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1.
Evidence is presented for Ca2+ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca2+ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca2+, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca2+ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca2+) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.  相似文献   

2.
Streamer F mutants have been found to be useful tools for studying the pathway of signal transduction leading to chemotactic cell movement. The primary defect in these mutants is in the structural gene for the cyclic GMP specific phosphodiesterase. This defect allows a larger and prolonged peak of cyclic GMP to be formed in response to the chemotactic stimulus, cyclic AMP. This characteristic aberrant pattern of cyclic GMP accumulation in the streamer F mutants has been correlated with similar patterns of changes in the influx of calcium from the medium, myosin II association with the cytoskeleton, myosin phosphorylation and a decrease in speed of movement of the amoebae. From these studies a sequence of events can be deduced that leads from cell surface cyclic AMP stimulation to cell polarization prior to movement of the amoebae in response to the chemotactic stimulus.  相似文献   

3.
Brain type II Ca2+/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca 2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2+-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC20 Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC17 Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid  相似文献   

4.
—Depolarizing concentrations of K+ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K+ -induced accumulation of cyclic GMP. Barium and Sr2+, but not Mn2+ or Co2+, can substitute for Ca2+ in this process. Relatively high concentrations of Mg2+ inhibit the effect of Ca2+ on K+-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K+ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg2+ and no other divalent cations. High concentrations of Ca2+ (1 mm or greater) augment this Mg2+ -dependent, K+-induced accumulation of cyclic AMP, however. Strontium and Mn2+, but not Ba2+ or Co2+, can substitute for Ca2+ in this process, and high concentrations of Mg2+ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca2+ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca2+ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca2+-dependent mechanism(s) mediating release of intracellular substances.  相似文献   

5.
Calmodulin and the regulation of smooth muscle contraction   总被引:8,自引:0,他引:8  
Calmodulin, the ubiquitous and multifunctional Ca2+-binding protein, mediates many of the regulatory effects of Ca2+, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca2+]i transientvia the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca2+/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca2+. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca2+/calmodulin or indirectly by phosphorylation catalysed by Ca2+/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca2+ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca2+/calmodulin, e.g. the sarcolemmal Ca2+ pump and the ryanodine receptor/Ca2+ release channel, and other proteins which indirectly regulate [Ca2+]i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.  相似文献   

6.
Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca2+ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10?8 M and 5.0 × 10?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. 251, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca2+-dependently and independently.  相似文献   

7.
The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.  相似文献   

8.
Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-32P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca2+ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca2+ in digitonin-treated cells. In the presence of 1 M Ca2+, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca2+. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl2 - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution  相似文献   

9.
—Guanosine 3′,5’cyclic monophosphate (cyclic GMP) levels in incubated slices of mouse cerebellum are increased 10-fold by glutamate and two-to three-fold by glycine or γ-aminobutyric acid (GABA). Glutamate also produces a 10-fold increase in adenosine 3′,5’cyclic monophosphate (cyclic AMP) in the same tissue. However, GABA decreases cyclic AMP levels 30-40 per cent, and glycine produces only a transient 50 per cent accumulation of this cyclic nucleotide. Theophylline slightly augments the accumulation of cyclic GMP produced by all three amino acids but markedly attenuates the accumulation of cyclic AMP produced by glutamate. In the absence of Ca2+, none of the three amino acids has any effect on cyclic GMP levels, and glutamate produces only a 50 per cent rise in cyclic AMP levels. The decrease of cyclic AMP levels produced by GABA is not affected by theophylline or by the absence of Ca2+. These data suggest an involvement of both cyclic GMP and cyclic AMP in the neurochemical actions of glutamate, GABA and glycine.  相似文献   

10.
Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn2+ was up to several hundred-fold greater than with Mg2+ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn2+-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn2+ than with Mg2+. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.5  相似文献   

11.
The effect of Ca2+ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca2+ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E1 and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca2+, norephinephrine and adenosine.  相似文献   

12.
Summary Exogeneous cyclic AMP (cAMP, 10–8M), when added together with acetylcholine (ACh, 500 M) to dissociated chick embryo cells, blocked the ACh-stimulated increase in the level of cytosolic free Ca2+ ([Ca2+]i). This inhibiting action of exogeneous cAMP is probably mediated by intracellular cyclic GMP (cGMP) and cAMP.  相似文献   

13.
Signal transduction for chemotaxis in Dictyostelium amoebae   总被引:1,自引:0,他引:1  
The signal for chemotaxis in D. discoideum is cyclic AMP. This molecule binds to cell surface receptors and triggers the production of inositol (1,4,5)trisphosphate which releases Ca2+ from non-mitochondrial stores. The subsequent chain of signal transduction events brings about the polymerization of cytoskeletal actin (associated with pseudopodium formation) within five seconds and the formation of a peak of cyclic GMP within 10 s. Evidence from streamer F mutants indicates that the cyclic GMP regulates the association of myosin with the cytoskeleton that occurs at 25-50 s and that this phenomenon is concerned with elongation of the amoebae during chemotactic movement.  相似文献   

14.
Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca2+ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca2+ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca2+ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca2+ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.  相似文献   

15.
Cyclic nucleotide phosphodiesterase activity of porcine cerebral cortical extracts was measured with 0.1–100 μM-cyclic AMP and cyclic GMP and found to be dependent on both Ca2+ and added cyclic nucleotides. With decreasing substrate concentration activity with cyclic GMP became more dependent on Ca2+ whereas hydrolysis of cyclic AMP became less dependent. Cyclic GMP at 3 μM stimulated the hydrolysis of 0.1–10μM-cyclic AMP in the absence of Ca2+ (< 10-10M) but inhibited activity with 200 μM-Ca2+ present. This differential, substrate- and Ca2+-dependent regulation was attributed to the presence of at least two types of phosphodiesterase distinguishable by DEAE-column chromatography. In the absence of Ca2+, activity with 1 μM-cyclic GMP eluted in one minor peak followed by two major peaks, D-I and D-II. Activity with 1 μM-cyclic AMP eluted almost entirely in D-II. Hydrolysis of cyclic AMP in D-II was activated by cyclic GMP. With added Ca2+ plus a Ca2+-dependent regulator (CDR), activity with 1 μM-cyclic GMP was markedly increased and eluted entirely at D-I. Total activity with 1 μM-cyclic AMP was only moderately increased and eluted as D-I with a shoulder at D-II. Elution profiles with 100 μM-substrate were relatively independent of substrate, with D-I predominant with Ca2+·CDR present and D-II predominant in its absence. Kinetic analysis of rechromatographed D-I showed a 20- to 40-fold activation by Ca2+·CDR that was largely due to an increase in Vmax, with only 50% decreases in Km Both substrates competitively inhibited hydrolysis of the other with Ki values equal to their respective Km values (1.7 μM for cyclic GMP and 48 μM for cyclic AMP with Ca2+-CDR present). Studies with theophylline and trifluoperazine indicate differential, substrate-dependent inhibitions of both enzymes. These findings demonstrate that phosphodiesterase activity in neural tissue is subject to regulation by Ca2+, cyclic GMP, and inhibitors in a complex, substrate-specific and concentration-dependent manner.  相似文献   

16.
Incubation of slices of rat cerebral cortex with the calcium ionophore A23187 produced small increases in the accumulation of adenosine 3,5-monophosphate (cyclic AMP). While low concentrations of Ca2+ ions (e.g., 200 µM) were sometimes necessary, the presence of adenosine (e.g., 50 µM) was essential; no effect of ionophore was observed when isoproterenol or isobutylmethylxanthine was substituted for adenosine. These results are consistent with the previously advanced hypothesis that stimulation of -adrenergic receptors in this issue may cause calcium mobilization and thereby produce a calmodulin-mediated stimulation of adenylate cyclase. However, there is no apparent explanation for the requirement for adenosine. In addition, the possibility that additional mechanisms may be operating was suggested by experiments in which the incorporation of 3H-adenine into cyclic AMP was examined under steady-state conditions. While brief exposure to 3H-adenine after maximal adenosine- or isoproterenol-induced accumulations had been achieved led to small increases in the specific activity of cyclic AMP, the combination of norepinephrine and adenosine (plus propranolol) produced substantial decreases in the specific activity of cyclic AMP. Since the rate of incorporation of radioactivity did not keep pace with the expansion of the cyclic AMP pool, it is possible that norepinephrine also caused some reduction in the rate of cyclic AMP degradation under these conditions. Other interpretations of these results are discussed.  相似文献   

17.
The rate constant of modification of a specific thiol group, SH2, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH2 (SH2 region) of skeletal myosin as a structural probe. The rate of Mg2+-ATP-induced SH2 modification of subfragment-1 (S-l) isozymes was regulated by Ca2+ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca2+-dependent rate of SH2 modification. Due to the presence of this Ca2+ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca2+ for DTNB light chain, this Ca2+ regulation in the pCa range above 6.4 is possibly related to the Ca2+ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg2+-ATP-induced SH2 modification of S-1 isozymes equally and of myosin, and reduced the Ca2+ sensitivity with an increase in F-actin concentration.  相似文献   

18.
Fajmut A  Brumen M  Schuster S 《FEBS letters》2005,579(20):4361-4366
Active Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays an important role in the process of MLC phosphorylation and consecutive smooth muscle contraction. Here, we propose a mathematical model of a detailed kinetic scheme describing interactions among Ca2+, CaM and MLCK and taking into account eight different aggregates. The main model result is the prediction of the Ca2+ dependent active form of MLCK, which is in the model taken as proportional to the concentration of Ca4CaM · MLCK complex. Wegscheider’s condition is additionally applied as a constraint enabling the prediction of some parameter values that have not yet been obtained by experiments.  相似文献   

19.
The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca2+. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.  相似文献   

20.
We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca2+/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca2+/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca2+/calmodulin, respectively, but was unaffected in the presence of Ca2+/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.  相似文献   

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