Cyclic GMP-dependent Protein Kinase Activity Research Articles

Gap Junctions and NCA Cation Channels Are Critical for Developmentally Timed Sleep and Arousal in Caenorhabditis elegans.

An essential characteristic of sleep is heightened arousal threshold, with decreased behavioral response to external stimuli. The molecular and cellular mechanisms underlying arousal threshold changes during sleep are not fully understood. We report that loss of UNC-7 or UNC-9 innexin function dramatically reduced sleep and decreased arousal threshold during developmentally timed sleep in Caenorhabditiselegans UNC-7 function was required in premotor interneurons and UNC-9 function was required in motor neurons in this paradigm. Simultaneous transient overexpression of UNC-7 and UNC-9 was sufficient to induce anachronistic sleep in adult animals. Moreover, loss of UNC-7 or UNC-9 suppressed the increased sleep of EGL-4 gain-of-function animals, which have increased cyclic-GMP-dependent protein kinase activity. These results suggest C. elegans gap junctions may act downstream of previously identified sleep regulators. In other paradigms, the NCA cation channels act upstream of gap junctions. Consistent with this, diminished NCA channel activity in C. elegans robustly increased arousal thresholds during sleep bouts in L4-to-adult developmentally timed sleep. Total time in sleep bouts was only modestly increased in animals lacking NCA channel auxiliary subunit UNC-79, whereas increased channel activity dramatically decreased sleep. Loss of EGL-4 or innexin proteins suppressed UNC-79 loss-of-function sleep and arousal defects. In Drosophila, the ion channel narrow abdomen, an ortholog of the C. elegans NCA channels, drive the pigment dispersing factor (PDF) neuropeptide release, regulating circadian behavior. However, in C. elegans, we found that loss of the PDF receptor PDFR-1 did not suppress gain-of-function sleep defects, suggesting an alternative downstream pathway. This study emphasizes the conservation and importance of neuronal activity modulation during sleep, and unequivocally demonstrates that gap junction function is critical for normal sleep.

Rho-Kinase Accelerates Synaptic Vesicle Endocytosis by Linking Cyclic GMP-Dependent Protein Kinase Activity to Phosphatidylinositol-4,5-Bisphosphate Synthesis

Rho-kinase plays diverse roles in cell motility. During neuronal development, Rho-kinase is involved in neuronal migration, and in neurite outgrowth and retraction. Rho-kinase remains highly expressed in mature neurons, but its physiological roles are poorly understood. Here we report that Rho-kinase plays a key role in the synaptic vesicle recycling system in presynaptic terminals. Vesicles consumed by excessive exocytosis are replenished by accelerating vesicle endocytosis via a retrograde feedback mechanism involving nitric oxide released from postsynaptic cells. This homeostatic control system involves presynaptic cyclic GMP-dependent protein kinase (PKG) and a plasma membrane phospholipid, phosphatidylinositol-4,5-bisphophate (PIP2). We found that application of a Rho-kinase inhibitor, a PKG inhibitor or both, reduced the PIP2 content in Wistar rat brainstem synaptosomes to a similar extent. Likewise, application of the Rho-kinase inhibitor into the calyx of Held presynaptic terminal slowed vesicle endocytosis to the same degree as did application of the PKG inhibitor. This endocytic slowing effect of the Rho-kinase inhibitor was canceled by coapplication of PIP2 into the terminal. By contrast, a RhoA activator increased the PIP2 content and reversed the effect of the PKG inhibitor in brainstem synaptosomes. The RhoA activator, when loaded into calyceal terminals, also rescued the endocytic slowing effect of the PKG inhibitor. Furthermore, intraterminal loading of anti-PIP2 antibody slowed vesicle endocytosis and blocked the rescuing effect of the RhoA activator. We conclude that Rho-kinase links presynaptic PKG activity to PIP2 synthesis, thereby controlling the homeostatic balance of vesicle exocytosis and endocytosis in nerve terminals.

Alterations in ventricular myocyte contraction caused by C-type natriuretic peptide and nitric oxide in eNOS–/– mice

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS–/–) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS–/– and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10 –8 and 10 –7 M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10 –6 and 10 –5 M). Although sGC activity was increased by +71% in eNOS–/–, SNAP had similar effects in the two groups (%shortening –39% control vs. –37% eNOS–/–), suggesting that the cyclic GMP pathway was desensitized in eNOS–/– myocytes. CNP had significantly smaller effects on cell contraction (%shortening –34% control vs. –14% eNOS–/–) and pGC activity was not changed in eNOS–/– myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca 2+ transients were also attenuated in eNOS–/– myocytes. SNAP did not alter Ca 2+ transients in eNOS–/– or control cells. In the eNOS–/– mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS–/– mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.

Mechanisms involved in the relaxation of bovine aortic endothelial cells

The importance of endothelial cell contraction in the regulation of vascular biology is being increasingly recognized. Our group has demonstrated that reactive oxygen species, particularly hydrogen peroxide, which are released in pathological conditions such as ischemia-reperfusion, are able to induce contraction in bovine aortic endothelial cells (BAEC). The cGMP-dependent relaxation of contractile cells depends on the ability of the cyclic nucleotide to interfere with intracellular calcium; however, this is not the only mechanism involved. The present experiments were designed to analyse the mechanism by which cGMP induces relaxation in BAEC. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, as well as atrial natriuretic (ANP) and C-type natriuretic (CNP) peptides, activators of particulate guanylate cyclase, blunted the hydrogen peroxide-induced contraction of BAEC and myosin light chain phosphorylation. The inhibitory effect was more marked with SNP and CNP than with ANP, and the action of SNP and CNP were partially reversed by blocking soluble and particulate guanylate cyclases, respectively. Dibutyryl cGMP (db-cGMP), a cGMP analogue, mimicked the effect of SNP and CNP. Cyclic GMP-dependent protein kinase (cGK) protein levels and activity were measured. Hydrogen peroxide induced a significant reduction in cGK activity without any change in protein level. This effect was completely reversed by preincubation with db-cGMP. Calyculin A, a myosin light chain phosphatase inhibitor, prevented the cGMP-induced relaxation of BAEC. SNP, CNP and db-cGMP also partially prevented the hydrogen peroxide-induced increase in intracellular calcium levels. Catalase completely blocked this effect. In summary, the present results support a role for those metabolites which activate guanylate cyclases in the relaxation of BAEC, and suggest that the cGMP-induced BAEC relaxation could be due, at least partially, to the stimulation of cGK and/or myosin light chain phosphatase activity, and to calcium blockade.

Negative functional effects of cGMP mediated by cGMP protein kinase are reduced in T 4 cardiac myocytes

We tested the hypothesis that in isolated rabbit cardiac myocytes, the negative functional effects of cyclic GMP are partly mediated by cyclic GMP-dependent protein kinase activity, and that these effects are altered in thyroxine (T 4, 0.5 mg/kg/day for 16 days)-induced hypertrophic myocytes. Using isolated ventricular myocytes from control ( N=8) and T 4 ( N=8) hypertrophic hearts, data for percent cell shortening (%) and maximum rate of contraction (μm/s) were collected using a video edge detector at baseline, after the addition of 10 −6 M 8-bromo-cyclic GMP (8-Br-cGMP), 10 −5 M 8-Br-cGMP, and 10 −6 M KT5823 (10-methoxy-10-methoxycarbonyl-9, 10, 11, 12-tetrahydro-9, 12-epoxy-(1 H)-diinidolo [1, 2, 3, f–g: 3′, 2′, 1′-k-j]-pyrrolidino-[3,4-i] [1,6]-benzodiazocin-2-methyl-1-one, cyclic GMP protein kinase inhibitor). Protein phosphorylation was determined autoradiographically after gel electrophoresis. In both control and T 4 myocytes, 8-Br-cGMP caused a significant decrease in percent shortening (5.56±0.49% to 3.02±0.47% in control and 4.34±0.33% to 3.13±0.17% in T 4 myocytes) and maximal rate of contraction 57.35±6.05 to 36.82±3.17 μm/s in control and 58.49±3.28 to 42.88±2.29 μm/s in T 4 myocytes). KT5823 significantly increased percent shortening to 3.77±0.28% and rate to 48.68±4.71 μm/s after 8-Br-cGMP only in control myocytes. In T 4 myocytes, the changes in percent shortening and rate after KT5823 were not significant. Protein phosphorylation was increased by 8-Br-cGMP in control and to a lesser extent in T 4 myocytes, but the increment was reduced by KT-5823 in control only. These data demonstrated that cyclic GMP had negative functional effects partially mediated by cyclic GMP protein kinase in control myocytes. Cyclic GMP also exerted negative functional effects in thyroxine-induced hypertrophic myocytes, but cyclic GMP protein kinase activity was not an important regulator of these effects in T 4 ventricular myocytes.

Cyclic GMP-Dependent Protein Kinase Activity in Rat Pulmonary Microvascular Endothelial Cells

Cyclic GMP-dependent protein kinase (cGPK) activity was determined in rat pulmonary microvascular endothelial cells (RPMVEC) using cGMP-stimulated phosphorylation of BPDEtide and histone F2B substrates in the presence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAPK)]. RPMVEC cGPK activity was localized to the 100,000 × g cytosolic fraction. The EC50 for cGMP activation in the presence of PKI was 0.16 μM and H-89 inhibition under similar conditions showed an IC50 value of 0.16 μM. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the cGMP-independent protein kinase activity and similar elution conductivities. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Preliminary studies reveal six potential substrates phosphorylated by cGPK in RPMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE).

Vasoactive intestinal peptide release and l-citrulline production from isolated ganglia of the myenteric plexus: Evidence for regulation of vasoactive intestinal peptide release by nitric oxide

Vasoactive intestinal peptide release and l-[ 3H]citrulline production were examined in ganglia isolated from the myenteric plexus of guinea-pig intestine. The nicotinic agonist, 1,1-dimethy l-4-phenylpiperizinium stimulated vasoactive intestinal peptide release and l-[ 3H]citrulline production; the latter was considered an index of nitric oxide production. Both vasoactive intestinal peptide release and l-[ 3H]citrulline production were abolished by tetrodotoxin, hexamethonium, and the nitric oxide synthase inhibitor, N G-nitro- l-arginine. Inhibition of vasoactive intestinal peptide release by N G-nitro- l-arginine was reversed by l-arginine but not by d-arginine. Exogenous nitric oxide stimulated vasoactive intestinal peptide release whereas exogenous vasoactive intestinal peptide had no effect on l-[ 3H]citrulline production. The pattern of stimulation by nitric oxide and inhibition by N G-nitro- l-arginine implied that vasoactive intestinal peptide release is facilitated by and may be dependent on nitric oxide production. Consistent with this notion, vasoactive intestinal peptide release in response to either 1,1-dimethyl-4-phenylpiperizinium or nitric oxide was abolished by KT 5823, an inhibitor of cyclic GMP-dependent protein kinase activity and by LY83583, an inhibitor of soluble guanylate cyclase activity. The study provides the first direct evidence of nitric oxide production from enteric ganglia.

Age-associated decrease in histamine-induced vasodilation may be due to reduction of cyclic GMP formation.

1. The effects of aging on histamine-induced vasodilatation and cyclic GMP production in rat thoracic aorta were investigated. 2. This histamine-induced dilatation of the aorta was mediated by H1-receptors and was dependent on the endothelium. 3. Histamine induced the greatest dilatation of arteries of 3-4 week old rats, progressively less of those of rats of 8 to 56 weeks old, and scarcely detectable dilatation of those of 100 week old rats. 4. Histamine induced cyclic GMP production in aorta from rats of 4 weeks old, with no change in the cyclic AMP level. This increase in the cyclic GMP level induced by histamine also decreased with age, being about 70% as great at 8 weeks, 50% as great at 50-60 weeks, and 10% as great at 130 weeks of age. 5. Removal of the endothelium completely abolished the histamine-induced increase in cyclic GMP. 6. The dilator effect of nitroprusside, which enhances cyclic GMP production by stimulating guanylate cyclase directly (not indirectly via the endothelium derived relaxing factor, EDRF), also showed age-related attenuation. 7. The dilator effect of 8-bromo cyclic GMP, which stimulates cyclic GMP-dependent protein kinase, also decreased during aging. 8. These results suggest that aging reduces the ability of the endothelium to produce EDRF, which stimulates guanylate cyclase, and so decreases cyclic GMP production. Thus the decreased dilator response of the arteries to histamine during aging is probably due to both loss of endothelial function and reduction of guanylate cyclase activity. Alteration of cyclic GMP-dependent protein kinase activity may also participate in the age-related changes.

Cyclic GMP‐Dependent Protein Kinase and Phosphorylation of the Endogenous Substrate Proteins in the Rabbit Superior Cervical Ganglion

When the homogenate of rabbit superior cervical ganglia (SCG) was incubated in the presence of [gamma-32P]ATP and Mg2+, two specific proteins were strongly labeled. Their apparent molecular weights were 90,000 and 54,000, respectively. The phosphorylation of the latter was significantly stimulated by 10-50 nM cyclic GMP but to a lesser extent by cyclic AMP, whereas that of the former was not stimulated significantly by either of the cyclic nucleotides. The purified protein kinase inhibitor from rabbit skeletal muscle did not inhibit the phosphorylation. These results indicated that the observed phosphorylation of 54K protein was dependent on cyclic GMP but not on cyclic AMP. When intact SCG was incubated in the presence of 32Pi, phosphorylation of 90K protein was stimulated by cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP (10 microM), whereas phosphorylation of 54K protein was not significantly stimulated by any of these substances. The present demonstration of endogenous cyclic GMP-dependent protein kinase activity and its endogenous substrate proteins raises a possibility that the physiological actions of cyclic GMP in SCG are mediated by the phosphorylation of these proteins.

Protein phosphorylation in cultured endothelial cells.

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.

Role of cyclic-GMP in Relaxations of Vascular mooth Muscle

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.

Intrinsic protein phosphorylation in synaptosomal plasma membrane fragments: a comparison of cerebral cortex tissue from several species, including human biopsy specimens.

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.

Adrenocortical cyclic GMP-dependent protein kinase: Purification, characterization, and modification of its activity by calmodulin, and its relationship with steroidogenesis

Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the M r of the enzyme was 145,000. This protein was composed to two identical subunits each with M r of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N 2-monobutyryl cyclic GMP > N 2, O 2-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis.

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg 2+ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20–30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.

Separation between modulator-dependent protein kinases I and II.

The separation of modulator-dependent protein kinase I from modulator-dependent protein kinase II obtained from the lungs of sexually premature male mice was accomplished by Sephadex G-200 gel filtration. After preincubation of a mouse lung cytosol fraction with arginine-rich histone, theophylline, cyclic GMP and crude protein kinase modulator a cyclic GMP-dependent protein kinase activity peak present in a non-preincubated sample completely disappeared and was replaced by a late-eluted modulator-dependent protein kinase II peak. There was a difference in substrate specificity between modulator-dependent protein kinase I and modulator-dependent protein kinase II despite their similar dependence on crude protein kinase modulator or partially purified stimulatory protein kinase modulator for their maximal activities.

Inhibition of protein secretion and protein kinase activity in the locust fat body by diamide (Azodicarboxylic acid-bis-dimethylamide)

Protein secretion from locust fat body incubated in vitro is instantaneously halted in the presence of diamide (azodicarboxylic acid-bis-dimethylamide). Independent inhibition of amino acid uptake, protein synthesis (verified in cell-free systems), and protein secretion is demonstrated. Diamide inhibits cyclic AMP- and cyclic GMP-dependent protein kinase activity in locust fat body.

Protein Kinase Activity of Isolated Rat Adipocytes as Related to Cell Size

SummaryWe have studied cellular cyclic AMP content and protein kinase activity in extracts of small and large adipocytes from two groups of rats differing in age and body weight. The data, expressed on a per cell basis, show that large adipocytes from larger rats present a significantly greater (p < 0.02) content of cyclic AMP and a 3-fold greater (p < 0.001) level of basal protein kinase activity than small adipocytes. Cyclic AMP (0.4 μM) stimulated the cyclic AMP-dependent protein kinase activity to a similar degree (about 3-fold) in both types of cells. No significant amount of cyclic GMP-dependent protein kinase activity was detected in extracts of adipocytes from either group of rats.The increased cellular content of cyclic AMP and the elevated protein kinase activity in large adipocytes are probably responsible for the greater basal rate of lipolysis observed in these cells.

Protein kinases in Tetrahymena cilia : II. Partial purification and characterization of adenosine 3′,5′-monophosphate-dependent and guanosine 3′,5′-monophosphate-dependent protein kinases

Adenosine 3′,5′-monophosphate (cyclic AMP)- and guanosine 3′,5′-monophosphate (cyclic GMP)-dependent protein kinase (EC 2.7.1.37) activities were detected in the Triton extract of Tetrahymena cilia. When the Triton extract was chromatographed on a DEAE-cellulose column, one peak of cyclic AMP-dependent protein kinase activity and three peaks of cyclic GMP-dependent protein kinase activity were detected, one of which was further purified with CM-Sephadex column chromatography. Two of the cyclic GMP-dependent protein kinases had quite similar properties, prefering histone and casein for substrate, and were specifically activated by a low concentration of cyclic GMP. The other one, cyclic GMP-dependent protein kinase, which preferentially phosphorylated histone and protamine, was less susceptible to cyclic GMP. The activity of cyclic AMP-dependent protein kinase was highly stimulated by a low concentration of cyclic AMP and showed preference for protamine or histone as substrate protein. The apparent molecular weights of all the cyclic GMP-dependent protein kinases were about 1·10 5, that of the cyclic AMP-dependent kinase being about 6·10 4.

Guanosine 3':5'-monophosphate-dependent protein kinases in mammalian tissues.

The mammalian protein kinase activity stimulated preferentially by low concentrations of guanosine 3':5'-monophosphate (cyclic GMP), but not by adenosine 3':5'-monophosphate (cyclic AMP), was readily assayed in a modified incubation system that contained a neutral phosphate buffer, protein kinase modulator, and arginine-rich histone. Cyclic GMP-dependent protein kinase activity assayed under these conditions was about two to three orders of magnitude higher than that previously detected. The enzyme activity occurred in all guinea pig and rat tissues (lung, heart, aorta, brain, liver, ileum, adipose, and pancreatic islets) examined. The activity can be separated from the cyclic AMP-dependent protein kinase activity, also present in the same tissues, by means of either Sephadex G-200 gel filtration or ammonium sulfate fractionation. The cyclic GMP-dependent enzyme preparations had K(a) values for cyclic GMP ranging from 0.03 to 0.12 muM, compared to the K(a) values for cyclic AMP ranging from 0.6 to 3.8 muM. The presence of phosphate and protein kinase modulator was essential for maximal cyclic GMP-dependent enzyme activity. The occurrence of high levels of cyclic GMP-dependent protein kinase activity in mammalian tissues clearly suggests that it may serve as a "target" enzyme for cyclic GMP, mediating many biological effects of this cyclic nucleotide.