Autophosphorylation of Mucor rouxii cAMP-dependent protein kinase and its role in holoenzyme activation.
Two phosphoproteins of 53,000 and 63,000 mol. wt detected in partially purified preparations of Mucor rouxii cAMP-dependent protein kinase submitted to phosphorylation conditions with [gamma-32P]ATP are demonstrated to be the result of the autophosphorylation of its regulatory subunit, according to the following criteria: (1) linearity of phosphate incorporation with enzyme sample; (2) independence of phosphate incorporation on temperature; (3) correlation of the phosphoproteins with enzymatic activity in a DEAE-Sepharose chromatography; (4) specific elution of the phosphorylated proteins from cAMP-agarose; (5) phosphorylation of the purified regulatory subunit. Antibodies specific against Mucor regulatory subunit detected an intact subunit of 72,000 mol. wt in crude extracts. Autophosphorylation of the fungal protein kinase A promotes activation of the holoenzyme by cAMP since: (1) under conditions of partial activation, increase of activity is observed when using the phosphoform of the enzyme; (2) release of free catalytic subunit from cAMP-agarose is enhanced when the holoenzyme is previously phosphorylated.
Rossi
S
S
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Guthmann
M
M
Moreno
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000906:Antibodies; D001665:Binding Sites; D002847:Chromatography, Agarose; D004789:Enzyme Activation; D005658:Fungi; D015151:Immunoblotting; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D000906:Antibodies; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D011494:Protein Kinases