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Biomedical subjects

K Beyer

Publications and source records attributed to K Beyer.

At least 73 records · Page 4Linked to original sources
PubMed1994

The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants.

Although the site-directed C73S mutation in the ADP/ATP carrier (AAC) AAC2 gene from Saccharomyces cerevisiae produced a glycerol-positive strain, indicating that the mutant AAC is active, on isolation and reconstitution in egg yolk phosphatidylcholine, the C73S AAC had no transport activity, whereas the wild-type AAC was fully active. Only on addition of cardiolipin was an exchange activity with the C73S AAC obtained. The AACs isolated from the other cysteine mutants did not (C244S) or only marginally (C271S) require cardiolipin for transport on reconstitution. [3H]Carboxyatractylate binding as a measure of incorporated AAC molecules was unchanged on addition of cardiolipin in all mutants, indicating that cardiolipin does not increase the incorporation of the AAC. It also shows that cardiolipin is required only for translocation and not for binding. The activity of the C73S mutant AAC shows half-saturation with cardiolipin at 2% by weight or at 1.15 mol % in the phosphatidylcholine vesicles. Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate. Among various cardiolipin derivatives, the selectivity for cardiolipin is high. Only monolysocardiolipin still retains 12% activity. After removal of the bulk of phospholipid, the content of bound phospholipids was assayed by 31P NMR. By unmasking with SDS, in the wild-type AAC and in the C73S AAC, 6.4 mol and only 1.3 and 2.9 mol of bound cardiolipin/mol of AAC dimer are found, respectively. Presumably, on isolation, cardiolipin is lost from the more labile C73S mutant AAC. Although the absolute requirement for cardiolipin is unique for the C73S AAC, it is concluded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC and of AACs from other sources.

Adenosine Diphosphate↗
PubMed1994

The role of eosinophils and eosinophil cationic protein in monitoring oral challenge tests in children with food-sensitive atopic dermatitis.

To investigate the role of peripheral blood eosinophils and eosinophil cationic protein as parameters in monitoring oral food challenges, we monitored 25 infants and children with atopic dermatitis for up to 48 hours after 47 placebo-controlled oral food challenges with cow's milk, hen's egg, cow's milk and hen's egg, or placebo for up to 48 hours. Six healthy young nonatopic adult volunteers served as control subjects. Compared with baseline values, peripheral blood eosinophils decreased significantly immediately after clinical reaction in positive challenges (p < 0.0004), independent of the kind of reaction. Eosinophil cationic protein increased significantly 8 hours after provocation, with a maximum at 24 hours (p < 0.03). This increase was predominantly related to eczematous reactions (p < 0.005). Blood sampling immediately after clinical reaction (for eosinophils) and at 24 hours (for eosinophil cationic protein) seems to be useful in monitoring oral food challenges in children with atopic dermatitis.

Adolescent↗
PubMed1994

Serum tryptase and urinary 1-methylhistamine as parameters for monitoring oral food challenges in children.

To study the usefulness of urinary 1-methylhistamine and serum tryptase concentration as monitoring parameters in clinical settings, we investigated 32 children with atopic dermatitis and suspected food allergy during oral food challenges with eggs and cow's milk. Urinary 1-methylhistamine (MH) excretion increased significantly within 1 h after positive oral food challenges (p < 0.006), but showed considerable variation in negative challenges. MH seems to be a sensitive parameter (92.8%), but its specificity is insufficient (37.7%). In the group of 16 positive oral food challenges serum tryptase concentration increased significantly (p < 0.02) directly after provocation and remained elevated up to 2 h after provocation. No variation was observed in negative challenges or nonatopic controls. Serum tryptase concentration seems to be specific for marked clinical reactions after oral food challenges (100%), but its sensitivity was low (25%) and not superior to evaluation by clinical means. We conclude that, despite positive results for the group of children, MH and serum tryptase concentrations are not useful parameters for monitoring oral food challenges in an individual child due to insufficient sensitivity and specificity.

Administration, Oral↗
PubMed1993

Monitoring of inflammation during challenge tests in children.

From our data we conclude, that the determinations of ECP and EPX do not add any useful information as monitoring parameters for bronchial allergen challenge tests. In contrast, eosinophils and eosinophil cationic protein may deserve further study as potentially useful monitoring parameters for oral food challenges in patients with food related atopic eczema.

Adolescent↗
PubMed1992

Interactions of phospholipids with the mitochondrial cytochrome-c reductase studied by spin-label ESR and NMR spectroscopy.

Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone:cytochrome-c oxidoreductase (bc1 complex) were studied by spin-label electron-spin resonance and by 31P-NMR spectroscopy. Spin-labelled phospholipids were employed to probe the relative binding affinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin-labelled cardiolipin (1,3-bisphosphatidyl-sn-glycerol) and with the spin-labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron-transport activity [Schägger, H., Hagen, T., Roth, B., Brandt, U., Link, T. A. & von Jagow, G. (1990) Eur. J. Biochem. 190, 123-130]. A simplified distribution model showed that neutral phospholipids have much lower protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin-label electron-spin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex. Considerable line narrowing was observed after phospholipase A2 digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.

Animals↗
PubMed1991

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.

Animals↗
PubMed1990

Lipid-protein interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy.

The stoichiometry and specificity of lipid-protein interaction, as well as the lipid exchange rates at the protein interface, have been determined from the electron spin resonance spectra of spin-labeled lipids in reconstituted complexes of the mitochondrial ADP-ATP carrier with egg phosphatidylcholine. With the exception of cardiolipin and phosphatidic acid, the lipids studied are found to compete for approximately 50 sites at the intramembranous surface of the protein dimer. This number of first-shell lipid sites is unusually large for a protein of this size. The specificity for the protein is in the order stearic acid approximately phosphatidic acid approximately cardiolipin greater than phosphatidylserine greater than phosphatidylglycerol approximately phosphatidylcholine, with the maximum association constant relative to phosphatidylcholine being approximately 4. The selectivity for anionic lipids was partially screened with increasing ionic strength, but to a lesser extent for cardiolipin and phosphatidic acid than for stearic acid. Only in the case of phosphatidylserine was the selectivity reduced at high ionic strength to a level close to that for phosphatidylcholine. The off rates for lipid exchange at the protein surface were independent of lipid/protein ratio and correlated in a reciprocal fashion with the different lipid selectivities, varying from 5 x 10(6) s-1 for stearic acid at low ionic strength to 2 x 10(7) s-1 for phosphatidylcholine and phosphatidylglycerol. The off rates for cardiolipin were unusually low in comparison with the observed selectivity, and indicated the existence of a special population of sites (ca. 30% of the total) for cardiolipin, at which the exchange rate was very low.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Rotational diffusion of mitochondrial ADP/ATP carrier studied by saturation-transfer electron spin resonance.

The rotational mobility of the mitochondrial ADP/ATP carrier has been studied solubilized in Triton micelles, reincorporated in phospholipid liposomes, and in mitochondria. Spin-labeled analogues of the noncovalent inhibitors carboxyatractyloside and atractyloside were found to be strongly immobilized when bound to the carrier [Munding, A., Beyer, K., & Klingenberg, M. (1983) Biochemistry 22, 1941-1947], such that saturation-transfer electron spin resonance spectroscopy could be used to study the rotational motion of the protein. Spin-labeled maleimide covalently bound to the carrier was found to have independent segmental motion and hence to be unsuitable for studies of protein rotation. The ESR spectra of the carboxyatractyloside and atractyloside spin labels were found to contain a second component from label in the lipid or detergent, necessitating the use of the saturation-transfer ESR integral method [Horváth, L. I., & Marsh, D. (1983) J. Magn. Reson. 54, 363-373] to analyze the rotational motion of the label component bound to the protein. Effective rotational correlation times obtained from integration of the high-field region of the spectrum were lower than those obtained from the total spectral integral, indicating strongly anisotropic rotational diffusion of the carrier in the vesicular and membrane systems, with the spin-label z axis oriented preferentially perpendicular to the rotation axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

[Abnormal gangliosides in the plasma of alcoholics].

Gangliosides were isolated from plasma of alcoholics. The pattern of gangliosides was compared with that of healthy humans. Up to seven fractions of gangliosides could be detected. In healthy subjects the following pattern was found: GM3 38.2%, SPG 6.0%, GD3 30.2%, GD1a 10.6%, GD1b 4.8%, GT1b 7.9%, GQ1b 2.2%. In alcoholics, significant differences were observed in the main ganglioside fractions GM3 (52.6%) and GD3 (17.5%).

Adult↗
PubMed1988

Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance.

The interaction of spin-labeled phospholipids with the detergent-solubilized ADP/ATP carrier protein from the inner mitochondrial membrane has been investigated by electron spin resonance spectroscopy. The equilibrium binding of cardiolipin and phosphatidic acid was studied by titration of the protein with spin-labeled phospholipid analogues using a spectral subtraction protocol for the evaluation of the mobile and immobilized lipid portions. This analysis revealed the immobilization of two molecules of spin-labeled cardiolipin per protein dimer. Phosphatidic acid has a similar affinity for the protein surface as cardiolipin. The lipid-protein interaction was less pronounced with the neutral phospholipids and with phosphatidylglycerol. The importance of the electrostatic contribution to the phospholipid-protein interaction shows up with a strong dependence of the lipid binding on salt concentration. Cleavage by phospholipase A2 and spin reduction by ascorbate of the spin-labeled acidic phospholipids in contact with the protein surface suggest that these lipids are located on the outer perimeter of the protein. At reduced detergent concentration, the protein aggregated upon addition of small amounts of cardiolipin but remained solubilized when more cardiolipin was added. This result is discussed with respect to the aggregation state of the protein in the mitochondrial membrane. It is also tentatively concluded that binding of spin-labeled cardiolipin does not displace the tightly bound cardiolipin of mitochondrial origin, which was detected previously by 31P nuclear magnetic resonance spectroscopy.

Binding, Competitive↗
PubMed1987

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels.

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.

Animals↗
PubMed1986

Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate.

The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.

1,2-Dipalmitoylphosphatidylcholine↗