Studies on bovine spleen cathepsin D.

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cathepsin D is a key aspartic protease involved in various cellular processes.
• Previous purification methods often resulted in degradation of the enzyme.
• Understanding the structural properties and substrate specificity of cathepsin D is crucial for its functional characterization.

Purpose of the Study:

• To develop an effective purification strategy for obtaining intact cathepsin D.
• To characterize the structural features of purified cathepsin D using biophysical techniques.
• To investigate the substrate specificity and inhibitor binding properties of cathepsin D.

Main Methods:

• Purification of cathepsin D utilizing affinity chromatography.
• Determination of molecular weight and N-terminal amino acid sequencing.
• Circular Dichroism (CD) spectroscopy for structural analysis.
• Enzyme specificity assays using synthetic substrates.
• Pepstatin binding studies at varying pH conditions.

Main Results:

• A robust purification method yielded a single polypeptide chain of 42,000 daltons, with glycine as the N-terminal amino acid.
• CD measurements indicated a predominantly unordered structure, with approximately 26% beta-structure and 5% alpha-helix.
• Pepstatin binding induced significant changes in the CD spectrum between 250-300 nm, with no observed interaction above pH 7.5.

Conclusions:

• Affinity chromatography is an effective method for purifying functional cathepsin D, preserving its single-chain integrity.
• Cathepsin D possesses a largely unstructured conformation with specific secondary structure elements.
• Pepstatin inhibition of cathepsin D is sensitive to pH, suggesting a role for protonation states in inhibitor binding.