Regulation of cyclic AMP phosphodiesterase from <i>Mucor rouxii</i> by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme

N, Kerner; Moreno, Sílvia; Passeron, Susana

doi:10.1042/bj2190293

Cited by 10 publications

(3 citation statements)

References 14 publications

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“…Harrison et al (11) Phosphorylation of other phosphodiesterases in purified or partially purified systems has been reported (11,(35)(36)(37)(38)(39)(40)(41). Different subunits of the purified Ca2+/calmodulin-sensitive phosphodiesterase from bovine brain were phosphorylated by cAMP-dependent protein kinase and by a Ca2+/calmodulin-sensitive protein kinase.…”

Section: Resultsmentioning

confidence: 99%

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Grant

Mannarino

Colman

1988

Proc. Natl. Acad. Sci. U.S.A.

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Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 jbM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-K., cGMPinhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMPdependent phosphorylation. When intact platelets that had been prelabeled with 32p; were treated with forskolin and the low-K., cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-K., cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-K., cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

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Section: Resultsmentioning

confidence: 99%

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Grant

Mannarino

Colman

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…We suppose that this microheterogeneity of PDE forms, detected through different and opposed sensitivity towards the inhibition of hydrolysis of 0.1 PM endogenous proteolysis (Kerner et al, 1984;Tomes et al, 1985). These different forms could not be detected by assay with the PDE inhibitors (not shown).…”

Section: Efect Cf Camp Analogs On Hydrolysis Of Camp By Mucor Pdementioning

confidence: 97%

“…Spores of M. rouxii (NRRL 1894) were produced and stored as described by Haidle and Storck (1966). Aerobic mycelium was grown until mid-exponential phase as previously described (Kerner et al, 1984) except that 1% acid hydrolysed casein replaced peptone in the culture medium. The harvested material was ground in a mortar with liquid N, and immediately processed.…”

Section: Culturesmentioning

confidence: 99%

cAMP analogs and selective inhibitors used to study low KmMucor rouxii cAMP phosphodiesterase

Tomes

Moreno

1990

International Journal of Biochemistry

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1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.

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

1985

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The 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 behavior 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.

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Regulation of cyclic AMP phosphodiesterase from Mucor rouxii by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme

Cited by 10 publications

References 14 publications

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

cAMP analogs and selective inhibitors used to study low KmMucor rouxii cAMP phosphodiesterase

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

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