Müller G, Korndörfer A, Kornak U, Malaisse W J
Hoechst Aktiengesellschaft Frankfurt am Main, Federal Republic of Germany.
Arch Biochem Biophys. 1994 Jan;308(1):8-23. doi: 10.1006/abbi.1994.1002.
The binding of hexo-/glucokinase and glycerol kinase to mitochondria via the channel forming protein, porin, in pancreatic islet beta-cells and adipocytes, was recently proposed to participate in nutritional signaling, glucose sensing, and the control of high-energy phosphate distribution and oxidative phosphorylation. In this study we demonstrate that polyclonal antisera against purified rat liver porin recognize unique proteins in rat pancreatic islets, adipocytes, and RINm5F cells, each with an apparent M(r) about 2000 smaller than that of liver porin. Immunoblotting of subcellular fractions, the purity of which has been controlled by the distribution of marker proteins, revealed the mitochondrial localization of the cross-reacting proteins. Their enrichment with a method used for the purification of porin proteins, the characteristic behavior during isoelectric focusing, and the specific binding of rat liver hexokinase and glycerol kinase to phospholipid vesicles containing the purified cross-reacting beta-cell or adipocyte proteins strongly suggest their identity with mitochondrial porin. The subtle differences in the apparent M(r) and charge heterogeneity raise the possibility of the existence of porin isoforms expressed in a tissue-specific manner. Anti-porin antisera coimmunoprecipitated hexo-/glucokinase from rat insulinoma cell (RINm5F) and adipocyte mitochondria as determined by subsequent immunoblotting of the immunoprecipitates with polyclonal antisera against yeast hexokinase and rat liver glucokinase, respectively. This indicates that some rat pancreatic glucokinase (54 kDa) and liver hexokinase (102 kDa), respectively, is bound to mitochondrial porin. The major portion of the bound fraction is released from mitochondria after treatment with glucose 6-phosphate. Incubation of RINm5F and fat cells with the insulin releasing sulfonylurea drug, glimepiride (20 nM and 5 microM, respectively) for 30 min reduces the amount of hexo-/glucokinase associated with mitochondria and porin to about 50-30%. The reduced kinase binding activity of porin is preserved after isolation of porin from glimepiride-treated cells, reconstitution into phospholipid vesicles and assaying for glucose 6-phosphate inhibitable binding of rat liver hexokinase. The sulfonylurea tolbutamide (20 microM and 5 mM) is significantly less effective. The sulfonylurea-induced inhibition of hexo-/glucokinase binding to mitochondrial porin does not require glucose metabolism or Ca2+ influx into the cells. These data suggest that the sulfonylurea glimepiride, which is thought to inhibit the ATP-regulated K(+)-channel in beta-cells, may have, in addition, an intracellular site of action in pancreatic islet and adipocyte cells at the level of regulation of gluco-/hexokinase binding to mitochondrial porin.
最近有人提出，己糖激酶/葡萄糖激酶和甘油激酶通过通道形成蛋白孔蛋白与胰腺胰岛β细胞和脂肪细胞中的线粒体结合，参与营养信号传导、葡萄糖感知以及高能磷酸分布和氧化磷酸化的控制。在本研究中，我们证明针对纯化的大鼠肝脏孔蛋白的多克隆抗血清可识别大鼠胰腺胰岛、脂肪细胞和RINm5F细胞中的独特蛋白质，每种蛋白质的表观分子量（M(r)）比肝脏孔蛋白小约2000。通过标记蛋白分布控制纯度的亚细胞组分免疫印迹显示，交叉反应蛋白定位于线粒体。它们通过用于纯化孔蛋白的方法富集，在等电聚焦过程中的特征行为，以及大鼠肝脏己糖激酶和甘油激酶与含有纯化的交叉反应β细胞或脂肪细胞蛋白的磷脂囊泡的特异性结合，强烈表明它们与线粒体孔蛋白相同。表观分子量和电荷异质性的细微差异增加了以组织特异性方式表达孔蛋白异构体的可能性。抗孔蛋白抗血清从大鼠胰岛素瘤细胞（RINm5F）和脂肪细胞线粒体中共免疫沉淀己糖激酶/葡萄糖激酶，随后分别用针对酵母己糖激酶和大鼠肝脏葡萄糖激酶的多克隆抗血清对免疫沉淀物进行免疫印迹测定。这表明大鼠胰腺葡萄糖激酶（54 kDa）和肝脏己糖激酶（102 kDa）的一部分分别与线粒体孔蛋白结合。用6-磷酸葡萄糖处理后，结合部分的主要部分从线粒体中释放出来。将RINm5F细胞和脂肪细胞分别与胰岛素释放磺脲类药物格列美脲（分别为20 nM和5 μM）孵育30分钟，可使与线粒体和孔蛋白相关的己糖激酶/葡萄糖激酶量减少至约50 - 30%。从格列美脲处理的细胞中分离孔蛋白，重新组装到磷脂囊泡中，并测定大鼠肝脏己糖激酶的6-磷酸葡萄糖抑制性结合后，孔蛋白降低的激酶结合活性得以保留。磺脲类药物甲苯磺丁脲（20 μM和5 mM）的效果明显较差。磺脲类药物诱导的己糖激酶/葡萄糖激酶与线粒体孔蛋白结合的抑制作用不需要葡萄糖代谢或Ca2+流入细胞。这些数据表明，被认为抑制β细胞中ATP调节的K(+)通道的磺脲类药物格列美脲，此外可能在胰腺胰岛和脂肪细胞的细胞内作用位点，在葡萄糖激酶/己糖激酶与线粒体孔蛋白结合的调节水平上发挥作用。
