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1.
Salt Activation of Sucrose-Phosphate Synthase from Darkened Leaves of Maize and Other C-4 Plants 总被引:1,自引:0,他引:1
Maize leaf sucrose-phosphate synthase (SPS) has been shown tobe inactivated by protein phosphorylation in vitro, which appearsto be the mechanism of light modulation in situ [Huber and Huber(1991) Plant Cell Physiol. 32: 319326]. The catalyticactivity of the inactivated enzyme (dark form or in vitro inactivatedform) was strongly stimulated by high ionic strength in theassay mixture and at 0.4 M KC1 reached activities similar tothose obtained from illuminated leaves. Numerous salts wereeffective, but for most studies, 0.3 M KC1 was used. The salt-stimulationof enzyme activity was rapid and readily reversible and wasantagonized by the presence of ethylene glycol in the assay.The presence of salt was also found to reduce the IC50 (concentrationrequired for 50% inhibition) for p-chloromercuribenzenesulfonicacid. We postulate that phosphorylation of maize SPS inducesa conformational change in the protein (that affects both maximumcatalytic activity and sensitivity to Pi either through electrostaticor hydrophobic interactions which are affected by high ionicstrength. Salt stimulation of the deactivated enzyme extractedfrom darkened leaves was observed for a variety of C-4 plants,but not for any of the C-3 species tested. (Received October 23, 1990; Accepted January 7, 1991) 相似文献
2.
Ogawa Noriyuki; Yabuta Naohiro; Ueno Yoshihisa; Izui Katsura 《Plant & cell physiology》1998,39(10):1010-1019
Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31
[EC]
] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca2+-requiring protein kinase from Ca2+-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa2+-dependent but calmodulin-independent protein kinase (CDPK).Although the Mr, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an Mr of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed.
1Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan.
2Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan.
4N.O. and N.Y. contributed equally to this work. 相似文献
3.
Three protein kinases which phosphorylate histone were isolatedfrom cellular extract of Lemna plants. They were separated byelution from DEAE-Sephacel column and referred to as PI, PITand PHI. The PI protein kinase activity was partially inhibitedby 10µM cyclic AMP, cyclic GMP or cyclic IMP, while thePII enzyme was activated in the presence of these cyclic nucleotides.The PIII enzyme was cAMPindependent, but slightly inhibitedby cyclic CMP and cyclic UMP. The molecular weights of thesethree protein kinases were 165,000, 85,000 and 145,000, respectively,as estimated from Sephacryl S-300 gel filtration. A single cyclicAMP-binding protein was detected in the PII enzyme fractionby using the photoaffinity cAMP-analogue, 8-N3-cAMP. The proteinwhich specifically bound [3H]-8-N3-cAMP had an apparent molecularweight of 48,000 as determined by SDS-polyacrylamide gel electrophoresis.The phosphorylation of cellular proteins in Lemna was examinedby SDS-polyacrylamide gel electrophoresis. Four phosphorylatedpolypeptides were detected, the phosphorylations of which werestimulated by cAMP. The molecular weights of these four polypeptideswere 59,000, 19,000, 16,000 and 14,000, respectively. (Received January 26, 1983; Accepted April 13, 1983) 相似文献
4.
Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle 总被引:4,自引:0,他引:4
Ca+/calmodulin-dependent protein kinase II(CaM kinase II) has been implicated in the regulation of smooth musclecontractility. The goals of this study were to determine: 1) towhat extent CaM kinase II is activated by contractile stimuli in intactarterial smooth muscle, and 2) the effect of a CaM kinase IIinhibitor (KN-93) on CaM kinase II activation, phosphorylation ofmyosin regulatory light chains (MLC20), and force. Bothhistamine (1 µM) and KCl depolarization activated CaM kinase II witha time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaMkinase II activation was inhibited by KN-93 pretreatment(IC50 ~1 µM). KN-93 inhibited histamine-induced tonicforce maintenance, whereas early force development andMLC20 phosphorylation responses during the entire timecourse were unaffected. Both force development and maintenance inresponse to KCl were inhibited by KN-93. Rapid increases in KCl-inducedMLC20 phosphorylation were also inhibited by KN-93, whereassteady-state MLC20 phosphorylation responses wereunaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did notactivate CaM kinase II and PDBu-stimulated force development wasunaffected by KN-93. Thus KN-93 appears to target a step(s) essentialfor force maintenance in response to physiological stimuli, suggestinga role for CaM kinase II in regulating tonic contractile responses inarterial smooth muscle. Pharmacological activation of protein kinase Cbypasses the KN-93 sensitive step. 相似文献
5.
Equilibration of Adenylates by Maize Leaf Adenylate Kinase: Effects of Magnesium on Apparent and True Equilibria 总被引:2,自引:1,他引:1
In this study, the effect of magnesium on equilibration of adenylatesby purified maize leaf adenylate kinase (AK) was investigated.The equilibration was expressed in terms of either apparentequilibrium constant, defined as Kapp=(ATPtotal)(AMPtotal)/(ADPtotal)2,or true equilibrium constant, defined as Ktrue=(Mg-ATP)(AMPfree)/(Mg-ADP)(ADPfree).At a fixed concentration of free magnesium (1?81?9 mM),the Kapp, and Ktrue were constant at 0?76?0?10 and 6?02?0?75,respectively. On the other hand, at the free magnesium rangeof 0?00l4 to 8?3 mM, the Kapp varied from 0?30 to 1?27, whileremained constant at 5?93?0?31. The data indicate that, contraryto previous speculations, leaf AK does not maintain an equilibriumof total adenylates. Rather, the enzyme governs an equilibriumof Mg-ADP, free ADP, Mg-ATP, and free AMP, which are the truesubstrates/products of the AK reaction. Some implications ofthis finding for studies on energy metabolism in plant tissuesare discussed. Key words: Adenylate kinase, adenylate energy charge, adenylates, C4-photosynthesis, magnesium 相似文献
6.
Cloning, Expression, and Characterization of a Root-Form Phosphoenolpyruvate Carboxylase from Zea mays: Comparison with the C4-Form Enzyme 总被引:2,自引:0,他引:2
Dong Long-Ying; Masuda Takao; Kawamura Takao; Hata Shingo; Izui Katsura 《Plant & cell physiology》1998,39(8):865-873
A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase(PEPC) was isolated. In the coding region, the root-form PEPCshowed 76 and 77% identity with the C4- and C3-form PEPCs ofmaize, respectively, at the nucleotide level. At the amino acidlevel, the root-form was 81 and 85% identical to the C4- andC3-form PEPCs, respectively. The entire coding region was insertedinto a pET32a expression vector so that it was expressed underthe control of T7 promoter. The purified recombinant root-formPEPC had a Vmax value of about 28 mol min1(mg protein)1at pH 8.0. The Km values of root-form PEPC for PEP and Mg2+were one-tenth or less of those of C4-form PEPC when assayedat either pH 7.3 or 8.0, while the value for HCO3 wasabout one-half of that of C4-form PEPC at pH 8.0. Glucose 6-phosphateand glycine had little effect on the root-form PEPC at pH 7.3;they caused two-fold activation of the C4-form PEPC. The Ki(L-malate) values at pH 7.3 were 0.12 and 0.43 raM for the root-and C4-form PEPCs, respectively. Comparison of hydropathy profilesamong the maize PEPC isoforms suggested that several stretchesof amino acid sequences may contribute in some way to theircharacteristic kinetic properties. The root-form PEPC was phosphorylatedby both mammalian cAMP-dependent protein kinase and maize leafprotein kinase, and the phosphorylated enzyme was less sensitiveto L-malate.
1These authors contributed equally to this work.
2Present address: Otsuka Chemical Co. Ltd., 463 Kagasuno, Kawauchi-cho,Tokushima, 771-0130 Japan.
3Present address: Sumitomo Pharmaceuticals Research Center,1-98, Kasugade, Naka 3-cho-me, Konohana-ku, Osaka, 554-0022Japan. 相似文献
7.
Buhagiar Kerrie A.; Hansen Peter S.; Gray David F.; Mihailidou Anastasia S.; Rasmussen Helge H. 《American journal of physiology. Cell physiology》1999,277(3):C461
Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa+-K+pump for Na+. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa+. When pipette solutions wereK+ free, pump current(Ip) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in Ipmeasured with pipettes containingK+. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in Ip wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofIp. We concludethat ANG II regulates the pump's selectivity for intracellularNa+ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade. 相似文献
8.
Brette F Lacampagne A Sallé L Findlay I Le Guennec JY 《American journal of physiology. Cell physiology》2003,285(2):C310-C318
Inactivation of the L-type Ca2+ current (ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only 60% in these cells. Cells infused with either N-methyl-D-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by 200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution. L-type calcium current; calcium-dependent inactivation; facilitation; phosphorylation; cesium 相似文献
9.
Commercially available cell wall-degrading enzymes frequentlyused for protoplast isolation inhibited CO2 fixation and photosyntheticO2 evolution, and stimulated dark respiration by leaf tissueand isolated mesophyll protoplasts of Nicotiana tabacum L. andAntirrhinum majus L. They also depolarized the membrane potentialof cells of leaf tissue, inhibited uptake of 86Rb by tobaccoleaf tissue and isolated mesophyll protoplasts, and stimulated36CI uptake by tobacco leaf tissue. Where studied, these effectswere found to be reversible. The depolarization effect on Antirrhinumleaf cells occurred even when the enzyme preparations had beendenatured, dialysed, or desalted, and the effect was greatestin those fractions of the enzyme preparation which showed thehighest cellulase activity. Plasmolysis of tobacco leaf tissue inhibited photosyntheticO2 evolution, CO2 fixation, and 86Rb uptake to levels belowthose exhibited by isolated protoplasts in media of the samecomposition and osmolarity. The implications of these resultsfor work with leaf tissue and isolated protoplasts are discussed. 相似文献
10.
11.
Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using 14CO2 showed that C4 acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO2 from the bundle sheath cells and decreasedeffectiveness of C4 photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity 相似文献
12.
The Influence of Nitrate and Ammonium Nutrition on the Growth of Wheat (Triticum aestivum) and Maize (Zea mays) Plants 总被引:3,自引:0,他引:3
The effects of NO-3 and NH+4 nutrition on hydroponically grownwheat (Triticum aestivum L.) and maize (Zea mays L.) were assessedfrom measurements of growth, gas exchange and xylem sap nitrogencontents. Biomass accumulation and shoot moisture contents ofwheat and maize were lower with NH+4 than with NO-3 nutrition.The shoot:root ratios of wheat plants were increased with NH+4compared to NO-3 nutrition, while those of maize were unaffectedby the nitrogen source. Differences between NO-3 and NH+4-fedplant biomasses were apparent soon after introduction of thenitrogen into the root medium of both wheat and maize, and thesedifferences were compounded during growth. Photosynthetic rates of 4 mM N-fed wheat were unaffected bythe form of nitrogen supplied whereas those of 12 mM NH+4-fedwheat plants were reduced to 85% of those 12 mM NO-3-fed wheatplants. In maize supplied with 4 and 12 mM NH+4 the photosyntheticrates were 87 and 82% respectively of those of NO-3-fed plants.Reduced photosynthetic rates of NH+4 compared to NO-3-fed wheatand maize plants may thus partially explain reduced biomassaccumulation in plants supplied with NH+4 compared to NO-3 nutrition.Differences in the partitioning of biomass between the shootsand roots of NO-3-and NH+4-fed plants may also, however, arisefrom xylem translocation of carbon from the root to the shootin the form of amino compounds. The organic nitrogen contentof xylem sap was found to be considerably higher in NH+4- thanin NO-3-fed plants. This may result in depletion of root carbohydrateresources through translocation of amino compounds to the shootin NH+4-fed wheat plants. The concentration of carbon associatedwith organic nitrogen in the xylem sap of maize was considerablyhigher than that in wheat. This may indicate that the shootand root components of maize share a common carbon pool andthus differences induced by different forms of inorganic nitrogenare manifested as altered overall growth rather than changesin the shoot:root ratios.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize, nitrogen, growth, photosynthesis, amino acids, xylem 相似文献
13.
Ding Y Schwartz D Posner P Zhong J 《American journal of physiology. Cell physiology》2004,287(2):C413-C421
It has been suggested that L-type Ca2+ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba2+ currents (IBa) through L-type Ca2+ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel activity but did not alter the voltage-dependent characteristics of Ca2+ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-318425 or Go-6983, prevented IBa enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased IBa under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on IBa when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current 相似文献
14.
A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption 总被引:3,自引:0,他引:3
Falin R Veizis IE Cotton CU 《American journal of physiology. Cell physiology》2005,288(5):C1003-C1011
Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (Isc) by 1525%, whereas the addition of ATP to the apical bathing solution decreased Isc by 4060%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive Isc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive Isc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in Isc, but did not alter the EGF- or ATP-induced inhibition of Isc. The DBHQ-mediated decrease in Isc was due to inhibition of basolateral Na+-K+-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na+ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel 相似文献
15.
When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 1045% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K+-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH+-ATPase of maize coleoptiles. Inhibition of the vacuolar H+-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H+-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg1 protein. BafilomycinA1 inhibited the vacuolar H+-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theVmax, of the H+ -ATPase, but had no effect on its Km towardsATP. Key words: Bafilomycin A1, coleoptile, H+-ATPase (vacuolar), maize, Zea mays L 相似文献
16.
Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration 总被引:1,自引:0,他引:1
Previous studies have shown that inhibition of L-type Ca2+ current (ICa) by cytosolic free Mg2+ concentration ([Mg2+]i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of ICa, rat cardiac myocytes and tsA201 cells expressing L-type Ca2+ channels were whole cell voltage-clamped with patch pipettes in which [Mg2+] ([Mg2+]p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca2+ channels (1C/2A/2), increasing [Mg2+]p from 0.2 mM to 1.8 mM decreased peak ICa by 76 ± 4.5% (n = 7). Mg2+-dependent modulation of ICa was also observed in cells loaded with ATP--S. With 0.2 mM [Mg2+]p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP2A) produced large changes in ICa amplitude; however, with 1.8 mM [Mg2+]p, these same manipulations had no significant effect on ICa. With mutant channels lacking principal PKA phosphorylation sites (1C/S1928A/2A/S478A/S479A/2), increasing [Mg2+]p had only small effects on ICa. However, when channel open probability was increased by 1C-subunit truncation (1C1905/2A/S478A/S479A/2), increasing [Mg2+]p greatly reduced peak ICa. Correspondingly, in myocytes voltage-clamped with pipette PP2A to minimize channel phosphorylation, increasing [Mg2+]p produced a much larger reduction in ICa when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg2+ modulates the extent to which channel phosphorylation regulates ICa. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca2+ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A 相似文献
17.
cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct 总被引:3,自引:0,他引:3
It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(GCl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR GCl is independent ofprotein kinase A. We activated G proteins and monitored CFTRGCl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRGCl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR GCl requiredMg2+ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRGCl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa2+, suggesting that Ca2+-dependent proteinkinase C and Ca2+/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with106 M forskolin plus 106 M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR GCl endogenously via a cAMP-independent pathwayin this native absorptive epithelium. 相似文献
18.
Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes 总被引:4,自引:0,他引:4
Hansen PS Buhagiar KA Gray DF Rasmussen HH 《American journal of physiology. Cell physiology》2000,278(3):C546-C553
Insulin enhancesNa+-K+ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na+-K+ pump function in rabbit ventricularmyocytes. Pump current (Ip) was measured using thewhole-cell patch-clamp technique at test potentials(Vms) from 100 to +60 mV. When theNa+ concentration in the patch pipette([Na]pip) was 10 mM, insulin caused aVm-dependent increase in Ip.The increase was ~70% when Vm was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK+ and K+-congeners were excluded from thepatch pipettes. When [Na]pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inIp involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved. 相似文献
19.
In myogenic C2C12 cells, 5 mM creatine increased the incorporation of labeled [35S]methionine into sarcoplasmic (+20%, P < 0.05) and myofibrillar proteins (+50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+40%, P < 0.001). Expression of myosin heavy chain type II (+1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and -alanine did not mimic the effect of creatine, ruling out an osmolarity-dependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70s6k) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70s6k and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70s6k (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (–50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (–55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70s6k pathways in the enhanced differentiation induced by creatine in C2C12 cells. protein synthesis; insulin-like growth factor; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2; 70-kDa ribosomal S6 protein kinase 相似文献
20.
SCAGLIARINI SANDRA; PUPILLO PAOLO; VALENTI VINCENZO 《Journal of experimental botany》1988,39(8):1109-1119
Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.J. exp. Bot. 39: 11091119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40
[EC]
) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min1 mg1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min1 mg1 protein (3.2 ? 0.5 µmolmin1 mg1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min1 mg1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C4, isozymes, malic enzyme, photodifferentiation, Zea mays 相似文献