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Isolation and characterisation of cAMP-dependent protein kinase from Candida albicans . Purification of the regulatory and catalytic subunits
- Source :
- European Journal of Biochemistry. 252:245-252
- Publication Year :
- 1998
- Publisher :
- Wiley, 1998.
-
Abstract
- cAMP-dependent protein kinase (PKA) from Candida albicans yeast cells was isolated and characterised. Structural parameters of the holoenzyme and those of its subunits suggested that C. albicans PKA is a tetramer of 287 kDa composed of two regulatory (R) subunits of 64 kDa and two catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent Km for ATP and Kemptide were 30 microM and 60 microM respectively. The [A]0.5 for cAMP activation was 150 nM with a Hill coefficient of 1.6. The holoenzyme undergoes autophosphorylation on the R subunit, a characteristic of the type-II R subunits. Photoaffinity labeling with 8-azido-[32P]cAMP of crude extracts from yeast and mycelial cells strongly suggests that only one type of R subunit is present in the fungus. The R subunit was purified to apparent homogeneity as a protein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64-kDa protein from crude extracts, indicating that the purified R subunit very probably represents the native form of the protein. The 78-kDa form of the C subunit was detected in crude extracts and in Mono Q Sepharose column fractions with heterologous anti-C Ig. It could be isolated by cAMP treatment of the holoenzyme immunoprecipitated from crude extracts with anti-R serum, but this form could not be purified further. Instead, a 60-kDa protein with the main characteristics of C subunit was purified to near homogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78-kDa form by proteolytic degradation.
- Subjects :
- Fungal protein
Photoaffinity labeling
Protein Conformation
Immunoprecipitation
Protein subunit
Autophosphorylation
Affinity Labels
Cyclic AMP-Dependent Protein Kinase Type II
Biology
Cyclic AMP-Dependent Protein Kinases
Biochemistry
Molecular biology
Fungal Proteins
Molecular Weight
Sepharose
Adenosine Triphosphate
Protein structure
Candida albicans
Cyclic AMP
Phosphorylation
Protein kinase A
Oligopeptides
Subjects
Details
- ISSN :
- 14321033 and 00142956
- Volume :
- 252
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry
- Accession number :
- edsair.doi.dedup.....89280ad1519cd0b4898e3121f63a8d61
- Full Text :
- https://doi.org/10.1046/j.1432-1327.1998.2520245.x