Wehren A, Meyer H E, Sobek A, Kloetzel P M, Dahlmann B
Diabetes Forschunginstitut, Düsseldorf, Germany.
Biol Chem. 1996 Jul-Aug;377(7-8):497-503.
Proteasomes, the major catalysts of the non-lysosomal proteolytic pathway in eukaryotic cells, were analyzed for their content of phosphoamino acids using polyacrylamide gel electrophoresis and subsequent detection on Western blots by phosphoamino acid antibodies. No specific binding to proteasome subunits was observed with phosphoserine or phosphothreonine antibodies, whereas phosphotyrosine antibodies were bound by a single proteasome subunit, which was identified in rat as well as in human proteasomes as subunit C7-1. Since dephosphorylation of the subunit by phosphatases was not possible, analysis of phosphoamino acid content of all proteasome subunits was performed using another method. All proteasome subunits were isolated from 2D-polyacrylamide gels and subjected to partial acid hydrolysis. Phosphoamino acids were subsequently detected by capillary electrophoresis after their derivatization with phenylisothiocyanate. This analysis revealed no phosphorylated amino acid in subunit C7-1, however, subunit C3 contained phosphotyrosine and phosphothreonine, and phosphoserine was detected in subunits zeta, C5, C8 and C9.
蛋白酶体是真核细胞中非溶酶体蛋白水解途径的主要催化剂，利用聚丙烯酰胺凝胶电泳对其磷酸化氨基酸含量进行了分析，并随后通过磷酸化氨基酸抗体在蛋白质印迹上进行检测。用磷酸丝氨酸或磷酸苏氨酸抗体未观察到与蛋白酶体亚基的特异性结合，而磷酸酪氨酸抗体与单个蛋白酶体亚基结合，在大鼠和人类蛋白酶体中该亚基均被鉴定为C7-1亚基。由于无法通过磷酸酶对该亚基进行去磷酸化，因此使用另一种方法对所有蛋白酶体亚基的磷酸化氨基酸含量进行了分析。从二维聚丙烯酰胺凝胶中分离出所有蛋白酶体亚基，并进行部分酸水解。磷酸化氨基酸在用异硫氰酸苯酯衍生后，随后通过毛细管电泳进行检测。该分析显示C7-1亚基中没有磷酸化氨基酸，然而，C3亚基含有磷酸酪氨酸和磷酸苏氨酸，并且在ζ、C5、C8和C9亚基中检测到了磷酸丝氨酸。
