Hydrophobic binding domains of rat intestinal maltase-glucoamylase.
Rat intestinal microvillus maltase-glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Sephadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Sepharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with ethanol--formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26-28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase-glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.
D000345:Affinity Labels; D000818:Animals; D001389:Azirines / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D005959:Glucosidases / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology*; D007457:Iodine Radioisotopes; D008051:Lipid Bilayers / Q000378:metabolism; D046911:Macromolecular Substances; D010782:Photolysis; D051381:Rats; D000520:alpha-Glucosidases / Q000378:metabolism*
D000345:Affinity Labels; D001389:Azirines; D007457:Iodine Radioisotopes; D008051:Lipid Bilayers; D046911:Macromolecular Substances; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D005959:Glucosidases; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase