Characterization of cyclic GMP-binding sites of cyclic GMP-dependent protein kinase by rapid filtration assay.
The kinetics of cyclic [3H]GMP binding to the purified cyclic GMP-dependent protein kinase (cG kinase) were studied by using the rapid filtration assay method with polyethyleneimine-treated glass filters (method A), and the data were compared with those of the (NH4)2SO4 precipitation procedure (method B), which has been used for many previous studies on cyclic GMP binding to cG kinase. Each method gave a similar stoichiometry of approx. 2 mol of cyclic GMP/mol of cG kinase subunit; however, other binding kinetics obtained with these two methods were different. The dissociation of bound cyclic [3H]GMP from the kinase showed a single slow component when method A was used, whereas rapid and slow dissociation components were observed with method B. The Scatchard plot of cyclic [3H]GMP binding with method A was linear with a Kd value of 11 +/- 2 nM, suggesting that the two intrachain binding sites have similar high affinity for cyclic GMP. Results obtained on cyclic nucleotide analogue specificity of the two intrachain cyclic GMP-binding sites were also different between these two methods. These findings suggest that cG kinase has two high-affinity cyclic GMP-binding sites per subunit in the native state, and that when (NH4)2SO4 is added, ostensibly to stop the binding reaction, one low-affinity site is created from one high-affinity site.
Hirai
M
M
Department of Pharmacology, Kobe University School of Medicine, Japan.
Hashimoto
S
S
Kuno
T
T
Tanaka
C
C
D000818:Animals; D001665:Binding Sites; D011232:Chemical Precipitation; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D005374:Filtration; D007700:Kinetics; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
C016276:8-bromocyclic GMP; D011494:Protein Kinases; D006152:Cyclic GMP