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J Brunner

Publications and source records attributed to J Brunner.

At least 109 records · Page 6Linked to original sources
PubMed1986

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid.

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.

Actins↗
PubMed1986

Tetanus toxin is labeled with photoactivatable phospholipids at low pH.

The mechanism of cell penetration by tetanus toxin is unknown; it has been suggested that the toxin may penetrate into the lipid bilayer from a low-pH vesicular compartment. In this work, the interaction of tetanus toxin with liposomal model membranes has been studied by following its photoinduced cross-linking with either a nitrene or a carbene photolytically generated from corresponding light-sensitive phosphatidylcholine analogues. The toxin was labeled only at pHs lower than 5.5. The low pH acquired hydrophobicity of tetanus toxin appears to be confined to its light chain and to the 45-kDa NH2-terminal fragment of the heavy chain. Negatively charged lipids promote the interaction of this toxin with the hydrocarbon chain of phospholipids. The relevance of the present findings to the possible mechanism of nerve cell penetration by tetanus toxin is discussed.

Carbon Radioisotopes↗
PubMed1985

Membrane topology of light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+. Amino acid residues in contact with the lipid bilayer as inferred from labeling with photogenerated carbenes.

The amino acid residues of the light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+ that in the chromatophore membranes are in contact with the lipid phase were identified by hydrophobic photolabeling. Three reagents have been used which all contained the trifluoromethyldiazirinylphenyl group as a photo-sensitive precursor of a carbene but which otherwise differed in shape, molecular structure, and in the way they interact with membranes. 3-Trifluoromethyl-3-(m-[125I]iodophenyl)diazirine is a highly lipid-soluble compound, 11-[4-[(trifluoromethyl)diazirinyl]-phenyl]-[10-3H] 9-oxaundecanoic acid is an analogue of a fatty acid, and 1-palmitoyl-2-[11-[(trifluoromethyl)diaziri-nyl] phenyl]-[10-3H]9-oxaundecanoyl]-sn-glycero-3-phosphorylcholine one of a lecithin. Following labeling of chromatophores with these reagents, B870-alpha was isolated and subjected to (solid phase) Edman degradations in order to determine individual amino acid residues labeled. The main features of these results are as follows. 1) Labeling occurred both within the N-terminal segment (residues 1-8) and within the predominantly hydrophobic transmembrane stretch (residues 14-33). 2) Label distribution patterns within segments are indicative of helical structures to which the reagents had access to one face only of the cylindrical envelopes. 3) With regard to the transmembrane segment, the label distribution patterns were similar for all reagents whereas striking differences were noticed within the N-terminal portion. The labeling patterns are consistent with previous models proposing tight association of the transmembrane helix with that of the B870-beta chain. They also suggest that the N-terminal segment forms an amphipathic helix which interacts with the water-lipid interface of the membrane.

Affinity Labels↗
PubMed1985

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

Animals↗
PubMed1985

Reliable detection of inspiration and expiration by computer.

A new computer assisted method is proposed to distinguish between the inspiratory and expiratory phases of breathing. The method is based on the analysis of both gas flow and CO2-concentration. The algorithm is effective and reliable and is most suitable in critical care patients when an uninterrupted sequence of breaths is to be analysed immediately at the bedside. Marked variations in tidal volume such as are seen in intermittent mandatory ventilation or spontaneous breathing during the phase of weaning from the ventilator, artefacts such as mechanical vibrations of the flow transducer or its connecting tubes do not disturb the analysis.

Computers↗
PubMed1985

[Comparative hemolysis studies of various dialysis technics].

In simultaneous experiments the haemolysis in the double-needle dialysis was investigated in comparison to the single-needle technique. As SN-technique the 2-clamp technique with permanently going and start-stop function of the blood pump, respectively, was used. Another technique without arterial and with retarded venous clamp was tested. Furthermore, the pressure limits in the SND were varied. A slight increase of the haemolysis in the SND in comparison to the DND was stated. In the different SN-techniques no essential differences in the haemolysis could be proved, whereas an increase of the pressure limits led to a clear increase of the haemolysis.

Hemolysis↗
PubMed1984

Structure of the membrane-embedded F0 part of F1F0 ATP synthase from Escherichia coli as inferred from labeling with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) is a photoactivatable carbene precursor designed to label selectively the hydrophobic core of membranes. We have used this reagent to obtain information on the topological organization of the membrane-embedded subunits of F1F0 ATP synthase from Escherichia coli. The study included [125I]TID labeling of F0 subunits in different structural (conformational) states and Edman degradations of the labeled polypeptides in order to assign the covalently bound radioactivity to individual amino acid residues. Released phenylthiohydantoin amino acids were analyzed by thin-layer chromatography, and the radioactive derivatives were visualized by autoradiography. The data suggest that labeling patterns can be correlated in a meaningful manner with reagent accessibility and hence with protein-lipid contact. Subunit b appears to be anchored to the membrane by a short N-terminal segment. As almost all of the amino acids of this part are accessible to the reagent, it is inferred that this segment has little interaction with the other subunits. In contrast, in the two segments of subunit c that were labeled with [125I]TID, only certain amino acids reacted with the label. The pattern of these labeled residues is compatible with that of tightly packed alpha-helices.

Adenosine Triphosphatases↗
PubMed1984

Hydrophobic labeling of the membrane binding domain of acetylcholinesterase from Torpedo marmorata.

Membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was labeled with the hydrophobic photoactivatable reagent 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ( [125I]TID). Labeling with [125I]TID was restricted to the membranous polypeptide segment of AChE as shown upon conversion of the amphiphilic form to the hydrophilic one by limited digestion with proteinase K. The labeled membranous segment, which has an Mr of approx. 3000 was isolated by gel filtration on Sephadex LH-60 in ethanol/formic acid.

Acetylcholinesterase↗
PubMed1984

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a hydrophobic, photoreactive probe, labels calmodulin and calmodulin fragments in a Ca2+-dependent way.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means to characterize calmodulin sites that play a role in the interaction with targets.

Amino Acids↗
PubMed1983

A chemical procedure for determining the sidedness of the NH2 terminus in a membrane protein. The small intestinal sucrase-isomaltase.

We have developed a chemical procedure for determining the sidedness of the NH2 termini of sucrase-isomaltase complex in the small intestinal brush-border membrane. The methodology involves amidination of sealed right-side-out brush-border membrane vesicles with the impermeant imidoester 3-[dimethyl-2-[( 3H]acetimidoxyethyl)ammonio]propanesulfonate with subsequent quantitation of this reaction at the NH2 termini of sucrase-isomaltase. It was found that amidination yields were every similar for the reaction of 3-[dimethyl-2-[( 3H]acetimidoxyethyl)-ammonio] propanesulfonate with intact membrane vesicles and with leaky, deoxycholate-extracted membrane fragments. This demonstrates that the NH2 termini were equally accessible to the impermeant reagent in both systems and, hence, are exposed on the outside of the sealed membrane vesicles. The methodology developed does not involve proteolysis and should be of wide applicability.

Amino Acid Sequence↗
PubMed1983

Labeling of intramembrane segments of the alpha-subunit and beta-subunit of pure membrane-bound (Na+ + K+)-ATPase with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine.

The photoactivatable carbene precursor 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID) was tested as a probe for labeling lipid-embedded segments of the proteins of pure membrane bound (Na+ + K+)-ATPase. The probe labeled the alpha-subunit (100 kDa), its major tryptic and chymotryptic fragments of 78 kDa, 58 kDa, and 46 kDa, and the beta-subunit (38 kDa) from within the lipid bilayer to nearly the same specific activity. The labeling was resistant to extensive proteolysis and the distribution of label among the proteolytic fragments and the two subunits was independent of a 47-fold variation in concentration of [125I]TID. The data show that several transmembrane segments are distributed along the sequence of the alpha-subunit and that also the beta-subunit traverses the bilayer. [125I]TID provides a more uniform labeling of the transmembrane segments of the alpha-subunit and beta-subunit than that obtained with other hydrophobic reagents. This will facilitate further studies of the primary structure and folding pattern of the Na+,K+-pump proteins in the membrane.

Animals↗
PubMed1983

[The impossibility of direct flow measurements in lung function studies. Analysis of errors and compensation].

The authors demonstrate that the conditions for correct flow measurement are not fulfilled when a resistive flow transducer (Fleisch pneumotachograph, screen pneumotachograph etc.) is connected directly to the mouth or to the end of the endotracheal tube. This is because the composition, temperature and water content of the respiratory gas varies markedly within a respiratory cycle, the mechanically ventilated patient exhales with a huge expiratory initial peak flow, and laminar flow tends to switch over to turbulent flow in this system. Methods are proposed of continuously compensating the effects of changing gas composition, reducing expiratory peak flow without an increase in expiratory resistance, and preventing the occurrence of turbulent flow. The improvement of measuring accuracy to 2% makes the estimation of respiratory volumes more reliable. Secondly, the increased quality of primary data enables one to analyze these data in a more complex and sophisticated manner (N2-washout compartment analyses, VDS, investigation of complicated modes of ventilation such as IMV, etc.).

Humans↗
PubMed1983

[Behavior of functional residual capacity in acute respiratory insufficiency].

Variations of functional residual capacity (FRC, estimated by the N2-washout technique) and oxygenation (PaO2/FIO2) were investigated in patients mechanically ventilated for acute respiratory failure (ARF, caused by pneumonia). The various ventilatory modes were compared. The results were as follows: 1. If FRC is reduced due to ARF, the reduction is diminished by PEEP. The quantitative amount of this effect cannot be predicted in the individual patients. 2. If CPPV is switched to IMV or CPAP with an equal PEEP value, FRC was not usually changed when the clinical course was favourable; however, FRC decreased if clinical signs of insufficient spontaneous respiration were present. The proportion of FRC reduction following such a change of respiratory mode was equal to the effect of removal of PEEP from 10 cm H2O to zero. 3. FRC and oxygenation do not undergo a parallel change in every situation. 4. Treatment and further research should focus not only on increasing reduced lung volume but mainly on diverting ventilation to perfused lung regions.

Acute Disease↗
PubMed1983

Hydrophobic labeling of a single leaflet of the human erythrocyte membrane.

The photoactivatable phospholipid 1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[9-3H] -8-oxadecanoyl]-sn-glycero-3-phosphocholine [( 3H]PTPC) was synthesized with high specific radioactivity. When a sonicated dispersion of [3H]PTPC was incubated with human erythrocyte membranes (ghosts), the radiolabel was inserted spontaneously into the erythrocyte membrane. Photo-cross-linking of [3H]PTPC to membrane components and subsequent analysis of the distribution of radiolabel among polypeptide fragments of glycophorin allowed conclusions concerning the transbilayer distribution of [3H]PTPC in the erythrocyte membrane. Thus, [3H]PTPC was inserted exclusively into the outer leaflet of resealed ghosts, whereas with unsealed (leaky) ghosts, the photosensitive lipid was incorporated into both halves of the membrane simultaneously. These results are incompatible with fusion of [3H]PTPC liposomes with the erythrocyte membrane being responsible for the lipid transfer observed and suggest instead that PTPC exchanges spontaneously between membranes. This property of PTPC could be related to the critical micelle concentration of this lipid [(6 +/- 2) X 10(-9) M at 23 degrees C].

Affinity Labels↗
PubMed1983

Biosynthesis and assembly of the largest and major intrinsic polypeptide of the small intestinal brush borders.

The sucrase-isomaltase complex (SI) of the small intestinal brush border membrane accounts for approximately 9-10% of the intrinsic protein. The isomaltase subunit alone interacts with the membrane directly, via a highly hydrophobic segment at its N-terminal region. This segment has a helical conformation for more than 85% and crosses the membrane twice, the N-terminus being located at the outer, luminal side of the membrane. The sucrase subunit is attached to the membrane solely via its interactions with the isomaltase subunit. The sucrase-isomaltase complex is synthesized as a single, very long (Mr approximately 260 000) polypeptide chain (pro-SI, carrying the two sites of sucrase and isomaltase in an already enzymically active form), with the isomaltase portion corresponding to the N-terminal part of pro-SI. Pro-SI is processed into 'final' SI by pancreatic proteases. Recently the cell-free translation of pro-SI has been achieved in vitro. From a detailed knowledge of the anchoring of SI (and pro-SI) in the membrane it has been possible to suggest one particular mechanism as the most likely for the synthesis, insertion and assembly of pro-SI.

Amino Acid Sequence↗