In vitro phosphorylation of 4-aminobutyrate aminotransferase by cAMP dependent protein kinase.
Highly purified 4-aminobutyrate aminotransferase from pig brain is susceptible to phosphorylation by the purified cAMP-dependent protein kinase catalytic subunit. Up to 0.7 moles of phosphate from ATP-(gamma)-32P can be incorporated per mole of dimeric holoenzyme. Maximum phosphorylation was observed within about 90 minutes at 30 degrees C. Despite the extensive degree of phosphorylation observed, no kinetic property of the enzyme was perceptibly altered. Removal of cofactor had no detectable impact on the extent of phosphorylation but thermal inactivation of the enzyme increased and mild reduction with sodium borohydride decreased the phosphorylatability of the aminotransferase. It was possible to separate the enzyme into phospho and dephospho forms by the use of DEAE chromatography. Validation that the two fractions represented genuine aminotransferase was obtained by proteolytic peptide mapping. The phospho form of the enzyme was found to possess little or no aminotransferase activity while that of the dephospho form exhibited higher specific activity than the purified enzyme prior to phosphorylation. Furthermore, the dephospho form of the enzyme could not be detectably phosphorylated by reincubation with the kinase following DEAE chromatography unless it was subjected to thermal inactivation. The stoichiometry of phosphorylation of the fraction containing 32P from DEAE chromatography was approximately 1 mole/mole of dimer. These results suggest that the substrate for phosphorylation by the kinase is a form of the aminotransferase which is somehow inactivated during routine purification even when extensive precautions are taken to maximally preserve catalytic activity.
R K
RK
Carr
D
D
Schlichter
C
C
Spielholz
W D
WD
Wicks
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000818:Animals; D001894:Borohydrides / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D006358:Hot Temperature; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D013997:Time Factors
D001894:Borohydrides; D000612:4-Aminobutyrate Transaminase; D011494:Protein Kinases