Analysis of Structure and Function of Proteasome Derived from Rat Liver Microsome

我们首次从大鼠肝微粒体中分离到ERb蛋白酶体作为内质网膜结合的蛋白酶体，本研究旨在对ERb蛋白酶体的结构和酶学特性进行表征，并确定ERb蛋白酶体的特异性结构元件，我们澄清了以下内容：1。在反相HPLC上，除了20S蛋白酶体的β2和α5外，ERb还含有20S蛋白酶体中对应β2和α5的额外亚基，我们称之为B和L.2。二维凝胶电泳显示，由于磷酸化，B亚基比β2酸性更强，L亚基与α5基本相同，但迁移率略有不同。在MALDI-TOF/MS上，B的赖氨酸肽酶消化模式与β2.4相同。n端氨基酸分析表明α5含有Thr， L被阻断。结合MALDI-TOF/MS结果，推测L的n端序列为ac - met - ph - leu -Thr，其中Thr与α5的n端相同。总之，ERb包含两个不同的亚基（称为B和L），而这两个亚基在20S中是不存在的。B在20S中是β2的对应物，在极性上与β2不同，等电点L是a5的对应物，在α5的n端有3个附加残基，n端残基Met被乙酰化。这些结构特征可以解释ERb与内质网膜结合的特性。

This investigation was aimed to characterize structural and enzymic properties of the ERb proteasome which we have isolated, for the first time in the world, from rat liver microsome fraction as an ER membrane-bound proteasome and to determine the structural element specific in the ERb proteasome, and we clarified the followings :1. On reverse-phase HPLC, in addition to β2 and α5 of 20S proteasome, ERb was found to contain extra subunits corresponding to β2 and α5 in 20S proteasome, which we referred as to B and L.2. TWO dimension gel electrophoresis revealed that B subunit is more acidic than β2 due to phosphorylation, and L subunit was essentially the same as α5, although slight difference in mobility was observed.3. On MALDI-TOF/MS, lysylendopeptidase digest of B showed the same pattern as that of β2.4. N-terminal amino acid analysis revealed that α5 has Thr and that of L was blocked. Taken together with the results of MALDI-TOF/MS, The N-terminal sequence of L was suggested as Ac-Met-Phe-Leu-Thr-where Thr was the same as N-terminal of α5.In summary, ERb contains one each of two distinctive subunits (referred to as B and L) which is absent from 20S. B is a counterpart of β2 in 20S and distinctive from β 2 in polarity and isoelectric point L is a counterpart of a5, having 3 additional residues to N-terminal of α5 and the N-terminal residue Met was acetylated. These structural feature may explain the property of ERb to bind to ER membrane.