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 Vmaxvalues (maximal reaction rates at high substrate concentrations) to in vivo conditions, hence comparison w...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1998-03, Vol.95 (5), p.2111-2116
Main Authors: Lee, Eric A., Weiss, Stacey L., Lam, Mandy, Torres, Rosa, Diamond, Jared
Format: Article
Language:English
Subjects:
Animals
Biochemistry
Biological Sciences
Biological Transport
Body tissues
Digestive system
Enzymes
Facilitative glucose transport proteins
Female
Glucose - metabolism
In Vitro Techniques
Intestinal Mucosa - enzymology
Intestinal Mucosa - metabolism
Intestine, Small
Intestines
Kinetics
Metabolism
Mice
Microvilli - enzymology
Microvilli - metabolism
Monosaccharide Transport Proteins - metabolism
Reagents
Sleeves
Small intestine
Sucrase - analysis
Sucrase - metabolism
Tissue samples
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Summary:Small intestinal brush-border hydrolases usually are assayed in intestinal mucosal homogenates resuspended in solutions of unphysiological ionic composition. Thus, extrapolation of measured Vmaxvalues (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 Vmaxis 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.95.5.2111