Purification and properties of cyclic-3',5'-GMP-dependent protein kinase from the nematode Ascaris suum.
A cyclic-3',5'-GMP-dependent protein kinase was purified 7400-fold from the reproductive tract of female ascarids to a specific activity of 718 nmol min-1 mg-1 (histone as substrate). The yield of the preparation was 25%. The enzyme protein obtained was homogeneous as judged by isoelectrofocusing and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme behaved as a dimer of two 82-kDa subunits in gel permeation chromatography on Superose 12. The protein kinase was inactive in the absence of cyclic purine nucleotides. Half-maximum velocity was obtained in the presence of 18 nM cGMP, whereas 400-fold higher concentrations of cAMP were required for the same activity. The enzyme underwent autophosphorylation in first-order kinetics (rate constant 0.054 min-1), leading to maximum incorporation of 0.96 phosphate per subunit. The autophosphorylation led to a 4-fold increase in Vmax, while the Km remained almost unchanged. In an extract from the reproductive tract, cGMP-stimulated phosphorylation was primarily observed in five proteins (molecular masses of 66, 60, 43, 30, and 25 kDa). These proteins also incorporated phosphate when isolated reproductive tracts were incubated in the presence of [32P]phosphate. The phosphate content in cellular proteins was enhanced when the incubation was performed in the presence of 10(-4) M of either octyl-cAMP or octyl-cGMP. In addition to the proteins mentioned above, however, six more electrophoretic bands containing radioactive phosphate were identified after in situ labeling of reproductive tracts with radioactive phosphate.
Thalhofer
H P
HP
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Hofer
H W
HW
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D013379:Substrate Specificity
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C022994:cyclic GMP-binding protein; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP