Date of Award
Doctor of Philosophy (PhD)
Dr. Brian J. Underdown
Intestinal disaccharidases are enzymes located in the apical membrane of enterocytes of the small intestine. These proteins are responsible for the breakdown of dietary carbohydrates. Despite the central importance of mouse genetics to modern biomedical research, little is known concerning the structure and regulation of the expression of murine intestinal disaccharidases. Moreover, limited information exists on the variations in molecular and genetic characteristics that the murine enzymes display between mouse strains.
In this thesis a partial characterization of murine intestinal disaccharidases expressed by seven different mouse strains was performed. Data obtained in these studies indicated the existence of two independent congenital deficiencies of murine intestinal disaccharidases. The two described murine deficiencies constitute the only cases in which normal and altered phenotypes are available in genetically related strains of animals. Therefore, these two deficiencies may serve as excellent models for the study of disaccharidase deficiencies.
One of the deficiencies consisting of relative low levels of sucrase activity, was found to be present in CBA/Ca mice and other two CBA/Ca-derived strains. These CBA/Ca mouse strains displayed aproximatedly 50% of intestinal sucrase activity compared to the other four mouse strains analysed. Experiments using papain-solubilized intestinal disaccharidases, molecular sieving HPLC, heat-inactivation and kinetic analysis of the sucrase activity, demonstrated that the deficiency was most likely caused by a structural alteration of the SucraseIsomaltase complex expressed by the CBA/Ca and derived mouse strains.
The second observed deficiency consisted in a virtual absence of y-Glucoamylase maltase activity exclusively in CBA/Ca mice. When analysed by heat-inactivation, molecular sieving HPLC and 2D-electrophoresis, the absence of y-Glucoamylase maltase activity was demonstrated to be caused by a lack of synthesis of y-Glucoamylase molecules in CBA/Ca mice.
The genetic background of the two disaccharidase deficiencies was analysed by cross-breeding CBA/Ca mice with CBA/J mice; the first strain displaying low sucrase and lack of y-GA expression and the second displaying normal phenotypes. The levels of activity of intestinal sucrase, maltase and palatinase expressed in the resulting F1 mice indicated that each of these deficiencies is most probably caused by a single defective gene, respectively. In addition, the defects were of a somatic nature and codominantly expressed with the respective normal phenotype.
Finally, data obtained from a partial molecular characterization of murine intestinal y-Gluccamylase indicated that the enzyme may be originally synthezised as a single polypeptide with approximated M.W. of 410 kDa. This polypeptide constitutes a precursor of four additional polypeptides with M.W. of 275, 260, 140 and 130 kDa, respectively, generated by proteolytic cleavage at two alternative points along the precursor molecule. The enzyme contains one single type of active site and variable proportion of N-linked carbohydrates. These data provide additional information on the inter-species structural variation of this enzyme and probable posttranslational processing experienced by y-Glucoamylase.
Quezada-Calvillo, Roberto, "Studies of Genetic Variation in Murine Intestinal Disaccharidases" (1994). Open Access Dissertations and Theses. Paper 1762.