共查询到20条相似文献,搜索用时 31 毫秒
1.
On the Role of Calcium Ions in the Regulation of Glycogenolysis in Mouse Brain Cortical Slices 总被引:7,自引:6,他引:1
D. Ververken P. Van Veldhoven C. Proost H. Carton H. De Wulf 《Journal of neurochemistry》1982,38(5):1286-1295
Abstract: Using mouse brain cortical slices, we investigated the relative roles of cyclic AMP and of calcium ions as the intracellular messengers for the activation of glycogen phosphorylase (EC 2.4.1.1; α-1,4-glucan:orthophosphate glucosyltransferase) induced by noradrenaline and by depolarization. Activation of phosphorylase by 100 μM noradrenaline is mediated by β-adrenergic receptors and does not require the copresence of adenosine. The role of the concomitant small increase in cyclic AMP is questioned. Short-term treatment with EGTA or LaCl3 abolishes the noradrenaline activation of phosphorylase, pointing to a critical role of extracellular calcium. Depolarization by 25 m M K+ or 100 μ M veratridine produces a rapid and large (fourfold) activation of phosphorylase. Only veratridine increases the cyclic AMP levels; exogenous adenosine deaminase essentially blocks this cyclic AMP accumulation but not the phosphorylase activation. A halfmaximal activation of phosphorylase occurs at about 12 m M K+ . Addition of EGTA or LaCl3 , reduces the effect of both depolarizations to a slight and transient activation of phosphorylase. These results indicate that activation of glycogen phosphorylase by K+ or veratridine occurs by a cyclic AMP-independent and calcium-dependent mechanism. The calcium dependency of brain phosphorylase kinase renders this kinase the prime target enzyme for regulation of glycogenolysis by calcium ions. 相似文献
2.
Rajeevkumar Raveendran Sudarsana Devi Suma Priya Madhavan Mayadevi Mathew Steephan Thankayyan R. Santhoshkumar John Cheriyan Rajendran Sanalkumar Kurup K. Pradeep Jackson James Ramakrishnapillai V. Omkumar 《Journal of neurochemistry》2009,110(1):92-105
Ca2+ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca2+ /calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca2+ /calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca2+ /calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca2+ /calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca2+ /calmodulin-independent binding whereas it allows the Ca2+ /calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca2+ -independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B. 相似文献
3.
R. Scott Turner C.-H. Jen Chou† Robert F. Kibler† J. F. Kuo 《Journal of neurochemistry》1982,39(5):1397-1404
Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+ , and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca2+ -dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca2+ -dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca2+ -dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7). 相似文献
4.
TOMOYUKI KUBOTA 《Development, growth & differentiation》1979,21(2):155-159
Injection of taurodeoxycholate (TDOC) alone or in combination with 10−5 M Ca2+ (Ca-EGTA buffer) into newt eggs induced a cup- or furrow-like depression on the egg surface. Reduction of the Ca2+ concentration inhibited the response. These findings imply that TDOC induces a cytoplasmic contraction associated with the membrane and that a micromolar Ca2+ concentration is required for this process. Injection of 10−5 M Ca2+ (Ca-EGTA buffer) alone had no effect. On the basis of these findings, the roles of TDOC and Ca2+ in induction of contraction were discussed. 相似文献
5.
Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis 总被引:4,自引:0,他引:4
Abstract: In fetal rat brain neurons, activation of voltage-dependent Na+ channels induced their own internalization, probably triggered by an increase in intracellular Na+ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na+ channel activation. An increase in intracellular Ca2+ concentration induced either by thapsigargin, a Ca2+ -ATPase blocker, or by A23187, a Ca2+ ionophore, was unable to provoke Na+ channel internalization. However, Ca2+ seems to be necessary because both neurotoxin- and amphotericin B-induced Na+ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca2+ /calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na+ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na+ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na+ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na+ channel internalization in neurons. 相似文献
6.
Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca2+ / phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca2+ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca2+ /phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca2+ /phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca2+ /phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca2+ /phospholipid-dependent protein kinase in chromaffin cell function. 相似文献
7.
Yasuyuki Kamata Masatoshi Mita Akiko Fujiwara Tadashi Tojo Jiro Takano Akira Ide Ikuo Yasumasu 《Development, growth & differentiation》1997,39(4):419-428
In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN− -insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+ ]i , (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN− -sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN− -insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2 , induced the envelope formation, acid production, augmentation of CN− -insensitive and sensitive respiration, but did not cause any increase in [Ca2+ ]i , the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN− -insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca2+ -triggered sequential reactions other than Ca2+ -caused activation of phospholipase A2 . 相似文献
8.
Abstract: The Na+ /Ca2+ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na+ /Ca2+ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na+ /Ca2+ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na+ /Ca2+ exchange activity and prolonged cytosolic Ca2+ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na+ /Ca2+ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na+ /Ca2+ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na+ /Ca2+ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na+ /Ca2+ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion. 相似文献
9.
Abstract A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca2+ /calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum . The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca2+ /calmodulin-dependence upon standing at 4°C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [γ-32 P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca2+ and calmodulin but not Ca2+ alone. Ca2+ /calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme. 相似文献
10.
EIGORO TAZAWA AKIKO FUJIWARA MASAYUKI KOMURAKI YUKIO FUJINO IKUO YASUMASU 《Development, growth & differentiation》1985,27(6):777-786
The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased 45 Ca2+ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca2+ level increases associated with fertilization-induced Ca2+ influx and that this stimulates Ca2+ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction. 相似文献
11.
Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca2+ /calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+ /calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca2+ /calmodulin-dependent kinase II such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+ /calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca2+ /calmodulin-dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca2+ /calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca2+ /calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation. 相似文献
12.
Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals 总被引:4,自引:1,他引:3
Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT3 receptor, a ligand-gated ion channel. The 5-HT3 receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca2+ concentration ([Ca2+ ]i ), K+ -induced depolarization resulted in variable but rapid increases in apparent [Ca2+ ]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+ ]i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+ ]i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT3 receptors induced slow increases in [Ca2+ ]i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+ . We conclude that significant increases in brain nerve terminal Ca2+ , rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels. 相似文献
13.
F. M. J. Heemskerk L. H. Schrama W. E. J. M. Ghijsen P. N. E. De Graan F. H. Lopes da Silva W. H. Gispen 《Journal of neurochemistry》1991,56(6):1827-1835
Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca2+ . Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca2+ concentration ([Ca2+ ]i ) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca2+ ]i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K+ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K+ still evoked an increase in [Ca2+ ]i , which was additive to the elevation caused by 4-AP. Several Ca2+ channel antagonists (CdCl2 , LaCl3 , and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca2+ ]i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na+ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca2+ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na+ channel activity. 相似文献
14.
Koh-ichi Sugiyama Izumi C. Mori Koji Takahashi Shoshi Muto Ikuko Shihira-Ishikawa 《Journal of phycology》2000,36(6):1145-1152
The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2 + . Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2 + , and a 40-kDa Ca2 + -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2 + -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2 + receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound. 相似文献
15.
Tomoyoshi Uchikawa Yuji Kiuchi Akihiko Yura Noriyuki Nakachi Yukako Yamazaki Chie Yokomizo Katsuji Oguchi 《Journal of neurochemistry》1995,65(5):2065-2071
Abstract: We studied effects of Ca2+ in the incubation medium on [3 H]dopamine ([3 H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca2+ (0–30 min) and Ca2+ concentration (0–10 m M ) in Krebs-Ringer medium affected [3 H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca2+ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V max of the [3 H]DA uptake process. On the other hand, [3 H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca2+ . The Ca2+ -dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca2+ -free medium following preincubation with 1 m M Ca2+ . Protein kinase C inhibitors did not affect apparently Ca2+ -dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca2+ /calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca2+ -dependent enhancement of [3 H]DA uptake is mediated by activation of calmodulin-dependent protein kinases. 相似文献
16.
Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca2+ -dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid-dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin- and KCI-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+ , it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCI- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C. 相似文献
17.
Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+ ]i ) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+ ]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S -nitrosocysteine (SNOC; 300 µ M ) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+ ]i , persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+ ]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+ ]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+ -activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+ ]i homeostatic mechanisms, and that the resulting persistent [Ca2+ ]i elevations contribute to the delayed neurotoxicity of NO. 相似文献
18.
Abstract: Purified rat brain Ca2+ /calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+ /calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+ -independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC50 =0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+ /CaM on native CaM-kinase II and on peptide CaMK 281-309. 相似文献
19.
Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca2+ - and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca2+ and phosphatidylserine. The apparent molecular mass of the Ca2+ - and phosphatidylserine-dependent protein kinase (Ca2+ /PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca2+ -dependent protein kinase (CDPK) in rice leaves in that Ca2+ /PS PK showed phospholipid dependency and the molecular mass of Ca2+ /PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca2+ /PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca2+ /PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation. 相似文献
20.
The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca2+ signalling by either amplifying Ca2+ entry through voltage-gated Ca2+ channels by Ca2+ -induced Ca2+ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca2+ release (IICR). The ER Ca2+ store emerges as a single interconnected pool, thus allowing for a long-range Ca2+ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca2+ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca2+ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca2+ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia. 相似文献