Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil.
It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.
Szmidt-Jaworska
Adriana
A
Department of Physiology and Molecular Biology of Plants, Nicolaus Copernicus University, Institute of General and Molecular Biology, Gagarina St. 9, PL 87-100 Torun, Poland. asjawors@biol.uni.torun.pl
Jaworski
Krzysztof
K
Tretyn
Andrzej
A
Kopcewicz
Jan
J
D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D027741:Ipomoea / Q000201:enzymology*; D009928:Organ Specificity; D010766:Phosphorylation; D013379:Substrate Specificity
D006657:Histones; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP