PURIFICATION OF MICROSOMAL SIGNAL PEPTIDASE AS A COMPLEX
PURIFICATION OF MICROSOMAL SIGNAL PEPTIDASE AS A COMPLEX
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DOI：
10.1073/pnas.83.3.581
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发表时间：
1986-02-01
影响因子：
11.1
通讯作者：
BLOBEL, G
中科院分区：
文献类型：
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作者：
EVANS, EA;GILMORE, R;BLOBEL, G
本文报道了从犬胰腺微粒体中纯化得到的信号肽酶。使用改进的翻译后测定监测纯化。通过在非离子洗涤剂/高盐缓冲液中选择性溶解，然后通过梯度筛吸阴离子和阳离子交换色谱，羟基磷灰石色谱，凝胶过滤和蔗糖梯度速度沉降，实现了42倍的富集超过起始膜。当通过NaDodSO 4/PAGE检查时，纯化的酶由表观分子量为25、23、22、21、18和12 kDa的六种多肽的复合物组成。基于其对内切糖苷酶H的敏感性和结合伴刀豆球蛋白A的能力，22-和23-kDa亚基被证明是糖蛋白。我们认为，只有一个亚基的这种复合物进行信号肽切割。化学计量的其他亚基的结构关联可能反映了它们在跨微粒体膜的链易位中的需要。
We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved post-translational assay. A 42-fold enrichment over starting membranes was achieved by selective solubilization in nonionic detergent/high-salt buffer followed by gradient sievorptive anion and cation exchange chromatography, hydroxylapatite chromatography, gel filtration, and sucrose gradient velocity sedimentation. When examined by NaDodSO4/PAGE, the purified enzyme consisted of a complex of six polypeptides with apparent molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa subunits were shown to be glycoproteins based on their sensitivity to endoglycosidase H and their ability to bind concanavalin A. We suggest that only one subunit of this complex carries out signal peptide cleavage. The structural association of the other subunits in stoichiometric amounts may reflect their requirement in chain translocation across the microsomal membrane.