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R A Demel

Publications and source records attributed to R A Demel.

At least 19 recordsLinked to original sources
PubMed1997

Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity.

Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

Amino Acid Sequence↗
PubMed1997

The C-terminal region of nisin is responsible for the initial interaction of nisin with the target membrane.

The interaction of nisin Z and a nisin Z mutant carrying a negative charge in the C-terminus ([Glu-32]-nisin Z) with anionic lipids was characterized in model membrane systems, and bacterial membrane systems. We focused on three possible steps in the mode of action of nisin, i.e., binding, insertion, and pore formation of nisin Z. Increasing amounts of anionic lipids in both model and natural membranes were found to strongly enhance the interaction of nisin Z with the membranes at all stages. The results reveal a good correlation between the anionic lipid dependency of the three stages of interaction, of which the increased binding is probably the major determinant for antimicrobial activity. Maximal nisin Z activity could be observed for negatively charged lipid concentrations exceeding 50-60%, both in model membrane systems as well as in bacterial membrane systems. We propose that the amount of negatively charged lipids of the bacterial target membrane is a major determinant for the sensitivity of the organism for nisin. Nisin Z induced leakage of the anionic carboxyfluorescein was more efficient as compared to the leakage of the potassium cation. This lead to the conclusion that an anion-selective pore is formed. In contrast to the results obtained for nisin Z, the binding of [Glu-32]-nisin Z to vesicles remained low even in the presence of high amounts of negatively charged lipids. The insertion and pore-forming ability of [Glu-32]-nisin Z were also decreased. These results demonstrate that the C-terminus of nisin is responsible for the initial interaction of nisin, i.e., binding to the target membrane.

Amino Acid Sequence↗
PubMed1997

Dependence of the activity of phospholipase C beta on surface pressure and surface composition in phospholipid monolayers and its implications for their regulation.

We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was more active than PLC beta 2, even at high surface pressure. Reduced enzyme activity was accompanied by an increase in reaction induction times, suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 4-fold when the mol % of PIP2 in the monolayer increased from 17.9% to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity.

Enzyme Activation↗
PubMed1996

Influence of the signal sequence and chaperone SecB on the interaction between precursor protein prePhoE and phospholipids.

To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 [corrected] nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers. It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB. Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers. The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB-prePhoE complex upon binding of the precursor to the bilayer. The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made.

Bacterial Proteins↗
PubMed1996

Nisin Z, mutant nisin Z and lacticin 481 interactions with anionic lipids correlate with antimicrobial activity. A monolayer study.

Monomolecular layers of lipids at the air/water interface have been used as a model membrane to study membrane interactions of the lantibiotic nisin. The natural lantibiotics nisin A and nisin Z proved to have a high affinity for the anionic lipids phosphatidylglycerol and bis(phosphatidyl)glycerol (cardiolipin). The interaction with zwitterionic phopholipids or neutral lipids is very low at surface pressures higher than 32 mN/m. Nisin, nisin mutants and lacticin 481 show a remarkable correlation between anti-microbial activity and anionic lipid interaction. The results indicate that primarily the N-terminal part (residues 1-22) penetrates into the lipid phase. Reduction of the flexibility at positions 20-21 has a negative effect on monolayer interaction and activity. The C-terminal part is probably responsible for ionic interactions of nisin in monomeric or oligomeric form with anionic lipids. In mixtures of anionic and zwitterionic lipids maximal interactions are found at approximately 70 mol/100 mol anionic lipid. Gram-positive bacteria, which form the main target for nisin, are characterized by a high content of anionic lipids in the membrane. Monolayers formed of lipid extracts of bacteria sensitive to nisin were more strongly penetrated than those of bacteria relatively insensitive to nisin.

Amino Acid Sequence↗
PubMed1996

Insertion of pea lectin into a phospholipid monolayer.

Pea lectin (PSL) is a secretory sugar-binding protein, readily soluble in aqueous solutions of low osmolarity. However, PSL also appears to be associated with the plasma membrane at the tip of young pea root hairs. By using the Wilhelmy plate method, we found that PSL can insert into a lipid monolayer. This property appeared to be independent of the sugar-binding ability of the protein. This result suggests that PSL may be directly involved in membrane-mediated interactions with saccharide ligands, for example during root hair infection by symbiotic rhizobia.

Carbohydrate Metabolism↗
PubMed1996

Phase behavior of stratum corneum lipids in mixed Langmuir-Blodgett monolayers.

The lipids found in the bilayers of the stratum corneum fulfill the vital barrier role of mammalian bodies. The main classes of lipids found in stratum corneum are ceramides, cholesterol, and free fatty acids. For an investigation of their phase behavior, mixed Langmuir-Blodgett monolayers of these lipids were prepared. Atomic force microscopy was used to investigate the structure of the monolayers as a function of the monolayer composition. Three different types of ceramide were used: ceramide extracted from pigskin, a commercially available ceramide with several fatty acid chain lengths, and two synthetic ceramides that have only one fatty acid chain length. In pigskin ceramide-cholesterol mixed monolayers phase separation was observed. This phase separation was also found for the commercially available type III Sigma ceramide-cholesterol mixed monolayers with molar ratios ranging from 1:0.1 to 1:1. These monolayers separated into two phases, one composed of the long fatty acid chain fraction of Sigma ceramide III and the other of the short fatty acid chain fraction of Sigma ceramide III mixed with cholesterol. Mixtures with a higher cholesterol content consisted of only one phase. These observations were confirmed by the results obtained with synthetic ceramides, which have only one fatty acid chain length. The synthetic ceramide with a palmitic acid (16:0) chain mixed with cholesterol, and the synthetic ceramide with a lignoceric acid (24:0) chain did not. Free fatty acids showed a preference to mix with one of these phases, depending on their fatty acid chain lengths. The results of this investigation suggest that the model system used in this study is in good agreement with those of other studies concerning the phase behavior of the stratum corneum lipids. By varying the composition of the monolayers one can study the role of each lipid class in detail.

Animals↗
PubMed1995

Neutralization of the positive charges of surfactant protein C. Effects on structure and function.

Pulmonary surfactant protein C (SP-C) is a small, extremely hydrophobic peptide with a highly conservative primary structure. The protein is characterized by two adjacent palmitoylated cysteine residues, two positively charged residues (one arginine residue and one lysine residue) in the N-terminal region, and a long hydrophobic stretch. SP-C enhances the adsorption of phospholipids into an air-water interface. To determine the importance of the positively charged residues, we carried out experiments with natural porcine SP-C and modified porcine SP-C (SP-Cm) in which the positive charges had been blocked by phenylglyoxal. Circular dichroism experiments showed that SP-Cm had an increased content of alpha-helix. Natural SP-C, but not SP-Cm, catalyzed insertion of phospholipids into a monolayer at the airwater interface. This reduced insertion was due to a strong reduction of binding of phospholipid vesicles to the monolayer. The insertion catalyzed by the natural porcine SP-C was decreased by an increased pH of the subphase. In contrast to natural SP-C, SP-Cm induced lipid mixing between phospholipid vesicles. The extent of lipid mixing was a function of the SP-C content. We conclude that the positively charged residues of SP-C are important for the binding of phospholipid vesicles to the monolayer, a process that precedes the insertion of phospholipids into the monolayer.

Animals↗
PubMed1995

Characterization of a dimeric canine form of surfactant protein C (SP-C).

Canine pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide which has one palmitoylated cysteine residue. SP-C enhances the insertion of phospholipids into a monolayer. Two forms of canine SP-C were isolated using Sephadex LH-60 chromatography. It was found that canine SP-C exists in a palmitoylated monomeric form of 3.5 kDa, and a non-acylated dimeric form of 7 kDa. Circular dichroism showed that both forms of SP-C exhibit similar secondary structures at the air/water interface. Both forms of SP-C were able to induce the insertion of phospholipids into a monolayer as measured with the Wilhelmy plate technique. In contrast to the palmitoylated monomeric form of SP-C, the non-acylated dimeric form of SP-C does not require calcium ions to insert phospholipids into a monolayer without the negatively charged phosphatidylglycerol. It is concluded that two forms of canine SP-C exist, but the physiological significance of these different forms remains to be established.

Animals↗
PubMed1994

Presequence-mediated intermembrane contact formation and lipid flow. A model membrane study.

The ability of a synthetic peptide corresponding to the presequence of cytochrome c oxidase subunit IV from yeast to cause intermembrane contacts was investigated using monolayer techniques. The presequence inserted efficiently into the monolayer with a specificity for the mitochondrial cardiolipin. In the inserted form, the peptide strongly promoted the formation of close contacts with large unilamellar lipid vesicles present in the subphase, a property which was also specific for cardiolipin. The contacts formed were stable and tight and resulted in the flow of lipids from the vesicles to the monolayer. These results led to new suggestions on the involvement of intermembrane contact formation in mitochondrial protein import and membrane biogenesis.

Amino Acid Sequence↗
PubMed1994

Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C.

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.

Animals↗
PubMed1993

Effect of acylation on structure and function of surfactant protein C at the air-liquid interface.

Pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide that is palmitoylated on 2 adjacent cysteine residues. SP-C enhances the adsorption of phospholipids into a monolayer. The function of the acylation is not clear yet. The experiments described in this article were carried out in order to investigate the function of SP-C acylation in (protein-catalyzed) lipid monolayer formation, and in bilayer interactions. Palmitoylated and nonpalmitoylated human recombinant SP-C were used. In addition, a nonacylated SP-C with a Cys-->Ser mutations was included in these studies. In Wilhelmy plate experiments using negatively charged, protein-containing phospholipid monolayers and negatively charged vesicles, CaCl2 was required to obtain a maximal insertion rate of lipids into the monolayer. If the negatively charged phospholipids in the monolayer were replaced by neutral phospholipids, CaCl2 was only required to show a maximal SP-C-catalyzed insertion rate (if the molecule is palmitoylated, but not if nonpalmitoylated proteins were added). In pressure area measurements, the palmitoylated protein showed a different change in pressure as a function of the surface area, as compared with the nonpalmitoylated proteins. Circular dichroism experiments showed that all three proteins had a high content of alpha-helix. All three proteins showed a preferential orientation at the air-water interface, but the palmitoylated protein has an orientation which is more parallel to the monolayer than that of the nonpalmitoylated proteins. It is concluded that acylation of SP-C alters structural and physical properties of this protein.

Acylation↗
PubMed1993

The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids.

The interaction of the precursor of the chloroplast protein ferredoxin with membrane lipids was studied in monolayer experiments in order to investigate the possible involvement of membrane lipids in the protein translocation process. The precursor efficiently and specifically inserts into a total lipid extract of its biological target the outer envelope membrane of chloroplasts. This interaction is mediated by the transit sequence as it can also be observed for the chemically prepared transit peptide of ferredoxin but neither for the ferredoxin apoprotein nor holoprotein. Interactions with the individual chloroplast lipids, monogalactosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, and phosphatidylglycerol are predominantly involved which corresponds to the results obtained for transit peptide fragments of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (van't Hof, R., Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol and phosphatidylcholine, suggesting that a loose lipid headgroup packing due to small lipid headgroups and/or electrostatic repulsions facilitates efficient insertion. The observed preferences for interaction of the precursor and transit peptide of ferredoxin for the chloroplast outer envelope membrane lipid extract and the presequence of cytochrome c oxidase subunit IV for the mitochondrial outer membrane lipid extract indicate that targeting sequence-lipid interactions contribute to organelle-specific protein targeting.

Amino Acid Sequence↗
PubMed1993

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane.

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.

Adenosine Triphosphatases↗
PubMed1992

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Colicins↗
PubMed1992

Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption.

In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatine > alpha-chaconine > alpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.

Calorimetry, Differential Scanning↗
PubMed1992

Insect apolipophorin III: interaction of locust apolipophorin III with diacylglycerol.

In the formation of low-density lipophorin (LDLp) by the loading of diacylglycerol onto high-density lipophorin (HDLp) in insect hemolymph, apolipophorin III (apoLp-III) plays an essential role by binding to the increasing surface of the expanding lipoprotein particle. The present data on the surface properties of apoLp-III from Locusta migratoria demonstrate a preferential interaction with diacylglycerol. Injection of apoLp-III underneath a diacylglycerol monolayer results in a rapid interaction with the lipid; interaction with a phosphatidylcholine monolayer was considerably less. Locust apoLp-III binds with high affinity (Kd = 7.9.10(-9) M) to 1,2-diacylglycerol, which is consistent with its function in the LDLp particle; affinity for phosphatidylcholine is considerably lower. While the molecular area of locust apoLp-III in a monolayer is 2080 A2/molecule at the collapse pressure, in mixed monolayers of apoLp-III and lipid, the mean molecular area is decreased. Deglycosylation of the apoLp-III did not affect its interfacial stability. ApoLp-III from the moth Manduca sexta, which we included for comparison, demonstrated a similar reduction in molecular area resulting from interaction with lipid. These data do not support the hypothesis that interaction of apoLp-III with a lipid surface will lead to doubling of the molecular area of the protein (Kawooya, J.K., Meredith, S.C., Wells, M.A., Kézdy, F.J. and Law, J.H. (1986) J. Biol. Chem. 261, 13588-13591). The area of locust apoLp-III of 12.9 A2/amino acid residue at the collapse pressure is consistent with monolayers of alpha-helical proteins; circular dichroic spectra confirm a high alpha-helix content. The surface properties of apoLp-III reported here enable a high surface concentration of diacylglycerol in the LDLp particle, allowing the lipoprotein to act as an efficient reutilizable lipid shuttle.

Animals↗