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.

N-acetyl-D-glucosamine induces germination in Candida albicans through a mechanism sensitive to inhibitors of cAMP-dependent protein kinase.

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-D-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.

Reassessment of the effect of glucagon and nucleotides on Candida albicans germ tube formation.

The role of cyclic AMP in the process of germ tube formation in Candida albicans was investigated. The exogenous supply of the nucleotide or of agents that raise its intracellular levels stimulated germination induced by N-acetyl-D-glucosamine; glucagon showed this same stimulatory effect on yeast cell transition to the hyphal form. Compounds, included glucagon, that stimulated hyphal formation, also notably enhanced the development of hyphae. The stimulatory effect of glucagon on germination was blocked by the specific antagonist des His1 [Glu9] glucagon amide, probably indicating an interaction of the hormone with a glucagon-like receptor on the membrane of the cells. Indirect immunofluorescence experiments showed that glucagon binds to the yeast cell surface. When N-acetyl-D-glucosamine was replaced by serum as inducing agent of germination, the stimulatory effect of glucagon was substantially augmented, the resulting of germination being more than 2.5-fold greater than that attained in the presence of N-acetyl-D-glucosamine; moreover, the glucagon concentration needed for half maximal stimulatory activity with serum as inducing agent was at least 50-fold lower than with N-acetyl-D-glucosamine. Monoclonal and polyclonal anti-glucagon antibodies blocked the effect of the hormone. An interesting result observed during these experiments was the fact that a definite period of incubation of C. albicans yeast cells with N-acetyl-D-glucosamine as inducer commits them to hyphal development. When serum was used as inducer, only yeast cells evaginated during the initial incubation period evolved to the hyphal form upon further incubation in the absence of serum.

Steroidogenesis in immature chicken ovary. Hormonal stimulation of adenylyl cyclase system by luteinizing hormone and beta-adrenergic agonists.

The presence of a hormonally responsive adenylyl cyclase in the immature chicken ovary was investigated. We found that there was a highly significant difference (P < 0.05) between basal and LH and catecholamine activatable activities. In addition, the basal activity was stimulated by NaF, forskolin and the non-hydrolyzable GTP analogue guanosine-5'-(beta,gamma-imido)-triphosphate (GMP-P(NH)P. The action of catecholamines on cyclic AMP and progesterone production was also investigated and compared to that of LH. The stimulatory effect of isoproterenol on cyclic AMP and progesterone production was significantly higher (P < 0.05) than that of LH. The beta-adrenergic antagonist propranolol caused complete inhibition of the stimulatory action of catecholamines. Progesterone accumulation induced by LH or isoproterenol was synergistically augmented by the simultaneous presence of both inducers.

Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis.

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.

Cyclic AMP as an intracellular messenger of gonadotrophin-induced steroidogenesis during the development of the embryonic chick ovary.

(1) Ovine luteinizing hormone (LH) stimulates cyclic AMP (cAMP) and progesterone production (P) throughout late ontogeny of the chick ovary and cAMP mimicks LH in stimulating P secretion but: (2) P/cAMP ratios are lower at the earliest stages than at hatching, LH enhancing this tendency. (3) Immediately before hatching, on day 19 time-courses of LH stimulations of cAMP and P are different. (4) cAMP and P respond differently to increasing doses of LH but similarly to increasing doses of forskolin. (5) 1.5 mM dibutyryl cAMP (I) and 100 ng LH (II) increase P maximally, 2.5- and 3.3-fold respectively, but a mixture (I + II) increases P 7.5-fold.

A radioactive method for the measurement of trypsin and trypsin-like activities.

A simple and highly sensitive method for the assay of trypsin has been developed by making use of the phosphorylated synthetic peptide Leu-Arg-Arg-Ala-Ser-(32P)-Leu-Gly as substrate. The technique has been adapted from the phosphocellulose method of R. Roskoski, Jr. (in Methods in Enzymology (Corbin, J., and Hardman, J., Eds.), Vol. 99, pp. 3-6, Academic Press, New York) used for measuring of protein kinases. In addition to measuring the activity of trypsin at the microgram level, the 32P-labeled peptide method can be used for measuring other trypsin-like enzymes. It has been successfully utilized for the identification of a new peptidase from the fungus Saccobolus platensis.

cAMP levels and in situ measurement of adenylate cyclase and cAMP phosphodiesterase activities during yeast-to-hyphae transition in the dimorphic fungus Mucor rouxii.

Intracellular levels of cAMP and specific activities of adenylate cyclase and cAMP phosphodiesterase were measured during yeast-to-hyphae transition in the dimorphic fungus M. rouxii. Enzymatic activities were measured in permeabilized cells under conditions preventing protein dephosphorylation and proteolysis. A two-fold decrease in intracellular cAMP levels occurred shortly after exposure of the yeast to air quite before morphological changes became evident. Morphogenesis to hyphae after exposure to air was inhibited by the addition of 10 mM dibutyryl cAMP to the culture medium, and the yeast morphology was maintained for at least 24 hours. The decrease in cAMP levels that occurs shortly after exposure of yeast culture to air was mainly accounted for by variations in the state of activation of cAMP phosphodiesterase while the specific activity of adenylate cyclase did not vary significantly during yeast-to-hyphae transition.