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1.
Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents 总被引:9,自引:0,他引:9
Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43 000-Mr, 50 000-Mr and 66 000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40 000-Mr and 43 000-Mr chains. The microheterogeneity of the 43 000-Mr chain, but not that of the 40 000-Mr chain, might result from a selective phosphorylation of this particular chain. 相似文献
2.
Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+ 总被引:1,自引:0,他引:1
In particulate fractions from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 microM and reaches a maximum at 100 microM Zn2+. Among other divalent cations tested, Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes. 相似文献
3.
Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn 总被引:30,自引:0,他引:30
Epidermal growth factor stimulates a cyclic AMP-independent protein kinase associated with membrane vesicles derived from human epidermoid carcinoma cells (Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Biol. Chem. 254,. 4884-4891). The kinase specifically phosphorylates tyrosyl residues in a Mr = 150,000 membrane protein (Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255, 8363-8365). We show that the reverse reaction, catalyzed by a phosphotyrosyl-protein phosphatase associated with the membrane, is inhibited by Zn2+. Dephosphorylation of phosphotyrosyl residues in the Mr = 150,000 protein is completely inhibited by Zn2+ at concentrations as low as 10 microM, whereas other divalent cations have no substantial effect. Inhibition of the phosphatase was reversed by EDTA and the activity in membrane preparations was increased by EDTA or fluoride, agents commonly thought to be phosphatase inhibitors. Acid hydrolysis of the membrane proteins followed by analysis of phosphoamino acids by two-dimensional electrophoresis revealed that the phosphatase hydrolyzed phosphotyrosyl in preference to phosphoseryl residues. The specific inhibition of this phosphatase activity by low concentrations of Zn2+ may be indicative of the physiological importance of Zn2+ in the regulation of cellular phosphotyrosyl-protein levels. 相似文献
4.
Phosphorylation Characteristics of Brain Clathrin-Coated Vesicle Endogenous Proteins 总被引:2,自引:2,他引:0
William J. Schook Carlo Parker Walter I. Silva D. Stave Kohtz Saul Puszkin 《Journal of neurochemistry》1987,49(2):434-441
Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components. 相似文献
5.
Starai VJ Thorngren N Fratti RA Wickner W 《The Journal of biological chemistry》2005,280(17):16754-16762
Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion. 相似文献
6.
Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100 总被引:4,自引:0,他引:4
G M Alder W M Arnold C L Bashford A F Drake C A Pasternak U Zimmermann 《Biochimica et biophysica acta》1991,1061(1):111-120
Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer. 相似文献
7.
Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane. 相似文献
8.
Tyrosine phosphorylation of a 55- and 60-kDa protein was observed when EDTA-treated P2 membrane fraction from monkey basal ganglia was incubated with [gamma-32P]-ATP in the presence of Zn2+. Other metal ions were less effective in this phosphorylation. The effect of Zn2+ did not appear to be due to its inhibition of a tyrosine phosphatase. In the presence of Mg2+/Triton X-100 instead of Zn2+, phosphorylation on tyrosine residues of a 17-kDa protein and the external substrate poly(Glu, Tyr) 4:1 copolymer was observed. Both Mg2+ and Triton X-100 were essential for this and Zn2+ inhibited both of these phosphorylations. Convincing evidence for the existence of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases was obtained when the two kinases could be separated by extraction of the membranes by Triton X-100. The Zn2+-dependent phosphorylation was present exclusively in the Triton-solubilized supernatant whereas the Mg2+/Triton X-100-dependent phosphorylation was found associated with the Triton-insoluble membrane fractions. Externally added histone could also be phosphorylated on tyrosine residues in a Zn2+- or Mg2+/Triton X-100-dependent manner by the supernatant or membrane fraction, respectively. 相似文献
9.
Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli. 总被引:4,自引:1,他引:3
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The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli. 相似文献
10.
Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein 总被引:4,自引:0,他引:4
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《The Journal of general physiology》1993,102(4):693-712
We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein. 相似文献
11.
Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons
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We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins. 相似文献
12.
The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca2+-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca2+-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca2+-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes. 相似文献
13.
The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding. 相似文献
14.
Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods 总被引:9,自引:3,他引:6
《The Journal of general physiology》1993,101(1):1-25
The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway. 相似文献
15.
The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg2+, Ca2+, Mn2+, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg2+, 7 mM for Sr2+, 6 mM for Ca2+, 3.5 mM for Ba2+ and 1.8 mM for Mn2+. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn2+ > Ca2+ > Mg2+ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn2+ and Ca2+, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The 相似文献
16.
The broadening of spin-label absorption lines resulting from spin-exchange reactions that occur during collision with paramagnetic Ni2+ is diminished when Ni2+ binds to phospholipid vesicles. Subsequent addition of non-paramagnetic ions that compete for binding sites releases Ni2+ into solution and restores the line-broadening. The concentrations of various ions required to achieve this effect was used to order the ions with respect to their binding to vesicles containing phosphatidylethanolamine and phosphatidylglycerol. The relative strengths of binding for those ions studied were: Ca2+ > Mg2+ > Zn2+ > Sr2+ > Ba2+. The spin-broadening assay was also used to study the effects of two proteins on the availability of Ni2+-binding sites on the vesicles. Ribonuclease, which is thought to associate electrostatically as an extrinsic protein on the surface of vesicles, completely blocked the Ni2+-binding sites at comparatively low protein concentrations. Quantitative considerations of these data suggest the possibility that Ni2+ may bind preferenetially to phosphatidylglycerol, and that these binding sites are aggregated in the ribonuclease-containing vesicles. In contract to ribonuclease, cytochrome c does not block Ni2+-bindings sites on the phospholipid vesicles, but rather contains sites of its own that bind Ni2+, both when the protein is in solution and when it is associated with the vesicles. These results are consistent with other studies which suggest that cytochrome c becomes partially embedded in membrane bilayers and associates with phospholipid molecules through hydrophobic interactions. 相似文献
17.
Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli
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Anton A Weltrowski A Haney CJ Franke S Grass G Rensing C Nies DH 《Journal of bacteriology》2004,186(22):7499-7507
CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons. 相似文献
18.
N V Chichkova A G Evstafieva I G Lyakhov A S Tsvetkov T A Smirnova R N Karapetian E M Karger A B Vartapetian 《European journal of biochemistry》2000,267(15):4745-4752
The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant. 相似文献
19.
Protein kinase and its endogenous substrates in coated vesicles 总被引:3,自引:0,他引:3
Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction. 相似文献
20.
F Hayashi M Sokabe M Takagi K Hayashi U Kishimoto 《Biochimica et biophysica acta》1978,510(2):305-315
A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+. 相似文献