Quan R, Santiago N A, Tsuboi K K, Gray G M
Department of Medicine (Gastroenterology), Stanford University School of Medicine, California 94305.
J Biol Chem. 1990 Sep 15;265(26):15882-8.
The regulatory mechanism of decline in catalytic activity for intestinal lactase (lactase-phlorizin hydrolase, beta-galactosidase) as mammals mature has not been defined. Solubilized intestinal brush-border membranes from adult male rats (greater than 4 months of age, 200-400 g) were examined by high performance liquid Zorbax GF-450 chromatography, subjected to denaturing acrylamide electrophoresis, blotted to nitrocellulose, and identified by specific polyvalent anti-lactase. Three major species were present within the 235-kDa active lactase peak (225, 130, and 100 kDa). The 100-kDa moiety was also prominent in the approximately 300-kDa region of the GF-450 effluent, suggesting it is a catalytically inactive oligomer. In vivo synthesis and assembly of lactase by intraintestinal pulse [( 35S]methionine, 5 min) and chase (15-120 min) revealed rapid (15 min of chase; maximum, 60 min) intracellular synthesis in the endoplasmic reticulum-Golgi fraction of multiple species (64, 100, 130, 175, and 225 kDa). The 64-kDa species disappeared from the intracellular membrane compartment and was not transferred to the brush-border surface. The 175-kDa moiety appeared to be processed to the 225-kDa unit prior to relocation to the surface membrane. By 120 min, the 100-kDa species became the predominant (approximately 60%) radiolabeled unit in both endoplasmic reticulum-Golgi and brush border. In the adult rat, lactase is assembled in multiple molecular forms that are differentially processed: (a) intracellular degradation (64-kDa unit) or (b) transfer to the brush-border surface as catalytically active (225 and 130 kDa) or inactive (100 kDa) species. Although substantial synthesis of lactase proteins prevails, major changes in processing appear to serve as an important regulatory mechanism producing the maturational decline of catalytic activity. The accompanying article (Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889-15893) extends our studies to synthesis and assembly during the neonatal period of maturation.
随着哺乳动物的成熟，肠道乳糖酶（乳糖 - 根皮苷水解酶，β - 半乳糖苷酶）催化活性下降的调节机制尚未明确。通过高效液相Zorbax GF - 450色谱法对成年雄性大鼠（年龄大于4个月，体重200 - 400克）的溶解肠道刷状缘膜进行检测，然后进行变性丙烯酰胺电泳，转印至硝酸纤维素膜上，并用特异性多价抗乳糖酶进行鉴定。在235 kDa的活性乳糖酶峰内存在三种主要成分（225、130和100 kDa）。100 kDa的部分在GF - 450流出物的约300 kDa区域也很突出，表明它是一种无催化活性的寡聚体。通过肠内脉冲[（35S]甲硫氨酸，5分钟）和追踪（15 - 120分钟）对乳糖酶进行体内合成和组装，结果显示在多个物种（64、100、130、175和225 kDa）的内质网 - 高尔基体部分中存在快速的（追踪15分钟；最大值，60分钟）细胞内合成。64 kDa的物种从细胞内膜区室消失，并且没有转移到刷状缘表面。175 kDa的部分似乎在重新定位到表面膜之前被加工成225 kDa的单元。到120分钟时，100 kDa的物种在内质网 - 高尔基体和刷状缘中均成为主要的（约60%）放射性标记单元。在成年大鼠中，乳糖酶以多种分子形式组装，这些分子形式经过不同的加工处理：（a）细胞内降解（64 kDa单元）或（b）作为具有催化活性的（225和130 kDa）或无活性的（100 kDa）物种转移到刷状缘表面。尽管乳糖酶蛋白的合成大量存在，但加工过程中的主要变化似乎是导致催化活性成熟性下降的重要调节机制。随附的文章（Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889 - 15893）将我们的研究扩展到成熟新生儿期的合成和组装。
