A METHOD FOR ASSAYING INTESTINAL BRUSH-BORDER SUCRASE IN AN INTACT INTESTINAL PREPARATION

Small intestinal brush-border hydrolases usually are assayed in intest inal mucosal homogenates resuspended in solutions of unphysiological i onic composition. Thus, extrapolation of measured V-max values (maxima l reaction rates at high substrate concentrations) to in vivo conditio ns, 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 tran sport inhibitor phlorizin. The coefficient of variation of V-max is 16 % for sleeves from the same mouse and 8% for mean values from differen t mice. Sleeve sucrase activity is abolished by the inhibitor castanos permine. Activity in sleeves and homogenates proves to be the same whe n measured under identical solution conditions, but variations in assa y conditions cause large activity changes from values measured in phys iological solutions.