Physicochemical characterization of human intestinal lactase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Human lactase is crucial for lactose digestion.
• Understanding its properties aids in diagnosing and treating lactose intolerance.
• Purification of active enzymes is essential for detailed biochemical analysis.

Purpose of the Study:

• To isolate and purify human lactase from small-intestinal brush-border membranes.
• To characterize the biochemical and physical properties of purified human lactase.
• To develop antibodies for immunoaffinity purification and radioimmunoassay of lactase.

Main Methods:

• Multi-step chromatography including concanavalin A-Sepharose, Bio-Gel 1.5m, and chromatofocusing.
• SDS/polyacrylamide-gel electrophoresis and gel filtration for molecular weight determination.
• Raising antisera against purified lactase for immunoaffinity purification and radioimmunoassay development.

Main Results:

• Human lactase was purified 750-fold with a 1% yield.
• The purified enzyme showed homogeneity on SDS-PAGE (Mr ~170,000) but aggregated on gel filtration (Mr ~380,000).
• The enzyme exhibited optimal lactase activity at pH 6.0 and hydrolyzed various beta-galactosides and other substrates.

Conclusions:

• A highly purified and characterized human lactase enzyme was obtained.
• The developed antibodies are effective for immunoaffinity purification and sensitive radioimmunoassay of lactase.
• These findings provide tools for further research into lactase function and related disorders.