Sharp A H, Snyder S H, Nigam S K
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1992 Apr 15;267(11):7444-9.
Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.
我们使用针对从大鼠小脑纯化的肌醇1,4,5 - 三磷酸受体（IP3R）制备的多克隆抗血清，研究了IP3R在犬胰腺匀浆中的亚细胞分布。IP3R主要存在于平滑微粒体部分（低密度）、先前显示由高度纯化的粗面内质网（RER）囊泡组成的（高密度）粗面微粒体（RM）部分，以及程度小得多地存在于不包含RER或质膜标志物的中等密度微粒体部分。当RM部分在蔗糖梯度上进行等密度离心时，IP3R在高蔗糖密度下达到平衡。当通过用嘌呤霉素/高盐处理从RM部分提取核糖体时，IP3R在明显更轻的蔗糖密度下达到平衡。这种密度变化表明存在于RM部分的IP3R与RER相关联。由于大量的IP3R分馏到平滑微粒体部分（其中除其他“平滑”膜外还包含质膜）以及存在于也富含质膜的核沉淀中的大量IP3R，我们研究了IP3R可能存在于质膜中的可能性。对来自大鼠肝脏的粗质膜沉淀的进一步亚分级显示，IP3R与质膜标志物共同富集，并强烈提示IP3R与质膜相关联。关于为什么在多个生化和形态学上不同的膜部分中发现相同受体的问题，根据RER亚区室化和IP3R亚型的可能性进行了讨论。胰腺中IP3R的分级模式与先前报道的钙（Ca2 +）结合蛋白和细胞内钙 - ATP酶的分级模式（Nigam, S. K.和Towers, T.（1990）J. Cell Biol. 111, 197 - 200）有显著差异，这引发了关于后一种蛋白质与IP3敏感Ca2 +池之间联系的问题。然而，尽管分级模式不同，但所有这些蛋白质显然都与RER相关联。
