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- 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 microM), 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.
Sorol
M R
MR
Departamento de Quimica Biológica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Pereyra
E
E
Mizyrycki
C
C
Rossi
S
S
Moreno
S
S
D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001681:Biological Assay; D002384:Catalysis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D009089:Mucor / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D010455:Peptides; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases