Susana Passeron

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

Abstract The germination of spores of Mucor rouxii into hyphae was inhibited by 2 m m dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 m m cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 m m ) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air. Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.

SEPARATION OF SUGAR NUCLEOTIDES, PHOSPHORIC ESTERS AND FREE SUGARS BY PAPER CHROMATOGRAPHY WITH SOLVENTS CONTAINING BORATES OF ORGANIC BASES.

Abstract Four different chromatographic solvents containing borates of organic bases are described. Two of them permit the separation of sugar nucleotides differing only in the sugar moiety and give also a good resolution for some sugar 1-phosphates. The other two systems permit, in a single run, the separation of a group of sugars which is usually difficult to achieve.

Further studies on cyclic adenosine 3':5'-monophosphate protein kinase from dimorphic fungus Mucor rouxii.

Abstract In this paper, cyclic adenosine-3′:5′-monophosphate-dependent protein kinase from yeast-like cells of Mucor rouxii is characterized. A scheme of partial purification is described together with K m for ATP (15 μ m ), histone (0.2 mg/ml), half-maximal activation constant for cyclic AMP (30 n m ), and dissociation constant for the binding of cyclic AMP (40 n m ). This enzyme is similar to type II protein kinases in two main aspects: the elution position in DEAE-cellulose chromatography and the readiness of its reassociation. But it has a singular characteristic: it does not dissociate completely with cyclic AMP alone (even at concentrations as high as 0.3 m m ) unless histone or NaCl is present. NaCl displays several roles: helps dissociation, prevents inactivation of the catalytic subunit, inhibits enzyme activity, and does not prevent reassociation as occurs with type II protein kinases. Once the holoenzyme is dissociated, cyclic AMP is essential to maintain the enzyme in the dissociated state.

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.

cAMP levels and in situ measurement of cAMP related enzymes during yeast-to-hyphae transition in Candida albicans

Intracellular levels of cAMP and specific activities of adenylate cyclase, cAMP phosphodiesterase and cAMP-dependent protein kinase were measured during filamentation in the dimorphic fungus Candida albicans. Enzymatic assays were performed in permeabilized cells under conditions prevented endogenous proteolysis. The variations observed in cAMP levels were mainly accounted for by variations in the specific activities of adenylate cyclase and cAMP phosphodiesterase at different stages during germ tube formation. cAMP-dependent protein kinase, measured with kemptide as exogenous substrate, was developmental regulated. Some properties of the enzymatic activities from cell-free extracts are described.

Relationship between cyclic adenosine 3′:5′-monophosphate and germination inCandida albicans

Abstract Germination of yeast-like cells of Candida albicans is preceded by a significant decrease in intracellular levels of cyclic AMP during the early stage of germ-tube induction. These levels increased thereafter as germ-tube formation proceeded. The intracellular concentration of cyclic AMP was measured with a cyclic AMP radioimmunoassay and with a competitive assay method using a cyclic AMP-binding protein. Under inducing conditions, germtube formation was inhibited by the addition of cyclic AMP or compounds that are known to elevate the intracellular cyclic nucleotide concentration.

Multiple protein kinase activities in the dimorphic fungus Mucor rouxii. Comparison with a cyclic adenosine 3',5'-monophosphate binding protein.

Protein kinase and cyclic adenosine 3′,5′-monophosphate (cAMP) binding activities have been detected in cell extracts of the dimorphic fungus Mucor rouxii. The subcellular distribution of both activities indicates that most of the binding protein is in the high-speed supernatant (S100), while about 70% of the total protein kinase activity remains in particulate fractions. S100 preparations have been analyzed by diethylaminoethyl cellulose column chromatography. Binding activity can be resolved in two peaks (A and B) and protein kinase in three peaks (I, II, and III). Peaks I and II are casein dependent and insensitive to cAMP. Peak III utilizes histone as substrate and is activated (two- to fourfold) by cAMP. Theophylline strongly inhibits cAMP binding activity and mimics the effect of cAMP on cAMP-dependent protein kinase. The possible relationship between cAMP binding activity and cAMP-dependent protein kinase is suggested.

Activation of pyruvate kinase of Mucor Rouxii by manganese ions.

Mucor rouxii, a filamentous fungus, is able to grow either anaerobically, exhibiting a yeast-like morphology, or aerobically as a filamentous myceliurn. It was also demonstrated that yeast-like cells will develop in a culture under any conditions favoring a high level of fermentation. [ I] . In order to gain insight into the process of morphogenesis a general study of one of the key glycolytic enzymes (pyruvate kinase) was undertaken. In the present communication some kinetic properties of the Mucor pyruvate kinase (ATP: pyruvate phosphotransferase, E.C. 2.7.1.40) are described. Evidence is presented that in addition to fructose-l ,6diphosphate (FDP), Mn*+ is also an allosteric activator of the Mucor pyruvate kinase at physiological concentrations of substrate (phosphoenol pyruvate, PEP).

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

SummaryThe polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R2C2 of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behaviour towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.

Control of Mucor rouxii adenosine 3′:5′-monophosphate phosphodiesterase by phosphorylation-dephosphorylation and proteolysis☆

Partially purified cAMP phosphodiesterase from Mucor rouxii can be reversibly activated from 1.5- to 3-fold by treatment with MgATP, cAMP, and cAMP-dependent protein kinase, without change in its sedimentation behavior. Deactivation of activated enzyme can be observed in crude extracts under conditions which promote dephosphorylation; deactivation is prevented by 20 mm phosphate. cAMP phosphodiesterase can also be irreversibly activated by treatment with trypsin. The extent of activation by proteolysis is similar to that obtained by phosphorylation, but is accompanied by a decrease in the sedimentation coefficient of the enzyme. Activation by phosphorylation and proteolysis are not additive, suggesting that both mechanisms involve the same region of the enzyme molecule.