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 Vmax 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 Vmax 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.
Lee
E A
EA
Physiology Department, University of California Medical School, Los Angeles, CA 90095-1751, USA.
Weiss
S L
SL
Lam
M
M
Torres
R
R
Diamond
J
J
D000818:Animals; D001692:Biological Transport; D005260:Female; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000378:metabolism; D007421:Intestine, Small; D007700:Kinetics; D051379:Mice; D008871:Microvilli / Q000201:enzymology* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D013393:Sucrase / Q000032:analysis / Q000378:metabolism*
D009004:Monosaccharide Transport Proteins; D013393:Sucrase; D005947:Glucose