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I Uno

Publications and source records attributed to I Uno.

At least 73 records · Page 4Linked to original sources
PubMed1983

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast.

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.

Chromatography, Ion Exchange↗
PubMed1983

Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast.

A mutation (pde1) was detected by suppressor activity on the CYR3 mutation which caused cAMP requirement for growth at 35 degrees C by the alteration of cAMP-dependent protein kinase. The pde1 mutant produced a significantly reduced level of cyclic nucleotide phosphodiesterase activity when assayed with 500 microM cAMP. Two cyclic nucleotide phosphodiesterases, I and II, were identified. Approximate molecular weights of these enzymes were 60,000 and 110,000, and the apparent Km values were 100 and 0.4 microM, respectively. The pde1 mutant was deficient in phosphodiesterase I activity. The cells carrying the pde1 mutation accumulated several times over the intracellular cAMP found in wild type cells. Phosphodiesterase I was not essential for growth of yeast cells, but controlled the intracellular cAMP levels in wild type and various mutant strains.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Control of cell division in Saccharomyces cerevisiae mutants defective in adenylate cyclase and cAMP-dependent protein kinase.

Examination of the proportion of unbudded cells, terminal nuclear phenotype and DNA content of nuclei indicated that cyr1 mutants of yeast defective in adenylate cyclase activity were arrested at the G1 phase of the cell cycle. The step of G1 arrest due to the cyr1 mutation preceded the step sensitive to the mating pheromone. The temperature-sensitive cyr1 cells did not continue growth, nor retain the capacity to conjugate at a restrictive temperature. The phenotypes of the cyr1 mutant mimicked those of nutritionally limited cells. The G1 arrest caused by the cyr1 mutation was overcome by the presence of a suppressor mutation, bcy1, that resulted in deficiency of a regulatory subunit of cAMP-dependent protein kinase and production of high level of cAMP-independent protein kinase. The bcy1 mutation suppressed G1 arrest caused by nutritional limitation, and continued bud emergence for multiple cycles without further nuclear division. The data suggest that cAMP works as a positive effector at the start of a yeast cell cycle via activation of cAMP-dependent protein kinase.

Adenylyl Cyclases↗
PubMed1983

Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase.

Control of the initiation of meiosis was examined in diploids of yeast homozygous for two temperature-sensitive mutations, cyr1 and CYR3, which are defective in adenylate cyclase and cAMP-dependent protein kinase, respectively. The cyr1 and CYR3 mutations permitted the initiation of meiosis, but resulted in the frequent production of two-spored asci at the restrictive temperature. Unlike the wild-type diploid cells, the cyr1 and CYR3 homozygous diploid cells were capable of initiating meiosis even in nutrient growth media. This unique feature of the cyr1 and CYR3 mutants suggests that these mutations relate to the choice between mitotic and meiotic processes. In diploids homozygous for the bcy1 mutation that results in deficiency of the regulatory subunit of cAMP-dependent protein kinase and production of a high level of the catalytic subunit of this enzyme, no premeiotic DNA replication and commitment to intragenic recombination occurred, and no spores were formed. We conclude that the initiation of meiosis may be dependent upon the repression of cAMP production and the inactivation of cAMP-dependent protein kinase.

Adenylyl Cyclases↗
PubMed1983

Cyclic AMP may not be involved in catabolite repression in Saccharomyces cerevisiae: evidence from mutants unable to synthesize it.

Yeast cells with a nonsense adenylate cyclase mutation, cyr1-3, required cyclic AMP for growth. This phenotype was suppressed by the byc1 mutation; however, cyr1-3 bcy1 cells produced no detectable level of adenylate cyclase or cyclic AMP. On induction, the bcy1 and cyr1-3 bcy1 mutant cells produced the same levels of galactokinase and alpha-D-glucosidase as did the wild-type cells and fourfold-higher levels of invertase. Since galactokinase synthesis was severely repressed by glucose in the constitutive GAL81 mutants, irrespective of the cyr1-3 bcy1 genotype, cyclic AMP may not be involved in catabolite repression.

Adenylyl Cyclases↗
PubMed1982

Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase.

The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.

Cyclic AMP↗
PubMed1982

Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase.

Mutants of Saccharomyces cerevisiae that require cAMP for growth have been isolated. Some of the mutants isolated were deficient in adenylate cyclase activity and mapped at a locus, cyr1, located near the centromere of chromosome X. Growth of cells carrying the cyr1 mutation was arrested at the G1 phase of the yeast cell cycle in the absence of cAMP. The cyr1 mutation was suppressed by a secondary mutation designated bcy1. The bcy1 mutation bypassed the need for cAMP for growth. The bcy1 mutants had extremely low levels of cAMP-binding protein and cAMP-dependent protein kinase but produced a high level of cAMP-independent protein kinase. The results indicate that cAMP is an essential factor for yeast cells to proceed through the cell cycle via the activation of protein kinase.

Adenylyl Cyclases↗
PubMed1982

Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Endogenous substrates for protein kinases in Coprinus macrorhizus.

The phosphorylation of cellular proteins in mycelia in strain Fisc in Coprinus macrorhizus was examined. The phosphorylation of two proteins, Protein A and B, was stimulated by cyclic AMP in the presence of Mg2+, and that of one protein, Protein C, was inhibited by cyclic AMP. The molecular weight of these proteins was determined, by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), to be 64000 (Protein A), 46000 (Protein B), and 18000 (Protein C), respectively. These proteins were quickly phosphorylated and the phosphorylation reached to the maximal levels in 5 min. The concentration of cyclic AMP required for the half-maximal stimulation of phosphorylation of Protein A and B, and for the half-maximal inhibition of phosphorylation of Protein C was approx. 1.0 x 10(-7) M. Cyclic GMP and cyclic IMP were slightly effective for stimulation and inhibition of these proteins.

Agaricales↗
PubMed1981

Control of adenosine 3',5'-monophosphate level and protein phosphorylation by depolarizing agents in Coprinus macrorhizus.

The treatment of mycelial cells with membrane-active antibiotics, uncouplers of oxidative phosphorylation and KCl leads to a transient increase in adenosine 3',-5'-monophosphate (cyclic AMP) levels in Coprinus macrorhizus. The maximal values and duration of increase in the cyclic AMP level depended on the kind and amount of these drugs. The treatment with these drugs simultaneously resulted in a rapid increase in the phosphorylation of three cellular proteins. The levels and time course of phosphorylation of these proteins were paralleled with the increase of cyclic AMP level in response to the drugs used. Thus, the treatment of these drugs causes the transient increase of cyclic AMP level and cyclic AMP stimulates the phosphorylation of particular proteins by activating protein kinases.

Agaricales↗
PubMed1981

Adenosine 3',5'-monophosphate-receptor protein and protein kinase in Coprinus macrorhizus.

A single cAMP-receptor protein could be detected in mycelial extracts of Coprinun macrorhizus by using the photoaffinity cAMP-analogue, 8-N3-cAMP. The protein which specifically bound 32P-labeled 8-N3-cAMP had an apparent molecular weight of 46,000 as determined by an SDS-polyacrylamide gel electrophoresis system. The 46,000-dalton protein was characterized by the dissociation constant for [32P]-8-N3-cAMP, and by the nucleotide specific inhibition of [32P]-8-N3-cAMP binding. The 46,000-dalton protein was co-chromatographed on a DEAE-cellulose column with cAMP-dependent protein kinase. The levels of [32P]-8-N3-cAMP-binding and protein kinase activities in mycelial extracts of strains used was always in parallel. The result indicated that the 46,000-dalton protein may be a regulatory subunit of protein kinase with the capacity to bind cAMP. cAMP-dependent protein kinase of this fungus was immunologically different from those of higher animals.

Azides↗
PubMed1980

Phosphorylation and dephosphorylation of the regulatory subunit of cyclic 3',5'-monophosphate-dependent protein kinase (type II) in vivo and in vitro.

A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [32P]phosphate-injected rats. A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively. Dephosphorylation was stimuated by cyclic nucleotides. The Ka values for cyclic AMP and cyclic IMP were 0.30 and 1.0 microM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar Ka values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl2, protected against dephosphorylation unless ADP or cyclic AMP were present.

Adenosine Diphosphate↗
PubMed1977

Study of autophosphorylation of isoenzymes of cyclic AMP-dependent protein kinases.

Type I and type II cyclic AMP-dependent protein kinases, present in the cytosol from each of five rat and two bovine tissues, were separated from one another by DEAE-cellulose column chromatography in order to study their possible autophosphorylation. In each of the tissues studied, autophosphorylation of the regulatory subunit of the cyclic AMP-dependent protein kinase by the catalytic subunit could be demonstrated with the type II enzyme but not with the type I enzyme.

Animals↗
PubMed1977

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.

Affinity Labels↗