Related Experiment Video
Updated: Aug 18, 2026
Purification and Reconstitution of TRPV1 for Spectroscopic Analysis
Published on: July 3, 2018
Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum
1Department of Pathology, University of California, San Francisco, 94143.
Abstract:
A proteinase of Mr 27,000 with a possible role in the metabolism and invasion of host tissues was purified from the conditioned medium of Trichophyton rubrum by concanavalin A and anion-exchange chromatography. Peaks of proteolytic activity were analyzed by substrate gel electrophoresis. The 27,000-Mr proteinase had a pH optimum of 8.0, a calcium dependence of 2 mM, and was inhibited by serine proteinase inhibitors, especially phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone. By polyacrylamide gel electrophoresis, the 27,000-Mr proteinase had a reduced molecular weight of 44,000 and reacted with [3H]diisopropyl fluorophosphate. The proteinase degraded azocoll, type III collagen, type IV procollagen, laminin, fibronectin, and the peptide substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (1,573 M-1 s-1) and t-butyloxy carbonyl-Ala-Ala-Leu-p-nitroanilide (1,614 M-1 s-1).
Insights
A novel proteinase from Trichophyton rubrum, crucial for host tissue invasion, was purified and characterized. This enzyme degrades key extracellular matrix components, highlighting its role in fungal pathogenesis.
Area of Science:
- Mycology
- Enzymology
- Biochemistry
Background:
- Trichophyton rubrum is a common dermatophyte responsible for superficial fungal infections.
- Fungal tissue invasion and metabolism are complex processes potentially involving secreted enzymes.
- Understanding these enzymes is key to developing targeted antifungal therapies.
Purpose of the Study:
- To purify and characterize a specific proteinase from Trichophyton rubrum.
- To investigate the enzyme's role in the degradation of host tissues.
- To elucidate the biochemical properties and substrate specificity of the purified proteinase.
Main Methods:
- Purification using concanavalin A and anion-exchange chromatography.
- Analysis of proteolytic activity via substrate gel electrophoresis.
- Biochemical characterization including pH optimum, cofactor dependence, and inhibitor profiling.
Main Results:
- A 27,000-Mr proteinase was isolated with optimal activity at pH 8.0 and requiring 2 mM calcium.
- The enzyme was identified as a serine proteinase, inhibited by phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone.
- Degradation of extracellular matrix proteins like collagen, laminin, and fibronectin was observed.
Conclusions:
- The purified 27,000-Mr proteinase plays a significant role in Trichophyton rubrum's ability to invade host tissues.
- Its substrate specificity suggests a mechanism for breaking down the extracellular matrix.
- This enzyme represents a potential therapeutic target for treating dermatophytosis.
