Expression, purification, and characterization of the cGMP-dependent protein kinases I beta and II using the baculovirus system.
Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.
Pöhler
D
D
Medizinische Universitätsklinik, Labor für Klinische Biochemie, Würzburg, Germany.
Butt
E
E
Meissner
J
J
Müller
S
S
Lohse
M
M
Walter
U
U
Lohmann
S M
SM
Jarchau
T
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D051381:Rats; D011994:Recombinant Proteins; D018411:Spodoptera / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP