Kumagai C, Kadowaki T, Kitagawa Y
Graduate Program for Biochemical Regulation, Graduate School of Agricultural Sciences, Chikusa-ku, Nagoya, Japan.
FEBS Lett. 1997 Jul 21;412(1):211-6. doi: 10.1016/s0014-5793(97)00780-1.
Assembly of Drosophila laminin alpha, beta and gamma chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain-specific antibodies followed by two dimensional electrophoresis in which non-reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric beta (or gamma) with anti-gamma (or -beta) antibody revealed that beta and gamma form stable dimer before they are disulfide-bonded to each other. In contrast, alpha associates with neither monomeric beta, monomeric gamma nor betagamma dimer without disulfide-bonding but only with disulfide-bonded betagamma dimer to form alpha betagamma trimers. These results thus demonstrated that the interchain disulfide-boding between beta and gamma is essential for alpha to form alpha betagamma trimer. We also found that the alpha betagamma trimer can be secreted with alpha chain either disulfide-bonded or not bonded to the disulfide-bonded betagamma dimer.
通过用链特异性抗体对经代谢放射性标记的Kc 167细胞裂解物进行免疫沉淀，然后进行二维电泳（其中将非还原和还原SDS凝胶电泳相结合），分析了果蝇层粘连蛋白α、β和γ链的组装情况。用抗γ（或-β）抗体沉淀单体β（或γ）表明，β和γ在彼此形成二硫键之前先形成稳定的二聚体。相比之下，α在没有二硫键结合的情况下既不与单体β、单体γ结合，也不与βγ二聚体结合，而是仅与形成二硫键的βγ二聚体结合形成αβγ三聚体。因此，这些结果表明，β和γ之间的链间二硫键结合对于α形成αβγ三聚体至关重要。我们还发现，αβγ三聚体可以在α链与形成二硫键的βγ二聚体结合或未结合的情况下分泌。
