Biomedical subjects

Ben de Kruijff

Publications and source records attributed to Ben de Kruijff.

62 records · Page 4Linked to original sources

PubMed2002

Importance of hydrophobic matching for spontaneous insertion of a single-spanning membrane protein.

In this study, we have investigated the effect of hydrophobic mismatch between the thickness of the membrane and a transmembrane segment of a protein that directly inserts into the membrane bilayer. For this purpose we used mutants of the single-spanning Pf3 coat protein that can spontaneously insert into Escherichia coli membrane vesicles and large unilamellar vesicles (LUVs). The thickness of the liposomal bilayer could be altered by using lipids with different acyl chain lengths or by incorporation of cholesterol. The insertion efficiency of the protein clearly depended on the bilayer thickness, with most efficient insertion under hydrophobic matching conditions. To discriminate between effects of length and hydrophobicity, mutants with different synthetic transmembrane segments were constructed. These mutants inserted into LUVs in a mismatch-dependent manner. However, in particular for longer and less hydrophobic mutants, most efficient insertion was generally observed in thinner bilayers than expected on the basis of hydrophobic matching.

Amino Acid Sequence↗

PubMed2002

Cooperative activity of phospholipid-N-methyltransferases localized in different membranes.

The possibility that the phospholipid-N-methyltransferases from yeast are capable of acting upon a phospholipid substrate, localized in a different membrane than in which the enzymes reside ('trans-catalysis' hypothesis), was investigated using cho2 and opi3 gene disruptant strains, which are defective in phosphatidylethanolamine transferase (PEMT) and phospholipid methyltransferase (PLMT), respectively. When cell homogenates or microsomes of the two disruptant strains are mixed, the combined methyltransferase activity, measured as the incorporation of [(3)H]methyl label from S-adenosyl methionine, exceeds that expected based on the separate activities of PEMT and PLMT. The increased incorporation implies that monomethylphosphatidylethanolamine generated by PEMT becomes available for PLMT, as evidenced by increased synthesis of dimethylphosphatidylethanolamine and phosphatidylcholine. The kinetics of the cooperativity suggest a collision-based process, enabling either transport of substrate or 'trans-catalysis'.

Methylation↗

PubMed2002

Domain formation in phosphatidylcholine bilayers containing transmembrane peptides: specific effects of flanking residues.

Lateral segregation in biological membranes leads to the formation of domains. We have studied the lateral segregation in gel-state model membranes consisting of supported dipalmitoylphosphatidylcholine (DPPC) bilayers with various model peptides, using atomic force microscopy (AFM). The model peptides are derivatives of the Ac-GWWL(AL)(n)WWA-Etn peptides (the so-called WALP peptides) and have instead of tryptophans, other flanking residues. In a previous study, we found that WALP peptides induce the formation of extremely ordered, striated domains in supported DPPC bilayers. In this study, we show that WALP analogues with other uncharged residues (tyrosine, phenylalanine, or histidine at pH 9) can also induce the formation of striated domains, albeit in some cases with a slightly different pattern. The WALP analogues with positively charged residues (lysine or histidine at low pH) cannot induce striated domains and give rise to a completely different morphology: they induce irregularly shaped depressions in DPPC bilayers. The latter morphology is explained by the fact that the positively charged peptides repel each other and hence are not able to form striated domains in which they would have to be in close vicinity. They would reside in disordered, fluidlike lipid areas, appearing below the level of the ordered gel-state lipid domains, which would account for the irregularly shaped depressions.

Amino Acid Sequence↗

PubMed2002

Insertion and glycosylation of Pf3-derived membrane proteins in microsomes.

To get insight into the insertion mechanism of small newly synthesized single-spanning membrane proteins, Pf3 coat protein mutants were constructed with potential glycosylation sites in the N-terminus. Some of these proteins, when synthesized in vitro in the presence of microsomes, became efficiently glycosylated, proving that they insert into the membrane and translocate their N-terminus to the lumenal side. Such Pf3 constructs also insert efficiently into Escherichia coli vesicles and even in pure lipid vesicles, suggesting a common mechanism, which might be spontaneous. Glycosylation was sensitive to changes in the amino acid sequence of the N-terminus, suggesting that it depends on the structure of the protein and/or its positioning with respect to the lipid-water interface.

Amino Acid Sequence↗

PubMed2002

Components required for membrane assembly of newly synthesized K+ channel KcsA.

An Escherichia coli in vitro transcription-translation system was used to study the components involved in the biogenesis of the homotetrameric potassium channel KcsA. We show that a functional signal recognition particle pathway is essential for tetramer formation, probably to direct correct monomer insertion in the membrane. In the absence of YidC or at reduced SecYEG levels, KcsA assembly occurs with lower efficiency. Strikingly, the highest efficiency of tetramerization was observed when transcription-translation was carried out in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal membrane requirement to form the KcsA tetramer. It is concluded that SecYEG and YidC are not required for the formation of tetrameric KcsA in vitro.

Bacterial Proteins↗

PubMed2002

Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.

Energy Metabolism↗

PubMed2002

Phospholipid flip-flop in biogenic membranes: what is needed to connect opposite sides.

Phospholipids are synthesized in biogenic membranes, but only on one leaflet of the bilayer. To support balanced growth of the membrane, phospholipid translocation, or flip-flop, has to occur. Though consensus has been reached that flip-flop is most likely mediated by (a) membrane-associated protein(s), a dedicated flippase has not been identified yet in any biogenic membrane. The characteristics of the flip-flop process are summarized, and possible mechanisms, including the need for a dedicated flippase, are discussed.

Bacteria↗

PubMed2002

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity.

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.

Antineoplastic Agents↗