Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2).
Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2).
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兔髓核中与可溶性高分子量酸性蛋白相关的低分子量磷脂酶 A(2) 的纯化及其与兔脾 IIa 组磷脂酶 A(2) 的比较。
DOI：
10.1093/oxfordjournals.jbchem.a022715
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发表时间：
2000
影响因子：
2.7
通讯作者：
K. Horiike
中科院分区：
文献类型：
--
作者：
N. Tanaka;T. Ishida;S. Hukuda;K. Horiike
椎间盘是一大块富含蛋白聚糖和水的无血管软骨，由胶状髓核和纤维环组成。兔髓核可溶性部分表现出异常高的Ca（2+）依赖性磷脂酶A(2) （PLA(2)）活性（约占PLA(2)总活性的70%）。可溶性PLA(2)活性比兔纤维环和脾脏高6-7倍。PLA(2)在pH 7.4下与阴离子交换柱结合，在含有0.1-0.2 M盐的缓冲液的TSKgel SuperSW3000柱上凝胶过滤，在空隙体积附近作为宽峰被洗脱。当凝胶过滤柱在1 M盐的存在下发展时，几乎所有的PLA(2)活性都在总有效体积附近被洗脱。可溶PLA(2)被纯化至接近均匀性。从髓核中以1 M KBr提取的组分中也纯化了Ca（2+）依赖性PLA(2)。为了比较，我们从脾脏的KBr部分纯化了Ca（2+）依赖性PLA(2)。从其n端氨基酸序列和质谱判断，脾PLA(2)属于IIa族PLA(2)。在sds -聚丙烯酰胺凝胶电泳上，从髓核的可溶性部分和KBr部分纯化的酶都得到了15。7-kDa带位于脾脏IIa组PLA相同位置(2)。这些结果表明，IIa族PLA(2)与兔髓核细胞外基质中的可溶性高分子量蛋白（最有可能是蛋白聚糖）有关。
An intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca(2+)-dependent phospholipase A(2) (PLA(2)) activity (about 70% of the total PLA(2) activity). The soluble PLA(2) activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA(2) was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW3000 column developed with a buffer containing 0.1-0.2 M salt. When the gel-filtration column was developed in the presence of 1 M salt, almost all the PLA(2) activity was eluted near the total available volume. The soluble PLA(2) was purified to near homogeneity. A Ca(2+)-dependent PLA(2) was also purified from the fractions extracted with 1 M KBr from nucleus pulposus. For comparison, we purified a Ca(2+)-dependent PLA(2) from the KBr fraction of spleen. The splenic PLA(2) was identical to a group IIa PLA(2), as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15. 7-kDa band at the same position as splenic group IIa PLA(2). These results suggest that group IIa PLA(2) is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.