Abstract
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.
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