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D Kamp

Publications and source records attributed to D Kamp.

At least 37 records · Page 2Linked to original sources
PubMed1984

Application of high-performance liquid chromatographic techniques to the separation of ribosomal proteins of different organisms.

The ribosomal proteins from Escherichia coli, Bacillus stearothermophilus and Methanococcus vannielii were separated by size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC), employing new column materials, different gradient systems, and preparative columns, respectively. The purity of the isolated proteins was analysed by one- and two-dimensional gel electrophoresis and by direct micro-sequencing. The separation of ribosomal proteins could be improved by employing propanol gradients in combination with Vydac reversed-phase columns. From the E. coli ribosome, fifteen S and twenty-three L proteins were isolated in sequencer purity by this method. In addition, ion-exchange HPLC was proven to be useful for isolating ribosomal proteins under native conditions: six S proteins and sixteen L proteins from E. coli could be purified. Some of these proteins were not isolated by the reversed-phase procedures, e.g. proteins L9, L14 and L21.

Amino Acid Sequence↗
PubMed1984

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.

Coliphages↗
PubMed1982

Capillary resistance to flow of hardened (diamide treated)red blood cells (RBC).

Pressure-flow curves for control and hardened (diamide treated) human RBC's were obtained in capillaries of the isolated rat mesentery, in order to evaluate resistance to flow of hardened RBC's. Blood vessels were maximally dilated by an infusion of 10(-5) mol/l acetylcholine and isoprenaline and perfused with freshly collected human RBC's as well as with RBC's hardened by a treatment (hct 40%; pH 8.0; 37 degree C) with 0.5 mmol/l or 1.5 mmol/l diamide, respectively, suspended in Albumin (0.05%) - Ringer solution. The mesentery was perfused via a hydrostatic pressure reservoir. Arterio-venous pressure difference was varied from 4-10 kPa, and corresponding arteriolo-venular pressure gradients changed from about 200-500 Pa/mm. No significant difference in resistance to flow was observed between control and diamide treated cells over the whole pressure range. However, the flow through the microvascular bed was inhomogeneous upon perfusion with diamide treated cells, caused by a deceleration and stoppage of the cells at capillary narrowing (ratio of cell to vessel diameter greater than 2). The time of stagnation increased with decreasing pressure gradient.

Animals↗
PubMed1982

Nucleotide sequence of the immunity region of bacteriophage Mu.

The leftmost 1590 bp of Mu DNA covering the immunity region have been sequenced. This region encodes the cI repressor, the cII or ner function and the beginning of gene A. An open reading frame extends from position 863 to 342 on the l-strand corresponding to cI protein with a molecular weight of 19212. It is preceded by a sequence resembling a promoter. To the right of the HindIII site an open reading frame extends from position 1099 to 1323 corresponding to cII or ner protein (molecular weight of 8505) followed by the beginning of gene A at position 1328. Between position 863 and 1099 promoters for leftward and rightward transcription and operator-like structures can be recognized in the sequence. The promoter for rightward transcription overlaps with the HindIII site and coincides with a RNA polymerase binding site as demonstrated by electron microscopy.

Bacteriophage mu↗
PubMed1981

Topology of membrane sulfhydryl groups in the human erythrocyte. Demonstration of a non-reactive population in intrinsic proteins.

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, N-ethylmaleimide. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations. These 'non-reacting' SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labeled N-ethylmaleimide or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with N-[ethyl-2-3H]ethylmaleimide. The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive. After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards N-ethylmaleimide. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.

Erythrocyte Membrane↗
PubMed1981

Penetration of 2,4,5-trinitrobenzenesulfonate into human erythrocytes. Consequences for studies on phospholipid asymmetry.

The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 mumol/l cells) at 37 degrees C. Even at 0 degrees C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway. The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate leads to GSSG + aminodinitrobenzenesulfonate. The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.

Erythrocytes↗
PubMed1981

The relationship of two invertible segments in bacteriophage Mu and Salmonella typhimurium DNA.

A Mu gin- mutant which lacks the function required for inversion of the G segment can be complemented by hybrid plasmids and phages carrying the invertible segment that controls flagellar phase variation in Salmonella typhimurium. This suggests that the same kind of site-specific recombination mechanism is responsible for these inversions. Based on the different features of the two invertible segments we propose a model for their evolutionary relationship.

Genetic Complementation Test↗
PubMed1980

Is "deformability" a parameter for the rate of elimination of erythrocytes from the circulation?

The "deformability" of rat erythrocytes can be gradually decreased by an in vitro treatment with the SH-oxidizing agent diamide. Despite of this reduced deformability the cells are retained in the circulation for many hours when reinfused into the rat. Cells rigidified with glutaraldehyde are even less deformable than diamide treated cells, but also survive for many hours. In contrast to rigidified cells of normal volumes swollen rigidified cells obtained by a heat treatment of erythrocytes are rapidly eliminated. The results support the notion that the "recognition" preceding the elimination of senescent and damaged cells of normal volumes by the reticulo-endothelial system is not solely based on their diminished deformability.

Animals↗
PubMed1980

Effect of reduced red cell "deformability" on flow velocity in capillaries of rat mesentery.

A graded reduction of "deformability" of red blood cells (RBC's) of rats was obtained by treatment with the SH-oxidizing agent, diamide. Rigidified RBC's were injected into rats by isovolemic exchange against 60% of the native RBC's and RBC flow velocities in capillaries of rat mesentery measured. At normal mean arterial pressure RBC flow velocity decreases by 29% in rats receiving cells rigidified with 0.5 mmol . 1(-1) diamide. Surprisingly a further rigidification of erythrocytes by 1.5 mmol . 1(-1) diamide results in a decrease of flow by only 15%. During hypotension RBC flow velocities dropped precipitously to 8 +/- 15% for the 0.5 nmol . 1(-1) and to 2 +/- 6% for the 1.5 mmol . 1(-1) diamide group compared to velocities during normotension. By microscopy we observed a stop of flow in many vessels. This result outlines the importance of a normal red cell "deformability" for the maintenance of sufficient perfusion of the microcirculation, in particular at low blood pressure gradients.

Animals↗
PubMed1978

Reversible inhibition of anion exchange in human erythrocytes by an inorganic disulfonate, tetrathionate.

Tetrathionate (S4O6--) markedly inhibits anion exchange across the human erythrocyte membrane. This phenomenon has been studied in order to obtain further insight into the mechanism of action of reversible inhibitors, in particular disulfonate inhibitors, of anion exchange. Anion fluxes were measured by tracer techniques at equilibrium. The following results were obtained: Tetrathionate, although an inorganic compound, inhibits the self-exchange of sulfate and of divalent organic anions (oxalate, malonate) noncompetitively at Ki values (less than or equal to 0.5 mM) as yet only observed for amphiphilic inhibitors. The inhibitor is effective only from the outside of the cell. The inhibition is temperature-dependent, Ki increasing by a factor of 5 between 5 and 35 degrees C, and instantaneously and fully reversible. The presence of small monovalent anions (fluoride, bromide, chloride, nitrate, acetate) counteracts inhibition by tetrathionate to a varying and concentration-dependent extent, divalent anions have only a minor effect at high concentrations. Chloride exchange is also inhibited, while glycolate and lactate fluxes are much less sensitive or almost insensitive, in agreement with their alleged transfer by a different transport system. Tetrathionate is unique in its inhibitory action, its structural congeners, peroxodisulfate (S2O8--) and ethanedisulfonate (C2H4S2O6--) are much less effective. The results can be interpreted by assuming that tetrathionate inhibits the movement of anions via the inorganic anion exchange system by binding--in a 1 : 1 stoichiometry--to inhibitory "modifier sites", for which it competes with other anions. These sites are located only on the exofacial surface of the membrane. The high affinity of tetrathionate is probably due to a local excess of pi electrons in the region of its central disulfide bond. These may stabilize the binding by their ability to form electron donor-acceptor complexes with membrane sites, thus compensating for the absence of a hydrophobic binding domain in tetrathionate.

Binding Sites↗
PubMed1978

Selective alteration of erythrocyte deformabiliby by SH-reagents: evidence for an involvement of spectrin in membrane shear elasticity.

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reaents on the deformability of human erythrocytes was studied. Deformability was quuantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N, N' -p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Anaalysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides. The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.

Diamide↗
PubMed1978

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane.

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.

Diamide↗