Abstract
Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide.
Highlights
Theprimarytranslationproductupontranslocation was due to the addition of carbohydrate; treatment of MATERIALS ANDMETHODS the 220-kDa polypeptide with endo-8-N-acetylglucos- Guanidinium thiocyanate was from Fluka (Switzerland)
All other aminidase H increased its electrophoretic mobility to chemicals were of analytical grade. ~-[~~S]Methion(itnraenslation that of the 200-kDa polypeptide which was obtained grade, 1000Ci/mmol), ~-[~H]leuci(n1e47Ci/mmol), anddogpancreas in the absence of membraPnaers.tial N-terminal amino microsomes were from New England Nuclear or were prepared acacid sequence of a translation product lwabitehle[3dH] cording to the method described in Ref. 12 and treated with micro
Since sucrase-isomaltase is a glycoprotein, containing both N- and 0-glycosidically linked sugars [32, 33] and since it is known that dog pancreatic vesicles are capable of cotranslational N-glycosylation [12], we investigated whether the decrease in mobility of the segregated polypeptide chain was due to glycosylation
Summary
The abbreviations used are: Endo-H, endo-P-N-acetylglucosaminidase H; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; TID, 3-trifluoromethyl-3-(miodopheny1)diazirine. For 3H the filter squares were wetted in a countingvial with 10 pl ofHzOz, digested with 100 pl of NCS tissue solubilizer (Amersham Corp.) a t room temperature under agitation, and counted in 5 ml of a toluene-based scintillator. SDS-PAGE andFluorography Electrophoresis was performed using a discontinuous sulfate-borate system modified from Ref. 21, as described in Ref. 6. After drying the gels wereexposed a t -80 "C on Kodak X-Omat AR films
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