Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms.

The research investigated the purification of neutral alpha-D-glucosidase, an enzyme from horse kidney membranes. The enzyme was purified using Emulphogene BC 720, observed through crossed immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and found to have amphipathic properties. Also, the enzyme responded differently to treatments with papain and trypsin.

Enzyme Purification and Observation

• The researchers initially solubilized neutral alpha-D-glucosidase from horse kidney brush-border membranes using Emulphogene BC 720.
• The enzyme was purified by an affinity chromatography technique, which resulted in an enzyme preparation that was 390-fold purified and free from other known microvillus hydrolases.
• The purified enzyme was then examined using crossed immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These tests showed that the enzyme preparation had a single band of precipitate, indicating it was homogenous.

Amphipathic Nature of the Enzyme

• Based on the analysis from charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the researchers concluded that the purified form of the enzyme exhibited amphipathic properties, meaning it had both hydrophobic and hydrophilic regions.

Enzyme Reaction to Papain and Trypsin

• The researchers further treated the enzyme with papain and trypsin, which both resulted in varied enzymatic forms.
• Papain treatment on the brush-border membrane vesicles or the detergent-solubilized form of the enzyme resulted in an enzymatic form that lacked amphipathic properties.
• Trypsin treatment resulted in two forms of the enzyme. The first and majority form maintained its amphipathic properties, while the second form exhibited the same properties as the papain-treated enzyme.
• An interesting observation was made when trypsin was used to solubilize the enzyme from the brush-border membrane vesicles, only a small amount was released and the enzyme lacked amphipathic properties. This was interpreted as the trypsin attack site on the detergent form of the enzyme had either a poor affinity for, or obstructed access to, the protease. In contrast, the proteolytic site for papain was consistently accessible.