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Hydrophobic binding domains of rat intestinal maltase-glucoamylase
Abstract:
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.
Insights
Rat intestinal maltase-glucoamylase anchors to the membrane via a 14 kDa apolar peptide. This peptide likely forms the terminal anchor of the enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Rat intestinal microvillus maltase-glucoamylase is a key digestive enzyme.
- Understanding its membrane anchoring is crucial for its function.
Purpose of the Study:
- To elucidate the membrane anchoring mechanism of rat intestinal maltase-glucoamylase.
- To identify the specific peptide segment responsible for bilayer insertion.
Main Methods:
- Enzyme isolation, purification, and incorporation into phospholipid vesicles.
- Radioactive labeling with [125I]TID and photolysis.
- Enzymatic digestion with papain and separation of components.
- Chromatographic and electrophoretic analyses (Sephadex LH-60, SDS-PAGE).
Main Results:
- A 14 kDa apolar peptide was identified as the membrane anchor.
- This peptide remained associated with vesicles after papain digestion.
- SDS-PAGE revealed a 26-28 kDa doublet, suggesting a dimer of the anchor peptide.
- The 145 kDa subunit was processed to 130 kDa upon solubilization, indicating cleavage of the anchor.
Conclusions:
- Maltase-glucoamylase inserts into the phospholipid bilayer via a specific 14 kDa apolar peptide anchor.
- This anchor segment is likely a terminal part of the enzyme's subunit.
- Papain digestion releases the active enzyme head from the membrane-bound anchor.