Isolation and characterisation of cAMP-dependent protein kinase from Candida albicans. Purification of the regulatory and catalytic subunits.
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.
Zelada
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A
Programa de Investigaciones Bioquímicas y Fisiológicas, Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, Argentina.
Passeron
S
S
Lopes Gomes
S
S
Cantore
M L
ML
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D002176:Candida albicans / Q000201:enzymology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation
D000345:Affinity Labels; D005656:Fungal Proteins; D009842:Oligopeptides; C037642:kemptide; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases