共查询到20条相似文献,搜索用时 15 毫秒
1.
F Meggio B Boldyreff O Marin F Marchiori J W Perich O G Issinger L A Pinna 《European journal of biochemistry》1992,205(3):939-945
The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either alone or in combination with the recombinant non-catalytic beta subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becomes a good substrate for CK2 (Km 14.2 microM, Kcat 4.6 mol.min-1.mol CK2-1). The recombinant alpha subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant alpha subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant beta subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the beta-subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8-fold, with 15 nM polylysine being required for half-maximal stimulation, a stimulation of only 1.9-fold, with 80 nM polylysine required for half-maximal stimulation, is attained with recombinant alpha subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate. 相似文献
2.
3.
Gerda M. Hübner Jane Nøhr Larsen Barbara Guerra Karsten Niefind Milka Vrecl Olaf-Georg Issinger 《Molecular and cellular biochemistry》2014,397(1-2):285-293
Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET2 pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP2. This BRET2 sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET2 sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET2 signal by up to 50 %. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50 %. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET2 sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors. 相似文献
4.
Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release. 相似文献
5.
Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, -subunits, and two regulatory, -subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is down regulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit, and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30°C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2 subunit and protein substrates share a common, or at least an overlapping site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine. 相似文献
6.
Benjamin Mentzel Eike Jauch Thomas Raabe 《Biochemical and biophysical research communications》2009,379(2):637-642
The role of CK2β has been defined as the regulatory subunit of protein kinase CK2, which is a heterotetrameric complex composed of two CK2β and two catalytic active CK2α subunits. The identification of other serine/threonine kinases such as A-Raf, Chk1, and c-Mos that interact with and are regulated by CK2β has challenged this view and provided evidence for functions of CK2β outside the CK2 holoenzyme. In this report we describe the first interaction of Drosophila CK2β outside the CK2 holoenzyme with p21-activated kinase (PAK) proteins. This interaction is seen for distinct PAK and CK2β isoforms. In contrast to the CK2α–CK2β interaction, dimer formation of the CK2β subunits is not a prerequisite for binding of PAK proteins. Our results support the idea that CK2β can bind to PAK proteins in a CK2α independent manner and negatively regulates PAK kinase activity. 相似文献
7.
CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster. 总被引:1,自引:0,他引:1
Alla I Kalmykova Yuri Y Shevelyov Oksana O Polesskaya Anna A Dobritsa Alexandra G Evstafieva Brigitte Boldyreff Olaf-Georg Issinger Vladimir A Gvozdev 《European journal of biochemistry》2002,269(5):1418-1427
An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis. 相似文献
8.
To clarify the control mechanism of the catalytic activity of casein kinase 2 (CK2) during early embryonic development in the silkworm, Bombyx mori, we attempted an in-vitro functional analysis by using the recombinant alpha and beta subunits of B. mori CK2 (rBmCK2alpha and rBmCK2beta) produced in a bacterial system. The renatured rBmCK2alpha possessed protein kinase activity. When rBmCK2alpha and rBmCK2beta were reconstituted in an approximate 1:1 molar ratio, the catalytic activity was almost the same as that of rBmCK2alpha alone. The catalytic activity of rBmCK2alpha was inhibited by polylysine, which is one of the activators of CK2 activity. However, when using the reconstituted rBmCK2alpha and rBmCK2beta (rBmCK2), activation by polylysine was observed. We examined the influence of sorbitol and 3-hydroxykynurenine (3-OHK), which are contained mainly in diapause eggs, on the phosphorylation activity of rBmCK2. Three-OHK inhibited rBmCK2 activity, but sorbitol had no effect on it. Furthermore, a functional analysis using rBmCK2alpha and beta subunits of Drosophila melanogaster CK2 revealed that a difference in the C-terminal amino acid of the CK2beta subunit influenced the phosphorylation activity of rBmCK2alpha. These results may provide new insights for clarifying the control mechanism of B. mori casein kinase 2 in eggs. 相似文献
9.
Arrigoni G Marin O Pagano MA Settimo L Paolin B Meggio F Pinna LA 《Biochemistry》2004,43(40):12788-12798
Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine. 相似文献
10.
Pagano MA Arrigoni G Marin O Sarno S Meggio F Treharne KJ Mehta A Pinna LA 《Biochemistry》2008,47(30):7925-7936
Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500-518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2alpha catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500-518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the beta-subunit. Such a stimulatory efficacy of the CFTR 500-518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2. 相似文献
11.
Chen Z Otto JC Bergo MO Young SG Casey PJ 《The Journal of biological chemistry》2000,275(52):41251-41257
After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation. 相似文献
12.
1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin. 相似文献
13.
Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed. 相似文献
14.
G Dobrowolska F Meggio J Szczegielniak G Muszynska L A Pinna 《European journal of biochemistry》1992,204(1):299-303
Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase-2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the alpha/alpha' subunits of animal CK2 and devoid of the autophosphorylatable 25-kDa alpha subunit of animal CK2, which display an heterotetrameric alpha 2 beta 2/alpha alpha' beta 2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39,000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38,000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical beta subunits; (3) the 39-kDa band immunoreacts with antisera that recognize the alpha subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the beta subunit could be detected in CKIIB by Western-blot analyses with antisera that recognize animal beta subunits; (5) the recombinant beta subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation. While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non-catalytic subunits. Its properties, compared to those of animal CK2, suggest that the beta subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit. 相似文献
15.
Raaf J Bischoff N Klopffleisch K Brunstein E Olsen BB Vilk G Litchfield DW Issinger OG Niefind K 《Biochemistry》2011,50(4):512-522
The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design. 相似文献
16.
In Drosophila, protein kinase CK2 regulates a diverse array of developmental processes. One of these is cell-fate specification (neurogenesis) wherein CK2 regulates basic-helix-loop-helix (bHLH) repressors encoded by the Enhancer of Split Complex (E(spl)C). Specifically, CK2 phosphorylates and activates repressor functions of E(spl)M8 during eye development. In this study we describe the interaction of CK2 with an E(spl)-related bHLH repressor, Deadpan (Dpn). Unlike E(spl)-repressors which are expressed in cells destined for a non-neural cell fate, Dpn is expressed in the neuronal cells and is thought to control the activity of proneural genes. Dpn also regulates sex-determination by repressing sxl, the primary gene involved in sex differentiation. We demonstrate that Dpn is weakly phosphorylated by monomeric CK2α, whereas it is robustly phosphorylated by the embryo-holoenzyme, suggesting a positive role for CK2β. The weak phosphorylation by CK2α is markedly stimulated by the activator polylysine to levels comparable to those with the holoenzyme. In addition, pull down assays indicate a direct interaction between Dpn and CK2. This is the first demonstration that Dpn is a partner and target of CK2, and raises the possibility that its repressor functions might also be regulated by phosphorylation. 相似文献
17.
Recombinant murine BID protein was used as an in vitro substrate for the CK2 holoenzyme and the catalytic CK2alpha subunit. The results obtained show that BID can only serve as a substrate for the catalytic CK2alpha subunit. Phosphorylation of BID using the CK2 holoenzyme was only possible in the presence of polylysine, supporting the notion that BID behaves similarly to calmodulin. Co-immunoprecipitation of BID and CK2 subunits revealed that BID is preferentially associated with the CK2alpha subunit. Enzyme kinetic analyses yielded a Km value for BID that is a level of magnitude lower than that measured for casein and the synthetic peptide, suggesting more specific and tight binding of BID to CK2alpha. In contrast are the Vmax values observed, with a significantly higher phosphorylation rate measured for casein and the synthetic peptide than for BID. When BID was phosphorylated by polylysine-stimulated CK2 holoenzyme prior to caspase-8 cleavage, the formation of tC-BID was reduced in comparison to treatment with caspase-8 in the absence of protein kinase. Mass spectrometric analysis of BID phosphorylated by CK2alpha before and after cleavage with caspase-8 showed phosphorylation of residues Thr58 and Ser76. 相似文献
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19.
The interaction of polylysine and partially substituted dansyl, fluorescein, and quinacrine conjugates of polylysine with cytological preparations of human metaphase chromosomes has been studied by fluorescence microscopy. The fluorescence intensity along chromosomes stained with the dansyl and fluorescein conjugates exhibits little variation, suggesting that regions capable of binding these polycations are nearly evenly distributed. In contrast, the quinacrine derivatives of polylysine stain the chromosomes in a banded fluorescence pattern resembling that observed following quinacrine or quinacrine mustard treatment. 相似文献
20.
The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule. 相似文献