The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini.
cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.
Ruth
P
P
Institut für Pharmakologie und Toxikologie, Technischen Universität München, Federal Republic of Germany.
Landgraf
W
W
Keilbach
A
A
May
B
B
Egleme
C
C
Hofmann
F
F
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005822:Genetic Vectors; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014132:Trachea / Q000201:enzymology; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D010770:Phosphotransferases; D011494:Protein Kinases