Human placental neuraminidase
Human placental neuraminidase
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人胎盘神经氨酸酶
DOI：
10.1111/j.1432-1033.1985.tb08928.x
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发表时间：
1985
期刊：
影响因子：
5.4
通讯作者：
H. Galjaard
中科院分区：
文献类型：
--
作者：
F. Verheijen;S. Palmeri;A. Hoogeveen;H. Galjaard
人胎盘匀浆上清液中几乎不含溶酶体神经氨酸酶活性。然而，通过浓缩部分纯化的糖蛋白部分可以产生非常高的活性。这种活性对稀释不稳定，但在37℃和酸性pH下孵育可以稳定下来。
利用β-半乳糖苷酶特异性亲和层析和免疫滴定，我们证明激活和稳定的人溶酶体神经氨酸酶存在于与β-半乳糖苷酶的络合物中。蔗糖密度梯度离心实验表明，神经氨酸酶活性仅以高密度多聚体形式的β-半乳糖苷酶存在。
已知形成多聚体的β-半乳糖苷酶需要32000-mR的“保护性”蛋白质。使用硝酸纤维素印迹免疫亲和纯化程序，从含有针对64000-mRβ-半乳糖苷酶蛋白和32000-mR‘保护’蛋白的混合抗体的常规抗血清中提纯了针对该‘保护性’蛋白的单特异性抗体。用这些抗体进行的免疫滴定实验表明，32000-mR‘保护性’蛋白既与β-半乳糖苷酶多聚体结合，也与神经氨酸酶一起与高密度多聚体结合。
我们的数据进一步表明，32000-mR‘保护性’蛋白和另一个尚未确定的亚基的结合对于溶酶体神经氨酸酶的催化活性是必不可少的。这些结果解释了常染色体隐性遗传性人类溶酶体储存障碍半乳糖化病缺乏神经氨酸酶活性的原因，其中32000-mR‘保护性’蛋白是已知缺失的。
Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37°C and acid pH.
Using β-galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with β-galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of β-galactosidase.
The formation of multimeric forms of β-galactosidase is known to require a 32000-Mr‘protective’ protein. Monospecific antibodies against this ‘protective’ protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000-Mrβ-galactosidase protein and against the 32000-Mr‘protective’ protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000-Mr‘protective’ protein is present both in association with the β-galactosidase multimer and with the high-density multimeric form together with neuraminidase.
Our data further suggest that association of the 32000-Mr‘protective’ protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000-Mr‘protective’ protein is known to be absent.