Ma Z, Ramanadham S, Kempe K, Hu Z, Ladenson J, Turk J
Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biochim Biophys Acta. 1996 Aug 14;1308(2):151-63. doi: 10.1016/0167-4781(96)00088-7.
Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet beta-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in beta-cells is of interest because that isoform is thought to govern glycolytic oscillations and to interact with a metabolically activated beta-cell phospholipase A2 enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of beta-cells prepared by fluorescence-activated cell sorting (FACS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet alpha-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FACS-purified islet beta-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.
磷酸果糖激酶（PFK）在调节糖酵解通量中起关键作用，哺乳动物的该酶是一种四聚体。三种单体同工型由不同基因编码，在特定组织中差异表达，并根据它们在其中最丰富的组织命名（A，肌肉；B，肝脏；C，大脑）。胰腺胰岛中葡萄糖诱导的胰岛素分泌需要葡萄糖转运到胰岛β细胞并进行糖酵解代谢。关于胰岛PFK同工酶知之甚少，但PFK - A在β细胞中表达的可能性令人感兴趣，因为该同工型被认为控制糖酵解振荡并与代谢激活的β细胞磷脂酶A2酶相互作用。我们使用从大鼠胰岛RNA产生的PCR产物作为探针，该引物由人PFK - A序列设计，我们从大鼠胰岛cDNA文库中克隆了全长PFK - A cDNA。大鼠PFK - A推导的氨基酸序列与人类PFK - A的序列有96%的同一性，并且认为参与底物或变构效应物结合的所有残基在两个序列之间是保守的。大鼠PFK - A氨基酸序列与大鼠PFK - B和大鼠PFK - C分别有69%和68%的同一性，并且在三种同工型中观察到参与变构效应物结合的残基存在差异。表达为谷胱甘肽 - S转移酶融合蛋白的大鼠PFK - A被针对PFK - A序列中的肽产生的抗体识别。通过逆转录 - 聚合酶链反应（RT - PCR）在大鼠胰岛、通过荧光激活细胞分选（FACS）制备的纯化β细胞群体以及RIN - m5F胰岛素瘤细胞中检测PFK同工型mRNA种类的表达，所有这些都表达了PFK - A、 - B和 - C同工型的mRNA种类。PFK - A mRNA在富含胰岛α细胞的群体中表达水平要低得多。白细胞介素 - 1损害胰岛葡萄糖代谢和胰岛素分泌，并被发现诱导胰岛PFK - A mRNA表达的特异性下降。这些发现确定了大鼠PFK - A的序列，证明它在FACS纯化的胰岛β细胞中表达，并表明其表达受影响胰岛素分泌的细胞因子调节。
