Soukri A, Valverde F, Hafid N, Elkebbaj M S, Serrano A
Laboratoire de Biochimie, Biologie Cellulaire et Moléculaire, Faculté des Sciences-Ain Chock, Casablanca, Morocco.
Gene. 1996 Nov 28;181(1-2):139-45. doi: 10.1016/s0378-1119(96)00494-5.
A cDNA clone which contains the near-complete open reading frame (ORF) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was obtained by screening a muscle cDNA library of jerboa (Jaculus orientalis), a true hibernating rodent, with a PCR-amplified 0.5-kb genomic DNA probe from an internal region of the gene. The 1.1-kb cDNA clone consists of a 927-bp ORF which codifies for 309 aa, about 93% of the original GapC gene encoding the 36-kDa protein, and a 3'-noncoding region of 167 bp. The full-length aa sequence of GAPDH was achieved by sequencing the N-terminal region of the purified protein completing the missing part in the cDNA clone. Both nt and aa sequences exhibit a high degree of homology to other mammalian GAPDHs. The expression of the GapC gene was studied in skeletal muscle and liver of euthermic and hibernating jerboas both on the mRNA level by Northern blot hybridization using the cDNA clone as a probe and on the protein level by Western blot immunodetection using an antibody raised against muscle GAPDH. A clear decrease (about threefold) in the amount of GapC mRNA, a single 1.2-kb transcript, was observed in muscle of hibernating jerboa when compared with the same tissue from the euthermic animal. This mRNA level decrease directly correlates with a reduction in both protein amount and specific activity in crude protein extracts. In contrast, both GAPDH protein and GapC mRNA levels remained unchanged in liver from euthermic and hibernating jerboas although the enzymatic activity was also about threefold lower in the hibernating tissue. These result, together with previous data obtained from protein studies [Soukri et al. (1995) Biochim. Biophys. Acta 1243, 161-168 and (1996) 1292, 177-187] indicate that jerboa GAPDH is regulated by different mechanisms during hibernation in these tissues, that is, at transcriptional level in muscle and at posttranslational level in liver. The reduced GAPDH activity should result in both cases in a decrease of the glycolytic flux that would eventually contribute to the dramatic metabolic depression of this dormant state.
通过用从该基因内部区域PCR扩增得到的0.5 kb基因组DNA探针筛选长爪沙鼠（一种真正的冬眠啮齿动物）的肌肉cDNA文库，获得了一个包含编码甘油醛-3-磷酸脱氢酶（GAPDH，EC 1.2.1.12）的近完整开放阅读框（ORF）的cDNA克隆。这个1.1 kb的cDNA克隆由一个927 bp的ORF组成，该ORF编码309个氨基酸，约占编码36 kDa蛋白质的原始GapC基因的93%，以及一个167 bp的3'非编码区。通过对纯化蛋白的N端区域进行测序，完成了cDNA克隆中缺失的部分，从而获得了GAPDH的全长氨基酸序列。核苷酸和氨基酸序列与其他哺乳动物的GAPDH都具有高度同源性。使用该cDNA克隆作为探针，通过Northern印迹杂交在mRNA水平上，以及使用针对肌肉GAPDH产生的抗体通过Western印迹免疫检测在蛋白质水平上，研究了正常体温和冬眠长爪沙鼠的骨骼肌和肝脏中GapC基因的表达。与来自正常体温动物的相同组织相比，在冬眠长爪沙鼠的肌肉中观察到GapC mRNA（单一的1.2 kb转录本）的量明显减少（约三倍）。这种mRNA水平的降低与粗蛋白提取物中蛋白质含量和比活性的降低直接相关。相反，正常体温和冬眠长爪沙鼠肝脏中的GAPDH蛋白和GapC mRNA水平均保持不变，尽管冬眠组织中的酶活性也降低了约三倍。这些结果，连同先前从蛋白质研究中获得的数据[Soukri等人（1995年）《生物化学与生物物理学报》1243，161 - 168和（1996年）1292，177 - 187]表明，长爪沙鼠GAPDH在这些组织的冬眠过程中受到不同机制的调节，即在肌肉中是转录水平调节，在肝脏中是翻译后水平调节。在这两种情况下，GAPDH活性的降低都应导致糖酵解通量的减少，这最终将导致这种休眠状态下显著的代谢抑制。
