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Microsomal chitinase activity from Candida albicans
K Dickinson1, V Keer, C A Hitchcock
1Department of Microbiology, University of Leeds, U.K.
Abstract:
Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.
Insights
Microsomal chitinase in Candida albicans yeast cells exhibits zymogenic properties and shares characteristics with its cytosolic counterpart. This enzyme
Area of Science:
- Biochemistry
- Mycology
- Enzymology
Background:
- Chitinase (E.C. 3.2.1.14) plays a role in fungal cell wall biology.
- Understanding chitinase localization and properties is crucial for targeting fungal pathogens.
- Candida albicans is a significant opportunistic fungal pathogen.
Purpose of the Study:
- To characterize microsomal chitinase activity in Candida albicans.
- To investigate the properties and localization of microsomal chitinase.
- To explore the potential of microsomal chitinase as an antifungal target.
Main Methods:
- Enzyme activity assays on microsomal fractions from Candida albicans.
- Washing protocols to assess enzyme activation and stability.
- Pre-incubation with trypsin to evaluate zymogenic properties.
- Determination of kinetic parameters (Km) and inhibition constants (Ki).
- Solubilization studies using detergents.
- Density gradient centrifugation for enzyme localization.
Main Results:
- Microsomal chitinase activity was low in unwashed fractions but increased with washing and trypsin activation.
- Microsomal chitinase exhibited kinetic and inhibition properties similar to cytosolic chitinase.
- Detergents differentially solubilized and activated the enzyme.
- Chitinase was distributed broadly within the microsomes, not exclusively bound to one component.
Conclusions:
- Microsomal chitinase in Candida albicans is likely zymogenic and shares properties with cytosolic chitinase.
- The enzyme's distribution suggests it is not restricted to a single membrane component.
- Microsomal chitinase represents a potential target for developing specific antifungal agents.