Mucor circinelloides PKA: interaction between R and C subunits.

OCAMPO, J., MORENO, S. AND ROSSI, S.

Rosario, Santa Fe, Argentina

Congreso; 42 Reunion Anual de SAIB,; 2006

SAIB

PKA is a tetramer of regulatory subunits(R) and catalytic subunits(C).The interaction between R and C in M.circinelloidesM.circinelloides PKA is higher than in PKA from S. cereviciae and higher eukaryotic organisms.In the range where the PKA activity was no longer proportional to enzyme concentration,NaCl could increse the activity of the holoenzyme, indicating that complete holoenzyme disociation in vitro is not achived upon incubation with cAMP without NaCl. In a cAMP-agarose purification of C subunit it was necessary to use ClNa to obtain this subunit.Our hypothesis is that the N-terminal region of RM,which has acidic residues,is responsible for the difference in the interaction.Wt R and R1 laking the N-terminal and R2 lacking a region of acidic residues were cloned.Interaction between these Rs with C subunits was evaluated by in vitro inhibition or dissociation assays.The IC50 for wtR was lower than those for mutant Rs.Dissociation of reconstituted holoenzyme showed that higher cAMP concentration were required to dissociate the wtR-C than to dissociate the mtR-C. The behaivour of CM or Cb was different.The results indicates that the acidic residues region may be important in the high affinity interaction R-C found M. circinelloides PKA. circinelloides PKA. eukaryotic organisms.In the range where the PKA activity was no longer proportional to enzyme concentration,NaCl could increse the activity of the holoenzyme, indicating that complete holoenzyme disociation in vitro is not achived upon incubation with cAMP without NaCl. In a cAMP-agarose purification of C subunit it was necessary to use ClNa to obtain this subunit.Our hypothesis is that the N-terminal region of RM,which has acidic residues,is responsible for the difference in the interaction.Wt R and R1 laking the N-terminal and R2 lacking a region of acidic residues were cloned.Interaction between these Rs with C subunits was evaluated by in vitro inhibition or dissociation assays.The IC50 for wtR was lower than those for mutant Rs.Dissociation of reconstituted holoenzyme showed that higher cAMP concentration were required to dissociate the wtR-C than to dissociate the mtR-C. The behaivour of CM or Cb was different.The results indicates that the acidic residues region may be important in the high affinity interaction R-C found M. circinelloides PKA. circinelloides PKA. S. cereviciae and higher eukaryotic organisms.In the range where the PKA activity was no longer proportional to enzyme concentration,NaCl could increse the activity of the holoenzyme, indicating that complete holoenzyme disociation in vitro is not achived upon incubation with cAMP without NaCl. In a cAMP-agarose purification of C subunit it was necessary to use ClNa to obtain this subunit.Our hypothesis is that the N-terminal region of RM,which has acidic residues,is responsible for the difference in the interaction.Wt R and R1 laking the N-terminal and R2 lacking a region of acidic residues were cloned.Interaction between these Rs with C subunits was evaluated by in vitro inhibition or dissociation assays.The IC50 for wtR was lower than those for mutant Rs.Dissociation of reconstituted holoenzyme showed that higher cAMP concentration were required to dissociate the wtR-C than to dissociate the mtR-C. The behaivour of CM or Cb was different.The results indicates that the acidic residues region may be important in the high affinity interaction R-C found M. circinelloides PKA. circinelloides PKA. M. circinelloides PKA.PKA.