Barone L M, Mu H H, Shih C J, Kashlan K B, Wasserman B P
Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520, USA.
Plant Physiol. 1998 Sep;118(1):315-22. doi: 10.1104/pp.118.1.315.
Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.
来自红甜菜（Beta vulgaris L.）贮藏组织的质膜囊泡含有两种主要的内在蛋白，分子量分别为31 kD和27 kD（X. Qi、C.Y Tai、B.P. Wasserman [1995] Plant Physiol 108: 387 - 392）。在本研究中，使用亲和纯化的抗体来研究它们的定位和生化特性。两种质膜内在蛋白（PMIP）亚组在蔗糖梯度中的分配相同；然而，在检测多聚体形成和有限蛋白酶解时，它们各自表现出不同的特性。通过在组织匀浆和囊泡分离过程中加入各种巯基试剂，研究了每种PMIP种类形成二硫键连接聚集体的倾向。在没有二硫苏糖醇和巯基试剂的情况下，PMIP27产生单体和聚集物的混合物。相比之下，产生PMIP31的单体种类需要加入二硫苏糖醇、碘乙酸或N - 乙基马来酰亚胺。未检测到PMIP31和PMIP27亚组之间的混合二硫键连接的异二聚体。基于用胰蛋白酶对外翻囊泡进行的定向蛋白酶解以及对源自编码PMIP27的基因预测氨基酸序列的亲水性分析，建立了PMIP27的拓扑模型。从PMIP27的蛋白酶解中鉴定出两个暴露的胰蛋白酶切割位点，每个位点都与先前在PMIP31的表面环C中鉴定的单个暴露位点不同。尽管PMIP31和PMIP27种类都含有似乎存在于单个囊泡群体中的整合蛋白，但这些结果表明，每个PMIP亚组对膜扰动的反应不同。
