Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats.
The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum).
Nestlé Research Center, Nutrition Department, Nestec Ltd., Vers-chez-les-Blanc, Lausanne, Switzerland. magali.faure@rdls.nestle.com
Magali
M
Faure
Denis
D
Moënnoz
Franck
F
Montigon
Laurent B
LB
Fay
Denis
D
Breuillé
Paul A
PA
Finot
Olivier
O
Ballèvre
Julio
J
Boza
D000818:Animals; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D007583:Jejunum / Q000378:metabolism; D008297:Male; D009077:Mucins / Q000096:biosynthesis* / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014633:Valine / Q000032:analysis / Q000378:metabolism
D002250:Carbon Radioisotopes; D009077:Mucins; D014633:Valine