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

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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Pereyra, Elba Nora, Mizyrycki, Cynthia L., Rossi, Silvia Graciela, Moreno, Silvia
Publicado:	2001
Materias:	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
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00144827_v271_n2_p337_Sorol http://hdl.handle.net/20.500.12110/paper_00144827_v271_n2_p337_Sorol
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_00144827_v271_n2_p337_Sorol
record_format	dspace
spelling	paper:paper_00144827_v271_n2_p337_Sorol2023-06-08T14:37:17Z Protein kinase A activity in permeabilized cells as an approximation to in vivo activity Pereyra, Elba Nora Mizyrycki, Cynthia L. Rossi, Silvia Graciela Moreno, Silvia 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 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. Fil:Pereyra, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mizyrycki, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rossi, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2001 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00144827_v271_n2_p337_Sorol http://hdl.handle.net/20.500.12110/paper_00144827_v271_n2_p337_Sorol
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	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
spellingShingle	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 Pereyra, Elba Nora Mizyrycki, Cynthia L. Rossi, Silvia Graciela Moreno, Silvia Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
topic_facet	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
description	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.
author	Pereyra, Elba Nora Mizyrycki, Cynthia L. Rossi, Silvia Graciela Moreno, Silvia
author_facet	Pereyra, Elba Nora Mizyrycki, Cynthia L. Rossi, Silvia Graciela Moreno, Silvia
author_sort	Pereyra, Elba Nora
title	Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
title_short	Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
title_full	Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
title_fullStr	Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
title_full_unstemmed	Protein kinase A activity in permeabilized cells as an approximation to in vivo activity
title_sort	protein kinase a activity in permeabilized cells as an approximation to in vivo activity
publishDate	2001
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00144827_v271_n2_p337_Sorol http://hdl.handle.net/20.500.12110/paper_00144827_v271_n2_p337_Sorol
work_keys_str_mv	AT pereyraelbanora proteinkinaseaactivityinpermeabilizedcellsasanapproximationtoinvivoactivity AT mizyryckicynthial proteinkinaseaactivityinpermeabilizedcellsasanapproximationtoinvivoactivity AT rossisilviagraciela proteinkinaseaactivityinpermeabilizedcellsasanapproximationtoinvivoactivity AT morenosilvia proteinkinaseaactivityinpermeabilizedcellsasanapproximationtoinvivoactivity
_version_	1768544805206360064

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

Ejemplares similares