Rodríguez-Meléndez R, Pérez-Andrade M E, Díaz A, Deolarte A, Camacho-Arroyo I, Cicerón I, Ibarra I, Velázquez A
Unidad de Genética de la Nutrición, Instituto de Investigaciones Biomédicas UNAM and Instituto Nacional de Pediatría, Mexico City, Mexico.
Mol Genet Metab. 1999 Jan;66(1):16-23. doi: 10.1006/mgme.1998.2777.
Although the role of vitamins as prosthetic groups of enzymes is well known, their participation in the regulation of their genetic expression has been much less explored. We studied the effect of biotin on the genetic expression of rat liver mitochondrial carboxylases: pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC). Rats were made biotin-deficient and were sacrificed after 8 to 10 weeks, when deficiency manifestations began to appear. At this time, hepatic PCC activity was 20% of the control values or lower, and there was an abnormally high urinary excretion of 3-hydroxyisovaleric acid, a marker of biotin deficiency. Biotin was added to deficient primary cultured hepatocytes. It took at least 24 h after the addition of biotin for PCC to achieve control activity and biotinylation levels, whereas PC became active and fully biotinylated in the first hour. The enzyme's mass was assessed in liver homogenates from biotin-deficient rats and incubated with biotin to convert the apocarboxylases into holocarboylases, which were detected by streptavidin blots. The amount of PC was minimally affected by biotin deficiency, whereas that of the alpha subunits of PCC and of MCC decreased substantially in deficient livers, which likely explains the reactivation and rebiotinylation results. The expression of PC and alphaPCC was studied at the mRNA level by Northern blots and RT/PCR; no significant changes were observed in the deficient livers. These results suggest that biotin regulates the expression of the catabolic carboxylases (PCC and MCC), that this regulation occurs after the posttranscriptional level, and that pyruvate carboxylase, a key enzyme for gluconeogenesis, Krebs cycle anaplerosis, and fatty acid synthesis, is spared of this control.
尽管维生素作为酶的辅基的作用已广为人知，但它们在基因表达调控中的参与情况却鲜有人研究。我们研究了生物素对大鼠肝脏线粒体羧化酶基因表达的影响：丙酮酸羧化酶（PC）、丙酰辅酶A羧化酶（PCC）和3-甲基巴豆酰辅酶A羧化酶（MCC）。使大鼠缺乏生物素，在8至10周后出现缺乏症状时将其处死。此时，肝脏PCC活性为对照值的20%或更低，且生物素缺乏的标志物3-羟基异戊酸的尿排泄异常高。将生物素添加到缺乏生物素的原代培养肝细胞中。添加生物素后至少需要24小时，PCC才能达到对照活性和生物素化水平，而PC在第一小时内就变得活跃并完全生物素化。在来自生物素缺乏大鼠的肝脏匀浆中评估酶的质量，并与生物素一起孵育，将脱辅基羧化酶转化为全羧化酶，通过抗生物素蛋白印迹法进行检测。生物素缺乏对PC的量影响最小，而在缺乏生物素的肝脏中，PCC和MCC的α亚基的量显著减少，这可能解释了再激活和再生物素化的结果。通过Northern印迹和RT/PCR在mRNA水平研究了PC和αPCC的表达；在缺乏生物素的肝脏中未观察到显著变化。这些结果表明，生物素调节分解代谢羧化酶（PCC和MCC）的表达，这种调节发生在转录后水平，并且糖异生、三羧酸循环回补和脂肪酸合成的关键酶丙酮酸羧化酶不受这种调控。
