大鼠肠黏膜对α-D-葡萄糖基氟化物碳氟键的酶促水解。肠麦芽糖酶的定位。

Enzymic hydrolysis of the carbon-fluorine bond of alpha-D-glucosyl fluoride by rat intestinal mucosa. Localization of intestinal maltase.

作者信息

Barnett J E, Jarvis W T, Munday K A

出版信息

Biochem J. 1967 Jun;103(3):699-704. doi: 10.1042/bj1030699.

DOI:10.1042/bj1030699

PMID:6049382

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1270470/

Abstract

alpha-d-Glucosyl fluoride was hydrolysed by an extract of rat intestinal mucosa. The pH optimum was 6.6 and the K(m) 0.4mm at 20 degrees . Activity was assayed by release of either glucose or fluoride. 2. The alpha-d-glucosyl fluoride-hydrolase activity of the extract was associated with both mutarotase and alpha-d-glucosidase activities. 3. Tris (5mm) inhibited both the alpha-d-glucosidase and alpha-d-glucosyl fluoride-hydrolase activities by 55% but did not inhibit mutarotase. The K(i) of tris for both enzyme activities was 2mm. 4. The extract did not hydrolyse melibiose and lactose. Mutarotase used both alpha-d-glucose and beta-l-arabinose as substrates but the glucosyl fluoride-hydrolase activity did not extend to beta-l-arabinosyl fluoride. 5. The thermal stability of alpha-d-glucosidase and alpha-d-glucosyl fluoride hydrolase was identical. Mutarotase was more thermolabile. 6. A preparation of the brush border of intestinal epithelial cells contained both alpha-d-glucosyl fluoride-hydrolase and alpha-d-glucosidase activities. In each precipitate and washing the ratio of the two activities was the same. All the mutarotase activity was in the first supernatant. 7. Agidex, a fungal amyloglucosidase, cleaved glucosyl fluoride in addition to maltose. Tris inhibited both activities and in each case the K(i) was 3mm. 8. The probable identity of alpha-d-glucosyl fluoride hydrolase with alpha-d-glucosidase is discussed and a possible mechanism for the reaction suggested. 9. Incubation of intestinal slices with alpha-d-glucosyl fluoride led to complete hydrolysis in 30min. The glucose rapidly entered the cell and was metabolized, leaving the fluoride in the incubation medium. This constitutes a further proof that the intestinal alpha-d-glucosidase, although on the brush border, is located outside the site of active transport of sugars.

摘要

α-D-葡萄糖基氟化物被大鼠肠黏膜提取物水解。最适pH为6.6，20℃时的米氏常数（K(m)）为0.4mmol/L。通过葡萄糖或氟化物的释放来测定活性。2. 提取物的α-D-葡萄糖基氟化物水解酶活性与变旋酶和α-D-葡萄糖苷酶活性相关。3. 三羟甲基氨基甲烷（Tris，5mmol/L）使α-D-葡萄糖苷酶和α-D-葡萄糖基氟化物水解酶活性均抑制55%，但不抑制变旋酶。Tris对两种酶活性的抑制常数（K(i)）为2mmol/L。4. 提取物不水解蜜二糖和乳糖。变旋酶以α-D-葡萄糖和β-L-阿拉伯糖为底物，但葡萄糖基氟化物水解酶活性不作用于β-L-阿拉伯糖基氟化物。5. α-D-葡萄糖苷酶和α-D-葡萄糖基氟化物水解酶的热稳定性相同。变旋酶的热稳定性较差。6. 肠上皮细胞刷状缘制剂含有α-D-葡萄糖基氟化物水解酶和α-D-葡萄糖苷酶活性。在每次沉淀和洗涤中，两种活性的比例相同。所有变旋酶活性都在第一次上清液中。7. 真菌淀粉葡萄糖苷酶Agidex除了能切割麦芽糖外，还能切割葡萄糖基氟化物。Tris抑制这两种活性，在每种情况下抑制常数（K(i)）均为3mmol/L。8. 讨论了α-D-葡萄糖基氟化物水解酶与α-D-葡萄糖苷酶可能的同一性，并提出了反应的可能机制。9. 用α-D-葡萄糖基氟化物孵育肠切片30分钟后完全水解。葡萄糖迅速进入细胞并被代谢，氟化物留在孵育培养基中。这进一步证明，肠α-D-葡萄糖苷酶虽然位于刷状缘，但位于糖的主动转运位点之外。