共查询到20条相似文献,搜索用时 15 毫秒
1.
Robert F. Steiner 《Biopolymers》1984,23(6):1121-1135
The interactive binding by calmodulin of Ca2+ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca2+, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca2+ ions. Little further change occurs on binding additional Ca2+ ions. 相似文献
2.
We measured the fluorescence static anisotropy and the time-resolved fluorescence anisotropy decay of F-actin labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine at 20°C in solutions containing 100 mM KCl and free Ca2+ at various concentrations. The average fluorescence anisotropy and the fluorescence rotational correlation time of actin decreased in the presence of micromolar concentrations of free Ca2+. The change of the rotational correlation time of labelled actin could not be explained by a variation of the actin critical concentration. We concluded therefore that F-actin undergoes a conformational change induced by Ca2+ binding. The binding constant was 6 × 106 M?1. 相似文献
3.
Peter K. Lambooy Robert F. Steiner Hal Sternberg 《Archives of biochemistry and biophysics》1982,217(2):517-528
Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca2+. This is diminished in the presence of Ca2+.2 相似文献
4.
The time decay of fluorescence anisotropy was monitored as a function of pH and temperature for complexes of 2,6-toluidinylnaphthalenesulfonate with calmodulin, with its proteolytic fragments, and with the 1:1 complex of calmodulin and melittin. For all the conditions examined the anisotropy decay of native calmodulin involved at least two rotational modes. These corresponded to a short correlation time of 2-3 ns, reflecting a localized motion in the vicinity of the binding site and a longer correlation time which arises from the rotation of a major portion of the molecule. The relative amplitudes of the two rotational modes were dependent upon temperature in the range 11-40 degrees C, the contribution of the more rapid mode increasing with temperature. The maximum immobilization of the probe occurred at pH 5.0 and 12 degrees C. While these results indicate the presence of internal rotations in Ca2+-liganded calmodulin, the magnitude of the longer correlation time is consistent with the crystallographic structure. 相似文献
5.
Isolation of brain Ca2+-calmodulin tubulin kinase containing calmodulin binding proteins 总被引:1,自引:0,他引:1
J R Goldenring B Gonzalez R J DeLorenzo 《Biochemical and biophysical research communications》1982,108(1):421-428
Ca2+-calmodulin tubulin kinase activity was isolated from brain cytosol and separated from its substrate protein, tubulin, and Ca2+ regulatory protein, calmodulin. Characterization of the Ca2+-tubulin kinase system revealed a Km of 4 μM, 0.5 μM, 60 μM for Ca2+, calmodulin and ATP, respectively. The tubulin kinase system bound to a calmodulin affinity column in the presence of Ca2+ and was released from the column by chelation with EGTA. A major 55,000 and a minor 65,000 dalton peptide were identified as the only calmodulin binding proteins in the enzyme fraction, indicating that one or both of these peptides represent the calmodulin binding subunit of the Ca2+-calmodulin tubulin kinase system. 相似文献
6.
In Vitro Stimulation of Protein Kinase C by Melatonin 总被引:2,自引:0,他引:2
Antón-Tay Fernando Ramírez Gerardo Martínez Isabel Benítez-King Gloria 《Neurochemical research》1998,23(5):601-606
It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins. 相似文献
7.
Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca2+/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca2+-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca2+-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca2+/-binding proteins: first calmodulin, which could bind to Ca2+ and the casein kinase-2 form to give a Ca2+/calmodulin-dependent kinase, which could become Ca2+/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit. 相似文献
8.
Parul DA Bokut SB Kisselev PA Milyutin AA Petrov EP Nemkovich NA Sobchuk AN Dzhagarov BM 《Biochemistry. Biokhimii?a》2001,66(4):390-396
Binding of 1,8-anilinonaphthalene sulfonate (1,8-ANS) with native human oxyhemoglobin (Hb) in 50 mM potassium phosphate buffer (pH 7.4) was studied by steady state fluorescence spectroscopy and by laser spectrofluorimetry with subnanosecond time resolution. The distribution of fluorescence decay times and parameters of two- and three-exponential deconvolution of the fluorescence kinetics of 1,8-ANS in Hb solution demonstrate that the emission at wavelengths em of 455-600 nm is not single-exponential and has components with mean decay times <0.5, 3.1-5.5, and 12.4-15.1 nsec with the amplitudes depending on the emission wavelength. Analysis of time-resolved fluorescence spectra shows that the shortest-lived component should be assigned to 1,8-ANS molecules in the aqueous medium, whereas the two longer-lived components are assigned to two types of binding sites for 1,8-ANS in the Hb molecule characterized by different polarity and accessibility to water molecules. 相似文献
9.
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans. 相似文献
10.
Fluorescence dynamics studies of troponin C 总被引:2,自引:0,他引:2
The time decay of fluorescence anisotropy for a dansylaziridine (DANZ) conjugate with Met-25, which lies within the N-terminal lobe of troponin C (TnC), shows at 10 and 25°C a longer correlation time characteristic of the entire molecule and a shorter correlation time arising from a more localized motion of the probe. In the absence of Ca2+, the amplitude of the shorter correlation time increases, suggesting an increased mobility of the probe. At 40°C, in both the absence and presence of Ca2+, a significant increase in probe mobility occurs. A 2,6-toluidinyl naphthalene sulfonate (2,6-TNS) complex with Ca2+-liganded TnC shows only the longer correlation time at 12 and 25°C. An N-(iodoacetylaminoethyl-5-naphthylamine-1-sulfonate) conjugate with Cys-98 shows both a long and a short correlation time; the amplitude of the shorter correlation time is greater than for the DANZ conjugate. At 9, 25, and 40°C in the presence of Ca2+, and at 9°C in its absence, the magnitude of the long correlation time is consistent with motion of the entire molecule; at higher temperatures in the absence of Ca2+ it is substantially smaller, suggesting the presence of internal rotation. For Ca2+-liganded TnC at temperatures of 25°C or lower, the results with all three labels are interpretable in terms of the crystallographic structure of TnC. 相似文献
11.
Calmodulin and the regulation of smooth muscle contraction 总被引:8,自引:0,他引:8
Michael P. Walsh 《Molecular and cellular biochemistry》1994,135(1):21-41
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
12.
Matti Lang 《生物化学与生物物理学报:生物膜》1976,455(3)
A 4% cholesterol diet fed to rats for four weeks was found to increase the phospholipid and cholesterol contents and the activities of drug metabolizing enzymes in rat liver microsomes.Microsomes from rats on a high cholesterol diet were able to enhance the fluorescence of membrane bound 1-anilinonaphthalene 8-sulphonate (1,8-ANS) and ethidium bromide more than microsomes from rats on a standard diet.In the case of 1,8-ANS, the enhanced fluorescence was found to be due to the increased affinity of the molecules for microsomes. In the case of ethidium bromide the fluorescence increased partly because of the larger amount of binding sites and partly because of the enhanced quantum yield of the molecules.P-nitrophenol was found to compete with 1,8-ANS for the same binding sites in microsomes. On the other hand, 1,8-ANS lowered the rate of drug metabolism when present in the incubation mixture.In vitro treatments of microsomes with trypsin, phospholipase A or digitonin altered the binding properties of 1,8-ANS and ethidium bromide to microsomes.It is concluded that the binding sites of 1,8-ANS in microsomes are important for the activity of drug-metabolizing enzymes. The mechanisms of dietary cholesterol in enhancing the drug metabolism and the role of microsomal phospholipids in regulating the activity of drug-metabolizing enzymes are discussed. 相似文献
13.
Alves GG Lima LM Fávero-Retto MP Lemos AP Peres-Sampaio CE Meyer-Fernandes JR Vieyra A Sola-Penna M 《Bioscience reports》2001,21(1):25-32
The plasma membrane (Ca2++Mg2+)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca2+ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca2+ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca2+-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca2+ is related to opposite long-term hydration effects on the substrate binding domain and the Ca2+ binding domain. 相似文献
14.
Genevive Dupont Albert Goldbeter 《BioEssays : news and reviews in molecular, cellular and developmental biology》1998,20(8):607-610
In many cell types, Ca2+ signals are organized in the form of repetitive spikes. The frequency of these intracellular Ca2+ oscillations increases with the level of stimulation, suggesting the existence of a frequency encoding phenomenon. The question arises as to how the frequency of Ca2+ oscillations can be decoded inside the cell. Ca2+/calmodulin kinase II has long been proposed as an attractive candidate, as it is a key target of Ca2+ signals. By immobilizing the Ca2+/calmodulin kinase II and subjecting it to pulses of Ca2+ of variable amplitude, duration, and frequency, De Koninck and Schulman(1) have shown for the first time that the autonomous activity of Ca2+/calmodulin kinase II is highly sensitive to the temporal pattern of Ca2+ oscillations. BioEssays 20 :607–610, 1998.© 1998 John Wiley & Sons Inc. 相似文献
15.
A soluble Ca2+- and Ca2+—calmodulin-activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca2+-dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3-fold and up to 16-fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca2+-dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca2+-dependent fashion. This type of Ca2+-activated protein kinase may be involved in stimulus—response coupling in plants. 相似文献
16.
Walter G. Gonzalez Jaroslava Miksovska 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(9):1472-1480
DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca2 +DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS–DREAM complexes increases upon Ca2 + association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca2 +DREAM were determined to be 195 ± 20 μM and 62 ± 4 μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins. 相似文献
17.
Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle 总被引:3,自引:0,他引:3
Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. 相似文献
18.
Robert J. DeLorenzo 《Cell calcium》1981,2(4):365-385
Ca2+ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca2+ binding protein in brain, mediates the effects of Ca2+ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca2+ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca2+-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca2+ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca2+ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca2+-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca2+ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca2+ signal into a motor force in the process of neurotransmission. 相似文献
19.
Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is a prominent mediator of neurotransmitters which elevate Ca2+. It coordinates cellular responses to external stimuli by phosphorylating proteins involved in neurotransmitter synthesis, neurotransmitter release, carbohydrate metabolism, ion flux and neuronal plasticity. Structure/function studies of CaM kinase have provided insights into how it decodes Ca2+ signals. The kinase is kept relatively inactive in its basal state by the presence of an autoinhibitory domain. Binding of Ca2+/calmodulin eliminates this inhibitory constraint and allows the kinase to phosphorylate its substrates, as well as itself. This autophosphorylation significantly slows dissociation of calmodulin, thereby trapping calmodulin even when Ca2+ levels are subthreshold. The kinase may respond particularly wel to multiple Ca2+ spikes since trapping may enable a spike frequency-dependent recruitment of calmodulin with each successive Ca2+ spike leading to increased activation of the kinase. Once calmodulin dissociates, CaM kinase remains partially active until it is dephosphorylated, providing for an additional period in which its response to brief Ca2+ transients is potentiated.Special issue dedicated to Dr. Paul Greengard. 相似文献
20.
J.David Johnson 《Biochemical and biophysical research communications》1983,112(2):787-793
Felodipine is a fluorescent dihydropyridine Ca2+-antagonist. It binds to calmodulin in a Ca2+-dependent manner, and undergoes a fluorescence increase which allows us to monitor its interaction with calmodulin. Hydrophobic ligands including the calmodulin antagonist, R24571 and Ca2+ antagonists, prenylamine and diltiazem, bind to calmodulin and potentiate felodipine binding by as much as 20 fold. These studies suggest that allosteric interactions occur among different drug binding sites on calmodulin. Our results are discussed in terms of the mechanism of action of calmodulin. 相似文献