A method for assaying intestinal brush-border sucrase in an intact intestinal preparation

Small intestinal brush-border hydrolases usually are assayed in intestinal mucosal homogenates resuspended in solutions of unphysiological ionic composition. Thus, extrapolation of measured V(max) values (maximal reaction rates at high substrate concentrations) to in vivo conditions, hence comparison with physiological substrate loads, is uncertain. We therefore have developed a sucrase assay in an intact preparation of mouse small intestine, an everted intestinal sleeve incubated in a physiological Ringer’s solution. As in homogenate studies, sucrase is assayed by glucose production measured colorimetrically, but uptake of liberated glucose into the intestinal sleeve is prevented by the transport inhibitor phlorizin. The coefficient of variation of V(max) is 16% for sleeves from the same mouse and 8% for mean values from different mice. Sleeve sucrase activity is abolished by the inhibitor castanospermine. Activity in sleeves and homogenates proves to be the same when measured under identical solution conditions, but variations in assay conditions cause large activity changes from values measured in physiological solutions.

小肠刷状缘水解酶（Small intestinal brush-border hydrolases）通常在以非生理离子组成的溶液重悬的小肠黏膜匀浆（intestinal mucosal homogenates）中进行活性测定。因此，将测得的V(max)（高底物浓度下的最大反应速率）外推至体内（in vivo）条件，进而与生理底物负荷（physiological substrate loads）进行比较，其结果往往缺乏可靠性。为此，我们开发了一种针对小鼠小肠完整制备物的蔗糖酶（sucrase）测定方案：将外翻肠囊（everted intestinal sleeve）置于生理林格液（physiological Ringer’s solution）中孵育。与匀浆类实验的测定逻辑一致，该蔗糖酶活性通过比色法（colorimetrically）检测的葡萄糖生成量来量化，但借助转运抑制剂根皮苷（phlorizin）可阻断释放的葡萄糖被外翻肠囊摄取。同一只小鼠的外翻肠囊的V(max)变异系数（coefficient of variation）为16%，不同小鼠的肠囊均值的变异系数为8%。抑制剂卡斯托斯巴明（castanospermine）可完全消除外翻肠囊的蔗糖酶活性。在相同溶液条件下开展检测时，外翻肠囊与小肠黏膜匀浆的酶活结果一致，但测定条件的变化会导致生理溶液中测得的酶活数值出现大幅波动。