Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclic-nucleotide-elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium-derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade. Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441-448) established that in human platelets cyclic-nucleotide-elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP- and cGMP-dependent protein kinases. Of particular interest was a membrane-bound 50-kDa protein whose phosphorylation was increased both by cAMP- and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP- and cGMP-dependent protein kinase in platelet membranes. Since the molecular mechanism of action of cyclic-nucleotide-elevating vasodilators is unknown, this 50-kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye-ligand chromatography. The purified protein migrated as a 46-kDa protein in SDS/PAGE, was an excellent substrate for both cAMP- and cGMP-dependent protein kinases and migrated in SDS/PAGE as a 50-kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylated by both cAMP- and cGMP-dependent protein kinases in platelet membranes and in response to cAMP- and cGMP-elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP- or cGMP-dependent protein kinase. The availability of this vasodilator-regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.
 ...
PMID:Purification of a vasodilator-regulated phosphoprotein from human platelets. 280 62

Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.
 ...
PMID:A catalytically active fragment of cGMP-dependent protein kinase. Occupation of its cGMP-binding sites does not affect its phosphotransferase activity. 282 99

Several vascular and nonvascular mammalian tissue extracts exhibited variable amounts of two peaks (peaks I and II) of cGMP-dependent protein kinase by NaCl elution of DEAE columns. When [3H]cGMP was added to the extracts before chromatography, a peak of protein-bound [3H]cGMP coeluted with peak II. [3H]cGMP was added to purified bovine lung cyclic nucleotide-free enzyme followed by chromatography on high performance liquid chromatography-DEAE. Two kinase peaks, the first of which represented mainly cGMP-free enzyme and the second of which represented cGMP-bound enzyme, eluted at the same positions as peaks I and II, respectively, of the crude extracts. The relative amount of peak II increased as a function of increasing the [3H]cGMP added before chromatography, and peak II could be converted partially to peak I by rechromatography. The holoenzyme is known to contain two slowly exchanging cGMP binding sites (sites 1) and two rapidly exchanging sites (sites 2). Some protein-bound [3H] cGMP found entirely in site 1 coeluted with peak I, although most of the enzyme in that peak was cGMP-free. When low [3H]cGMP was used for the initial incubation, relatively more of the protein-bound [3H] cGMP appeared in peak I and could represent binding of [3H]cGMP to only one of the two sites 1 of the kinase. The [3H]cGMP bound to the peak II enzyme completely filled both sites 1. Cyclic GMP binding to these sites caused the apparent conformational change which shifted the DEAE elution position of the enzyme. The peak II kinase was partially active and had a higher sensitivity to further cGMP activation of kinase than did the cGMP-free enzyme, suggesting that activation of kinase by binding of cGMP to site 2 was facilitated by prior binding at site 1. In fractions of the trailing edge of peak II, the kinase activity was virtually cGMP-independent, and both sites 1 and 2 were almost saturated with [3H]cGMP. These results suggested a further conformational change and direct increase in activity by binding of cGMP at site 2.
 ...
PMID:Interconvertible cGMP-free and cGMP-bound forms of cGMP-dependent protein kinase in mammalian tissues. 282 8

Nuclear and total cellular cyclic nucleotide-dependent protein kinase activity was assayed in corneal epithelium from carbamylcholine-treated and control eyes of rabbits with and without resurfacing acid burn defects. In epithelium from resurfacing corneas carbamylcholine significantly elevated both nuclear and total cellular cGMP-dependent protein kinase activity and reduced activity of cAMP-dependent protein kinase. The drug also increased the proportion of total cellular cGMP kinase activity represented by nuclear activity, suggesting that the drug may enhance nuclear translocation of cGMP-dependent protein kinase.
 ...
PMID:Effects of carbamylcholine on cyclic nucleotide-dependent protein kinase activity in corneal epithelium during resurfacing. 282 19

The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4 degrees C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 microM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 microM CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 microM and 2 microM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.
 ...
PMID:8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase. 283 64

The purpose of this study was to investigate the effects of the intracellular messenger cyclic GMP (cGMP) on sequestration of cytosolic calcium (Ca2+) into the intracellular Ca2+ store (the sarcoplasmic reticulum) of vascular smooth muscle. Using saponin-skinned primary cultures of rat aortic smooth muscle, we investigated the effect of cGMP on 45Ca uptake in monolayers of cells. The intracellular store was loaded with Ca2+ by exposing the skinned cells to a 45Ca-labeled 1-microM free Ca2+-containing solution for varying durations (0-20 minutes). Addition of 10 microM cGMP to six monolayers increased both the initial Ca2+ uptake at 2 minutes (control, 240 +/- 8 pmol Ca2+/10(6) cells; + cGMP 295 +/- 7; mean +/- SEM; n = 6, p less than 0.01) and the final steady-state uptake reached at 20 minutes (control, 0.96 +/- 0.03 nmol Ca2+/10(6) cells; + cGMP 1.12 +/- 0.03, p less than 0.02). This stimulation of uptake was quantitatively similar to that caused by 10 microM cyclic AMP. It occurred at varying ambient cytosolic Ca2+ concentrations (0.1-1.0 microM Ca2+) and was not further enhanced by addition of 10 microM cGMP-dependent protein kinase. The dose-response of stimulation of Ca2+ uptake with cGMP indicated an ED50 of 5 nM cGMP. The release of Ca2+ from the sarcoplasmic reticulum in response to inositol 1,4,5-trisphosphate or caffeine was unaffected by cGMP. We conclude that the relaxation of vascular smooth muscle with cGMP-producing vasodilators is mediated in part by sequestration of cytosolic Ca2+ by the sarcoplasmic reticulum.
 ...
PMID:Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle. 283 13

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates. 283 78

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.
 ...
PMID:Kinetics of protein phosphorylation in microvessels isolated from rat brain: modulation by second messengers. 283 36

Cardiac sarcolemmae from guinea pig ventricles were purified and incubated with cGMP-dependent protein kinase. In the presence of the purified kinase plus 10(-5) M cGMP or 8-Br-cGMP, a protein of approximately 50 kD, (Kilodalton) was phosphorylated. This membrane-associated cGMP-dependent protein kinase substrate is similar in MW to the regulatory subunit of the cAMP-dependent protein kinase, which is known to be a substrate for the cGMP-dependent protein kinase. Thus, this substrate, the identity of which remains to be proven, may be a possible mediator of cGMP-mediated control of cardiac function.
 ...
PMID:Cardiac sarcolemmal substrate of the cGMP-dependent protein kinase. 285 60

Cyclic GMP depresses Ba2+ current through high-voltage-activated Ca2+ channels (ICa) in acutely isolated hippocampal neurons. The effect is produced by intra-, but not extracellular, cGMP or by 5' GMP. The membrane-permeant derivative, 8-Br-cGMP, produces a reversible suppression. The effect of 8-Br-cGMP is similar to phorbol ester-induced ICa depression, except that ICa depression due to 8-Br-cGMP is not blocked by protein kinase inhibitors H-8 or H-7, whereas phorbol ester effects are. The data suggest that cGMP depresses ICa by a cGMP-kinase- and protein kinase C (PKC)-independent mechanism. Cyclic AMP, which enhances ICa, and the cyclic nucleotide phosphodiesterase inhibitor, IBMX, both antagonize ICa depression induced by 8-Br-cGMP, but not that due to phorbol esters. Cyclic IMP, a more potent activator of phosphodiesterase than of cGMP-dependent protein kinase, is also a powerful depressant of ICa. We conclude that cGMP-induced depression of ICa is mediated by activation of cyclic nucleotide phosphodiesterase with consequent reduction of intracellular cAMP.
 ...
PMID:Cyclic GMP depresses hippocampal Ca2+ current through a mechanism independent of cGMP-dependent protein kinase. 285 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>