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Candida albicans nucleoside-diphosphate kinase: purification and characterization
1Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Archives of Biochemistry and Biophysics
|October 20, 1995
Summary
This study purified and characterized soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albicans, revealing a hexameric structure and key kinetic properties. The research also identified a similar membrane-associated NDP kinase, suggesting a shared functional role.
Area of Science:
- Biochemistry
- Enzymology
- Mycology
Background:
- Nucleoside-diphosphate kinase (NDP kinase) plays a crucial role in nucleotide metabolism.
- Understanding NDP kinase from pathogenic fungi like Candida albicans is important for potential therapeutic targets.
Purpose of the Study:
- To purify and characterize the soluble NDP kinase from Candida albicans.
- To investigate the kinetic and physical properties of the enzyme.
- To explore the potential existence and characteristics of a membrane-associated NDP kinase.
Main Methods:
- Enzyme purification to electrophoretic homogeneity.
- Partial protein sequencing.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Isoelectric focusing (IEF).
- Kinetic assays (Km, acceptor preference).
- Immunodetection and autoradiography.
Main Results:
- Purified soluble NDP kinase consists of identical 17 kDa subunits, forming a 98 kDa hexamer with an acidic pI of 4.5.
- Enzyme catalysis proceeds via a phosphoenzyme intermediate, with rapid phosphate transfer.
- GTP and ATP exhibit the lowest Km values, and GDP is the preferred phosphate acceptor.
- Evidence suggests the presence of a similar membrane-associated NDP kinase in Candida albicans.
Conclusions:
- The soluble NDP kinase from Candida albicans is a hexameric enzyme with distinct substrate preferences.
- The enzyme's catalytic mechanism involves a phosphoenzyme intermediate.
- The findings indicate potential similarities between soluble and membrane-associated NDP kinases within the organism.