Stability of enterocytes and certain enzymatic activities in suspensions of cells from the villous tip to the crypt of Lieberkühn of the mouse small intestine.
A series of studies in this laboratory have focused on how an indigenous microbiota influences the activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in the enterocytes of the upper small intestine of mice. To draw conclusions about the role of the microflora in determining levels of enzymatic activity, we found it necessary to develop a procedure by which cell suspensions could be obtained containing enterocytes isolated sequentially from the villous tip to the crypt of Lieberkühn. The procedure was modified from the one developed for rats by Weiser (J. Biol. Chem. 248:2536-2541, 1973), involved a minimum number of interfering factors (e.g., proteolytic enzymes and mechanical agitation), and worked reproducibly for mice. During development of the procedure, some variables affecting the assays of the enzymes known to be present in enterocytes were also explored. Rods to which were tied everted segments of gut were incubated in a series of tubes containing a solution of EDTA the concentration of which was changed from 1.5 to 5.0 mM, thus giving a greater yield of enterocytes at every step. The cells incubating in the chelating solution were most stable when 0.23 M sucrose was included in the EDTA solutions. Success in assaying enzymatic activities in the cell suspensions depended on (i) how the cells were isolated, (ii) the assay procedure for thymidine kinase, and (iii) whether cellular suspensions or extracts were assayed.
Whitt
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DD
Department of Microbiology, University of Illinois, Urbana 61801.
Savage
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DC
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D005260:Female; D005856:Germ-Free Life; D007421:Intestine, Small / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008871:Microvilli / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D013696:Temperature; D013937:Thymidine Kinase / Q000032:analysis*
D011506:Proteins; D013937:Thymidine Kinase; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases