Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms.
Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.
Giudicelli
J
J
Boudouard
M
M
Delqué
P
P
Vannier
C
C
Sudaka
P
P
D000818:Animals; D002850:Chromatography, Gel; D000939:Epitopes / Q000032:analysis; D005959:Glucosidases / Q000302:isolation & purification*; D006736:Horses; D007123:Immunoelectrophoresis, Two-Dimensional; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011092:Polyethylene Glycols; D012995:Solubility; D000520:alpha-Glucosidases / Q000302:isolation & purification*
D000939:Epitopes; C007590:emulphogene BC 720; D011092:Polyethylene Glycols; D005959:Glucosidases; D000520:alpha-Glucosidases; D010447:Peptide Hydrolases