases in the fractions from the sucrose density gradient. These results indicate a coexistence of peptidase and alkaline phosphatase in matrix vesicles. On the other hand, there was generally no correlation between the peptidase and alkaline phosphatase activities in vesicular specimens from bovine liver obtained in the same way. Most of the peptidase activity and about half of the alkaline phosphatase activity were solubilized from bone matrix vesicles by detergents. The extracted alkaline phosphatase and alanyl beta-naphthylamidase activities were separated from each other on a DEAE-cellulose column.
通过差速离心法分离牛胎儿牙槽骨和犬骨肉瘤的细胞外基质囊泡，然后在不连续蔗糖密度梯度上进行分级分离。通过电子显微镜检查这些级分，并分析其蛋白质、碱性磷酸酶、氨基三肽酶和四种不同的β-萘胺酶活性。电子显微镜显示，酶活性的低密度峰比粗基质囊泡级分更均匀。存在两个主要的蛋白质和酶活性峰，一个在高密度层，一个在低密度层。蔗糖密度梯度级分中碱性磷酸酶和各种肽酶的活性之间存在良好的相关性。这些结果表明肽酶和碱性磷酸酶在基质囊泡中共存。另一方面，以同样方式获得的牛肝囊泡标本中，肽酶活性和碱性磷酸酶活性之间通常没有相关性。大多数肽酶活性和大约一半的碱性磷酸酶活性可通过去污剂从骨基质囊泡中溶解出来。在DEAE-纤维素柱上，提取的碱性磷酸酶和丙氨酰β-萘胺酶活性彼此分离。
