María L. Cantore

Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

ABSTRACT The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

Variations in the levels of cyclic adenosine 3′:5′-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3t:́5′-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Abstract Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn 2+ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K m (1 μ m ) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn 2+ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.

In situ measurement of cAMP related enzymes in the dimorphic fungus Mucor rouxii.

Abstract Yeast cells of the dimorphic fungus Mucor rouxii have been permeabilized by treatment with toluene:ethanol. The permeabilization allowed the in situ measurement of pyruvate kinase, cAMP phosphodiesterase and adenylate cyclase activities. Using a small peptide as substrate, cAMP dependent protein kinase activity could also be measured. Permeabilized cells showed higher cAMP phosphodiesterase and adenylate cyclase activities than cellular homogenates. The main catalytic properties of the enzymes were similar to that previously found in in vitro studies.

Kinetic properties, solubilization, and molecular characterization of Mucor rouxii adenylate cyclase☆

Abstract Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 0.5 m KCl. Homogenates prepared in the presence of Lubrol PX or KCl exhibit two- to threefold more activity compared to homogenates prepared in the absence of detergent or salt. The membrane-bound enzyme prepared in the presence of 2% Lubrol PX was studied in detail. The kinetic properties of this adenylate cyclase were as expected for an allosteric enzyme with the ATP · Mn 2− complex as substrate and free Mn 2+ ions as activator. Unchelated ATP inhibits the reaction by competing with the substrate. The KCl-solubilized enzyme was partially purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel filtration. Molecular parameters of the solubilized enzyme are: Stokes radius, 8.6 nm; S 20,w , 13S; M r 510,000; and frictional ratio, 1.60.

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.

Cyclic adenosine 3′, 5′-monophosphate phosphodiesterase from Mucorrouxii: Regulation of enzyme activity by phosphorylation and dephosphorylation

Abstract Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg 2+ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ 32 P]ATP resulted in a significant amount of [ 32 P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg 2+ and was accompanied by the release of [ 32 P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.

Nucleotides and nucleotide sugars in human blood cells. II. Group "O".

Abstract A study of nucleotides and nucleotide sugars in human blood cells has been carried out. Ten samples from healthy yound people of group “O” were analyzed by ion-exchange chromatography, and the following nucleotides and nucleotide sugars were found: NAD+; UDP-glucose, UDP-mannose, UDP-galactose, UDP-acetylglucosamine and UDP-acetylgalactosamine; AMP; NADP+; ADP-glucose, ADP-galactose and ADP-mannose; GDP-mannose and GDP-fucose; ADP and ATP. The sugar nucleotides were identified on the basis of chemical, enzymatic and chromatographic analysis. UDP-galactose, UDP-mannose, UDP-acetylgalactosamine, ADP-glucose, ADP-galactose, ADP-mannose, GDP-mannose and GDP-fucose have not been found before in human blood, and UDP-mannose was not known to occur in nature. In order to minimize the decomposition of unstable substances, mild techniques were applied: cold acid extraction and neutral (ethanol-chloroform) extraction were compared, and in both cases the aqueous phase was passed through a column of Dowex- i (Cl-form) resin, and the nucleotides eluted with a linear gradient of NH4Cl. Results showed that the extraction step is not very critical, but the use of ammonium salts and almost neutral pH, as compared with methods used in previous papers, permitted the isolation of ten nucleotide-linked sugars, eight of which have not been described before in human blood. An adenine nucleotide with very particular properties was extracted with the ethanol-chloroform technique; it was eluted by the concentration of NH4Cl necessary to elute ATP, but it behaved as AMP on paper chromatography in several solvent systems. It is very unstable and decomposes even at −20° in a few days.

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.

Steroidogenesis in immature chicken ovary. Hormonal stimulation of adenylyl cyclase system by luteinizing hormone and β-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′‐(β, gamma;‐imido)‐triphosphate (GMPP(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 β‐adrenergic antagonist propranolol caused complete inhibition of the stimulatory action of catecholamines. Progesterone accumulation induced by LH or isoproterenol was synergistically augmentated by the simultaneous presence of both inducers.

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.