A novel cGMP-dependent protein kinase from Paramecium.
An unusual monomeric cGMP-dependent protein kinase, enriched in cilia, was isolated from Paramecium cilia and whole cells. Cilia and whole cell extracts had relatively high ratios of cGMP-dependent to cAMP-dependent protein kinase activity (1:2). The calculated molecular weight of the native enzyme was 88,000. The enzyme was identified on sodium dodecyl sulfate-polyacrylamide gels as a 77,000 molecular weight band based on copurification of this protein with enzyme activity, 8-N3-[32P]cAMP labeling, and autophosphorylation. Based on the size of the native enzyme, it was concluded that the kinase is a monomer with cGMP-binding and catalytic activities on the same polypeptide. Dimer-sized cGMP-dependent protein kinase, like that of the well characterized mammalian enzyme, was never seen, despite stringent efforts to control proteolysis. The structure of the Paramecium cGMP-dependent protein kinase supports a model in which the dimeric vertebrate form of the enzyme evolved from an early monomeric form. The catalytic properties of the Paramecium enzyme differed in several respects from those of the mammalian enzyme: it could use GTP or ATP as the phosphoryl donor, it did not phosphorylate Kemptide effectively, and it had poor histone kinase activity with high Mg2+ concentrations. Quercertin, 5'-guanylyl imidodiphosphate, indomethacin, and the isoquinolinesulfonamide drug H7 inhibited Paramecium cGMP-dependent protein kinase activity. The enzyme had fast and slow binding sites (with kd values of 5-10 x 10(-3)s-1 and 0.44 x 10(-3)s-1) and showed an order of preference for cyclic nucleotides and cyclic nucleotide analogs similar to that of the mammalian enzyme.
Miglietta
L A
LA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002923:Cilia / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010247:Paramecium / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D011494:Protein Kinases