Cyclic AMP-dependent protein kinases of Paramecium. II. Catalytic and regulatory properties of type II kinase from cilia.

The type II cAMP-dependent protein kinase (cAMP-PK-II) from cilia of Paramecium, purified free of type I cAMP-PK (cAMP-PK-I) and of cGMP-dependent protein kinase (cGMP-PK), phosphorylated several basic proteins and a heptapeptide containing serine (Kemptide). The enzyme was partially inhibited by the protein kinase inhibitor (Walsh inhibitor), but only at relatively high inhibitor concentrations. Half-maximal activation of cAMP-PK-II occurred at 15-25 nM cAMP. Several cAMP analogs were tested for ability to bind and activate the enzyme. 8-bromo-cGMP, a potent activator of Paramecium cGMP-PK, was a poor activator of Paramecium cAMP-PK-II. Activation of cAMP-PK-II was influenced by the phosphorylation assay buffer. Phosphate buffers provided increased activation by cAMP but decreased total activity relative to that measured in Mops-Tris buffer. The kinase was cAMP-independent when the pH of the assay buffer was high. Preincubation of cAMP-PK-II with histones also activated the enzyme in the absence of cAMP. The cAMP-PK-II bound cAMP with a Kd of 23 nM, and bound cAMP was released with a biphasic time course, suggesting two non-identical binding sites. The properties of the cAMP-PK of this ciliated protozoan appear to be closely similar to those of vertebrates.