ISOLATION AND CHARACTERIZATION 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 holoenzym e and those of its subunits suggested that C. albicans PKA is a tetram er of 287 kDa composed of, two regulatory (R) subunits of 64 kDa and t wo catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent K-m for ATP and Kemptide were 30 mu M and 60 mu M respec tively. The [A](0.5) for cAMP activation was 150 nM with a Hill coeffi cient of 1.6. The holoenzyme undergoes autophosphorylation on the R su bunit, a characteristic of the type-II R subunits. Photoaffinity label ing with 8-azido-[P-32]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 pr otein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64-kDa protein from crude e xtracts, indicating that the purified R subunit very probably represen ts the native form of the protein. The 78-kDa form of the C subunit wa s 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 ser um, but this form could not be purified further. Instead, a 60-kDa pro tein with the main characteristics of C subunit was purified to near h omogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78-kDa form by prote olytic degradation.