Protein kinase A activity in permeabilized cells as an approximation to in vivo activity

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Permeabilized germlings from the dimorphic fungus Mucor rouxii were used for in situ measurement of protein kinase A (PKA) activation, to compare the results with those obtained in vitro at low or high (nonlinear) enzyme concentrations. The apparent total activity per cell when measured in situ is 5...

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Autor principal: Sorol, M.R
Otros Autores: Pereyra, E., Mizyrycki, C., Rossi, S., Moreno, S.
Formato: Capítulo de libro
Lenguaje:Inglés
Publicado: Academic Press Inc. 2001
Acceso en línea:Registro en Scopus
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Biblioteca Central Dr. Luis F. Leloir (FCEN) de Universidad de Buenos Aires
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024 7 |2 scopus  |a 2-s2.0-0035842299 
024 7 |2 cas  |a cyclic AMP, 60-92-4; cyclic AMP dependent protein kinase; sodium chloride, 7647-14-5 
040 |a Scopus  |b spa  |c AR-BaUEN  |d AR-BaUEN 
030 |a ECREA 
100 1 |a Sorol, M.R. 
245 1 0 |a Protein kinase A activity in permeabilized cells as an approximation to in vivo activity 
260 |b Academic Press Inc.  |c 2001 
270 1 0 |m Moreno, S.; Departamento de Quimica Biologica, Fac. de Ciencias Exactas y Nat., Ciudad Universitaria, Pabellon 2, 1428 Buenos Aires, Argentina; email: smoreno@qb.fcen.uba.ar 
506 |2 openaire  |e Política editorial 
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504 |a Døskeland, S.O., Maronde, E., Gjersten, B.T., The genetic subtypes of cAMP-dependent protein kinase: Functionally different or redundant? (1993) Biochim. Biophys. Acta, 1178, pp. 249-258 
504 |a Yang, S., Fletcher, W.H., Johnson, D.A., Regulation of cAMP-dependent protein kinase: Enzyme activation without dissociation (1995) Biochemistry, 34, pp. 6267-6271 
504 |a Hunter, T., Signaling - 2000 and beyond (2000) Cell, 100, pp. 113-127 
504 |a Pawson, T., Nash, P., Protein-protein interactions define specificity in signal transduction (2000) Genes Dev., 14, pp. 1027-1047 
504 |a Colledge, M., Scott, J.D., AKAPs: From structure to function (1999) Trends Cell Biol, 9, pp. 216-221 
504 |a Zaremberg, V., Donella-Deana, A., Moreno, S., Mechanism of activation of cAMP-dependent protein kinase: In Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit (2000) Arch. Biochem. Biophys., 381, pp. 74-82 
504 |a Haidle, C.W., Storck, R., Control of dimorphism in Mucor rouxii (1966) J. Bacteriol., 92, pp. 1236-1244 
504 |a Bartnicki-García, S., Nickerson, W.J., Nutrition, growth and morphogenesis of Mucor rouxii (1962) J. Bacteriol., 83, pp. 841-858 
504 |a Serrano, R., Gancedo, J.M., Gancedo, C., Assay of yeast enzymes in situ. A potential tool in regulation studies (1973) Eur. J. Biochem., 34, pp. 479-482 
504 |a Maggesse, M.C., Galvagno, M.A., Cantore, M.L., Passeron, S., In situ measurement of cAMP related enzymes in the dimorphic fungus Mucor rouxii (1982) Cell. Biol. Int. Rep., 6, pp. 1101-1108 
504 |a Paveto, C., Passeron, S., Corbin, J., Moreno, S., Two different intrachain cAMP sites in the cAMP dependent protein kinase of the dimorphic fungus Mucor rouxii (1989) Eur. J. Biochem., 179, pp. 429-434 
504 |a Roskoski, R., Assay of protein kinase (1983) Methods Enzymol., 99, pp. 3-6 
504 |a Rossi, S., Moreno, S., Regulation of protein kinase A subunits during germination of Mucor rouxii sporangiospores (1994) Eur. J. Biochem., 222, pp. 501-506 
504 |a Guthmann, M., Pastori, R., Moreno, S., Polyamines and basic proteins stimulate activation by cAMP and catalytic activity of Mucor rouxii cAMP-dependent protein kinase (1990) Cell. Signal., 2, pp. 395-402 
504 |a Moreno, S., Passeron, S., Further studies on cAMPdependent protein kinase from the dimorphic fungus Mucor rouxii (1980) Arch. Biochem. Biophys., 199, pp. 321-330 
504 |a Rannels, S.R., Corbin, J.D., Two different intra-chain cAMP binding sites of cAMP-dependent protein kinases (1980) J. Biol. Chem., 255, pp. 7085-7088 
504 |a Pereyra, E., Zaremberg, V., Moreno, S., Effect of dibutyryl-cAMP on growth and morphology of germinating Mucor rouxii sporangiospores (1992) Exp. Mycol., 16, pp. 93-101 
504 |a Pereyra, E., Mizyrycki, C., Moreno, S., Threshold level of protein kinase A activity and polarized growth in Mucor rouxii (2000) Microbiology, 146, pp. 1949-1958 
504 |a Beebe, S., Holloway, R., Rannels, S.R., Corbin, J.D., Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes (1984) J. Biol. Chem., 259, pp. 3539-3547 
504 |a Braunmann, T., Jastorff, B., Physicochemical characterization of cyclic nucleotides by reversed-phase high-performance liquid chromatography. II. Quantitative determination of hidrophobicity (1985) J. Chromatogr., 350, pp. 105-118 
504 |a Griffioen, G., Branduardi, P., Ballarini, A., Anghileri, P., Norbeck, J., Baroni, M.D., Ruis, H., Nucleocytoplasmic distribution of budding yeast protein kinase A regulatory subunit Bcy1 requires Zds1 and is regulated by Yak1-dependent phosphorylation of its targeting domain (2001) Mol. Cell. Biol., 21, pp. 511-523 
504 |a Faux, M.C., Scott, J.D., Molecular glue: Kinase anchoring and scaffold proteins (1996) Cell, 85, pp. 9-12 
504 |a Portela, P., Zaremberg, V., Moreno, S., Evaluation of in vivo activation of protein kinase A under non-dissociable conditions through the overexpression of wild type and mutant regulatory subunits in Saccharomyces cerevisiae (2001) Microbiology, 147, pp. 1149-1159 
520 3 |a Permeabilized germlings from the dimorphic fungus Mucor rouxii were used for in situ measurement of protein kinase A (PKA) activation, to compare the results with those obtained in vitro at low or high (nonlinear) enzyme concentrations. The apparent total activity per cell when measured in situ is 5- to 10-fold lower than the in vitro measured activity in crude extracts from those cells. Polyamines and NaCl stimulate the activity in situ. The apparent relative specific activity of the in situ measured PKA toward four peptide substrates is similar to the results obtained in vitro at high holoenzyme concentration and not to those obtained with the free catalytic subunit. Saturation in the activation of PKA by cAMP in situ is attained at low concentrations (2 to 10 μM), while in vitro, at high holoenzyme concentration, no saturation was attained up to 1 mM cAMP (V. Zaremberg et al. Arch. Biochem. Biophys. 381, 74-82, 2000). Activation of PKA by site-selective cAMP analogs is assayed in situ and in vitro at two enzyme concentrations. Site B-selective cAMP analogs are good activators of PKA at low enzyme concentration in vitro but poor activators either at high enzyme concentration in vitro or in permeabilized cells. A physiological correlation with the behavior of site-selective analogs in situ is demonstrated in vivo when assaying the effect of increasing concentrations of site-selective cAMP analogs on the impairment of polarized growth of M. rouxii spores. © 2001 Elsevier Science.  |l eng 
536 |a Detalles de la financiación: Ministerio de Salud de la Nación, MSAL 
536 |a Detalles de la financiación: Universidad de Buenos Aires 
536 |a Detalles de la financiación: Consejo Nacional de Investigaciones Científicas y Técnicas 
536 |a Detalles de la financiación: Agencia Nacional de Promoción Científica y Tecnológica 
536 |a Detalles de la financiación: This work was supported by grants from Agencia Nacional de Promoción Cientifica y Tecnológica, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Ministerio de Salud, through its fellowship Carrillo-Oñativia. M.R.S. received a fellowship from FOMEC and C.M. from CONICET. We thank P. Portela for helpful discussion. 
593 |a Departamento de Quimica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina 
690 1 0 |a ACTIVATION 
690 1 0 |a CAMP 
690 1 0 |a CAMP ANALOGS 
690 1 0 |a IN SITU PERMEABILIZATION 
690 1 0 |a MUCOR ROUXII 
690 1 0 |a NONLINEAR 
690 1 0 |a PROTEIN KINASE A 
690 1 0 |a CYCLIC AMP 
690 1 0 |a CYCLIC AMP DEPENDENT PROTEIN KINASE 
690 1 0 |a HOLOENZYME 
690 1 0 |a POLYAMINE 
690 1 0 |a SODIUM CHLORIDE 
690 1 0 |a ANIMAL CELL 
690 1 0 |a ARTICLE 
690 1 0 |a CATALYSIS 
690 1 0 |a CELL ACTIVITY 
690 1 0 |a CELL MEMBRANE PERMEABILITY 
690 1 0 |a CONCENTRATION RESPONSE 
690 1 0 |a CONTROLLED STUDY 
690 1 0 |a ENZYME ACTIVATION 
690 1 0 |a ENZYME ACTIVE SITE 
690 1 0 |a ENZYME ACTIVITY 
690 1 0 |a ENZYME ASSAY 
690 1 0 |a FUNGUS GROWTH 
690 1 0 |a MEASUREMENT 
690 1 0 |a MUCOR 
690 1 0 |a NONHUMAN 
690 1 0 |a PRIORITY JOURNAL 
690 1 0 |a AMYLOMYCES ROUXII 
690 1 0 |a FUNGI 
690 1 0 |a MUCOR 
690 1 0 |a MUCOR ROUXII 
700 1 |a Pereyra, E. 
700 1 |a Mizyrycki, C. 
700 1 |a Rossi, S. 
700 1 |a Moreno, S. 
773 0 |d Academic Press Inc., 2001  |g v. 271  |h pp. 337-343  |k n. 2  |p Exp. Cell Res.  |x 00144827  |w (AR-BaUEN)CENRE-797  |t Experimental Cell Research 
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