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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- 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.


Documento: Artículo
Título:Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
Autor:Sorol, M.R.; Pereyra, E.; Mizyrycki, C.; Rossi, S.; Moreno, S.
Filiación:Departamento de Quimica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Palabras clave:Activation; cAMP; cAMP analogs; In situ permeabilization; Mucor rouxii; Nonlinear; Protein kinase A; cyclic AMP; cyclic AMP dependent protein kinase; holoenzyme; polyamine; sodium chloride; animal cell; article; catalysis; cell activity; cell membrane permeability; concentration response; controlled study; enzyme activation; enzyme active site; enzyme activity; enzyme assay; fungus growth; measurement; Mucor; nonhuman; priority journal; Amylomyces rouxii; Fungi; Mucor; Mucor rouxii
Página de inicio:337
Página de fin:343
Título revista:Experimental Cell Research
Título revista abreviado:Exp. Cell Res.
CAS:cyclic AMP, 60-92-4; cyclic AMP dependent protein kinase; sodium chloride, 7647-14-5


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---------- APA ----------
Sorol, M.R., Pereyra, E., Mizyrycki, C., Rossi, S. & Moreno, S. (2001) . Protein kinase A activity in permeabilized cells as an approximation to in vivo activity. Experimental Cell Research, 271(2), 337-343.
---------- CHICAGO ----------
Sorol, M.R., Pereyra, E., Mizyrycki, C., Rossi, S., Moreno, S. "Protein kinase A activity in permeabilized cells as an approximation to in vivo activity" . Experimental Cell Research 271, no. 2 (2001) : 337-343.
---------- MLA ----------
Sorol, M.R., Pereyra, E., Mizyrycki, C., Rossi, S., Moreno, S. "Protein kinase A activity in permeabilized cells as an approximation to in vivo activity" . Experimental Cell Research, vol. 271, no. 2, 2001, pp. 337-343.
---------- VANCOUVER ----------
Sorol, M.R., Pereyra, E., Mizyrycki, C., Rossi, S., Moreno, S. Protein kinase A activity in permeabilized cells as an approximation to in vivo activity. Exp. Cell Res. 2001;271(2):337-343.