Search PubMed⌕ Search

Biomedical subjects

B de Kruijff

Publications and source records attributed to B de Kruijff.

At least 19 recordsLinked to original sources
PubMed1998

Fructans interact strongly with model membranes.

Bacterial fructans with a high degree of polymerisation cause a very large increase in surface pressure of lipid monolayers at the air-water interface with a broad range of lipids, including phosphatidylethanolamine and several types of phosphatidylcholines. The surface active effect of fructans contrasts strongly with the maximal effects observed for trehalose, sucrose and glucose under comparable conditions (20 and 0.6 mN/m for fructans and the other sugars, respectively). The results demonstrate a profound and specific membrane interaction of the fructans which is probably very different from the effect of the smaller carbohydrates. The fructan concentrations used in this study are within the physiological range observed in fructan-accumulating plants. The suggested water-stress protective effect of fructans may be induced by membrane-fructan interaction which prevent lipid condensation and phase transitions to take place.

Bacillus subtilis↗
PubMed1998

Translocase-bound SecA is largely shielded from the phospholipid acyl chains.

Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association.

Adenosine Triphosphatases↗
PubMed1998

Rapid transmembrane movement of newly synthesized phosphatidylethanolamine across the inner membrane of Escherichia coli.

For the first time the transmembrane movement of an endogenously synthesized phospholipid across the inner membrane of E. coli is reported. [14C]phosphatidylethanolamine (PE) was biosynthetically introduced into inner membrane vesicles from the PE-deficient strain AD93, by reconstitution with the enzyme phosphatidylserine (PS) synthetase. Upon addition of wild type cell lysate containing PS synthetase, and the metabolic substrates CTP and [14C]serine to inside-out vesicles from AD93, [14C]PS was synthesized, which was for the most part converted into [14C]PE. [14C]PE was introduced in right-side out vesicles by enclosing PS synthetase and CTP in the vesicle lumen and adding [14C]serine. The newly synthesized [14C]PE immediately equilibrated over both membrane leaflets (t1/2 less than one min), as determined by its accessibility toward the amino-reactive chemical fluorescamine. In both inside- out and right-side out vesicles, a 35-65% distribution was found of the newly synthesized PE over the cytoplasmic and periplasmic leaflet, respectively. The transport process of PE was not influenced by the presence of ATP or the proton motive force in inside out vesicles. Pretreatment of both types of vesicles with sulfhydryl reagents, or of right-side out vesicles with proteinase K, did not affect the rate and extent of the transmembrane distribution of the newly synthesized PE.

Biological Transport↗
PubMed1998

Phosphatidylethanolamine mediates insertion of the catalytic domain of leader peptidase in membranes.

Leader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes.

Amino Acid Sequence↗
PubMed1998

The orientation of nisin in membranes.

Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.

Acrylamide↗
PubMed1997

Charge-dependent insertion of beta-lactoglobulin into monoglyceride monolayers.

The interactions between beta-lactoglobulin and 1-monostearoyl-glycerol were studied in order to gain insight into protein-gel-phase monoglyceride interactions. Using a monomolecular layer at the air-water interface, we determined the insertion of beta-lactoglobulin into the monoglycerides under different conditions of protein and surface charge by varying the pH and/or incorporating charged amphiphiles into the monolayer, respectively, and using subphases with either a low or high ionic strength. The interactions were quantified by determining the binding of 14C-labeled beta-lactoglobulin to the monolayer. Our results show the importance of electrostatics for binding of beta-lactoglobulin to condensed monoglycerides. Moreover, electrostatic interactions were found to be important for specific insertion of beta-lactoglobulin into the monolayer. A negatively charged surface in particular allowed positively charged beta-lactoglobulin to insert in a surface charge density-dependent manner, even at surface pressures as high as 36 mN/m, whereas under other conditions, the limiting insertion pressure was 32 mN/m. The rheological properties of the monolayer were not affected by the interactions with beta-lactoglobulin.

Animals↗
PubMed1997

Cisplatin complexes with phosphatidylserine in membranes.

Upon incubation of the anticancer drug cisplatin [cis-diamminedichloroplatinum(II)] with model membranes composed of phosphatidylserine (PS), a stable product is formed that has been isolated after chloroform/methanol extraction of the sample. The product formation is specific for PS and does not occur with other major membrane phospholipids. The rate and extent of product formation is dependent on the pH, chloride ion concentration, and temperature, with the highest rate at pH 6.0, in the absence of Cl- and at 37 degrees C, indicating that positively charged aquated cisplatin is the reactive species. Over 80% of PS is converted within 15 h under these conditions with a halftime of 5 h. PS can be regenerated by an excess of glutathione. Mass spectrometry experiments demonstrate that interaction of cisplatin with PS involves a loss of two chloride ions and coordination of platinum to the amine and carboxyl group of the serine moiety. Cisplatin forms complexes specifically with PS not only in model membranes but also in the plasma membrane of human erythrocytes. Since PS is essential in several cellular processes, its interaction with cisplatin may have important physiological implications.

Antineoplastic Agents↗
PubMed1997

In vitro membrane integration of leader peptidase depends on the Sec machinery and anionic phospholipids and can occur post-translationally.

A cell-free system based on a lysate and membrane vesicles from Escherichia coli is used to study characteristics of the membrane integration reaction of the polytopic membrane protein leader peptidase (Lep). Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. Integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.

Adenosine Triphosphatases↗
PubMed1997

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.

Alkaline Phosphatase↗
PubMed1997

The anionic phospholipid-mediated membrane interaction of the anti-cancer drug doxorubicin is enhanced by phosphatidylethanolamine compared to other zwitterionic phospholipids.

The interaction of doxorubicin and lipids has been studied using large unilamellar vesicles (LUVET) composed of mixtures of anionic phospholipids and various zwitterionic phospholipids. Dilution of anionic lipids with zwitterionic lipids leads to decreased membrane association of the drug because electrostatic forces are very important in doxorubicin-membrane interaction. However, binding of doxorubicin to LUVET composed of anionic phospholipids combined with phosphatidylethanolamine (PE) is much higher than binding to LUVET made of anionic lipids plus a range of other zwitterionic lipids such as phosphatidylcholine (PC) and the N-methylethanolamine and N, N-dimethylethanolamine derivatives of PE. This preferential interaction is observed with all negatively charged phospholipids tested and is, in the case of phosphatidylserine (PS), confirmed in monolayer experiments. The increase in surface area observed in a monolayer composed of PS and PE (1/3) was 3 times higher than in a monolayer of PS/PC (1/3). The preferential interaction appears not to be due to the ability of PE to adopt inverted nonbilayer structures, but probably involves a combination of the ability of PE to form additional hydrogen bonds and of the intrinsic curvature of a bilayer containing PE because of its small headgroup. Implications of our finding for the in vivo membrane interaction and transport of the drug will be discussed.

Anions↗
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

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233-1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.

Animals↗
PubMed1997

A freeze-fracture study of the membrane morphology of phosphatidylethanolamine-deficient Escherichia coli cells.

Freeze-fracture electron microscopy was applied to study membrane morphology in a phosphatidylethanolamine-deficient E. coli strain. For growth, this strain requires millimolar concentrations of specific divalent cations like Mg2+ or Ca2+. These cations bring the bilayer to nonbilayer phase transition temperature of the lipids back to wild type levels by shifting the phase preference of cardiolipin in the membrane towards the inverted hexagonal (H(II)) phase. Under growth conditions, these cells show a bilayer based membrane with an intramembrane particle distribution as in wild type cells. Upon lowering the temperature, smooth areas are observed corresponding to gel state lipid bilayer domains. Ca2+ was used to manipulate the phase behavior of the membrane lipids in situ. Exposing the cells to Ca2+ up to 100 mM at 42 degrees C did not result in the appearance of nonbilayer structures, despite the fact that in total lipid extracts under these conditions the hexagonal H(II) phase was observed. However, the addition of a Ca2+ ionophore, which leads to exposure to Ca2+ of both faces of the plasma membrane, gives rise to formation of H(II) phase, stacked bilayer domains and blebbing upon addition of 50 mM CaCl2 at 42 degrees C. We conclude that the asymmetrical localization of divalent cations in the periplasm of this strain allows them to be functionally effective while membrane stability is maintained.

ATP-Binding Cassette Transporters↗
PubMed1997

Lipid polymorphism and biomembrane function.

In recent years, several major developments have taken place in the biology, physical chemistry and technology of polymorphism of membrane lipids. These include the identification of polymorphic regulation of membrane lipid composition in Escherichia coli, the importance of nonbilayer lipids for protein functioning, the special packing properties of bilayers containing these lipids, and the crystallization of a membrane protein out of three dimensional bilayer networks (lipid cubic phases). These exciting developments bring us closer to understanding the paradox of the lipid bilayer structure of biomembranes and the molecular basis of membrane protein structure and function.

Bacteriorhodopsins↗
PubMed1996

Transport of the anti-cancer drug doxorubicin across cytoplasmic membranes and membranes composed of phospholipids derived from Escherichia coli occurs via a similar mechanism.

An assay was developed to measure and directly compare transport of doxorubicin across right-side-out cytoplasmic membrane vesicles (ROV) and across model membranes (LUVET) composed of pure phospholipids, isolated from the corresponding cells. Escherichia coli was used as a model organism, since mutants are available which differ in phospholipid composition. Both in LUVET and ROV only passive diffusion across the bilayer is involved, because effects of drug concentration, pH, divalent cations, the phospholipid composition, and the active transport inhibitor verapamil were comparable. Permeability coefficients were about 2-3-times higher in ROV compared to LUVET. Furthermore, in LUVET an average activation energy of 87 kJ/mol and in ROV of 50 kJ/mol was observed. These differences are suggested to result from differences in membrane order between LUVET and ROV and differences in the temperature dependence of membrane order in LUVET and ROV, respectively. Because no background carrier-facilitated doxorubicin transport seems to be present, ROV are an excellent model system to study the effect of phospholipid composition on drug transport after expression of a multidrug resistance-conferring protein. Furthermore, data of passive diffusion of doxorubicin obtained with LUVET are representative for more complex, biologically relevant membrane systems.

Antibiotics, Antineoplastic↗
PubMed1996

The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration.

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.

Anions↗