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A Szewczyk

Publications and source records attributed to A Szewczyk.

50 records · Page 3Linked to original sources
PubMed1990

Purification of rat liver mitochondrial anion carriers by triazine dye affinity chromatography.

Mitochondria were prepared from rat liver, solubilized with Triton X-100 and submitted to chromatography on dry hydroxylapatite. The pass-through from the column, containing a mixture of mitochondrial carriers, was processed further by triazine dye affinity chromatography and optimal conditions for binding and elution of mitochondrial carriers were established. Activities of the mono-, di-, tricarboxylate and 2-oxoglutarate carriers were measured after reconstitution of various chromatographic fractions into asolectin vesicles, and a good resolution of different carriers was obtained. Thus the triazine dye affinity chromatography appears to be a potent new tool for purification of mitochondrial anion carriers.

Animals↗
PubMed1989

Azido derivatives of dicarboxylic acids for photoaffinity labeling of mitochondrial carriers.

New photoaffinity probes, N-(4-azidosalicylic)-aminosuccinic acid, 3-(4-azidophenylazo)-4-hydroxyphenylmalonic acid, (4-azido-2-nitroanilino)-N-succinic acid, 4-azidophenacylthiosuccinic acid and 4-azidophenylsuccinic acid, were synthesized and characterized chemically. They differ in the distance between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These reagents can be applied for photoaffinity labeling of mitochondrial anion carriers and enzymes interacting with dicarboxylic acids. Inhibition and labeling of the dicarboxylate carrier is presented.

Affinity Labels↗
PubMed1987

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria.

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Animals↗
PubMed1987

Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal.

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.

Anilino Naphthalenesulfonates↗
PubMed1986

Internalization of the spin-labelled surface potential probe CAT12 by energized mitochondria.

Rat liver mitochondria briefly incubated in the presence of 50 microM 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12) bind 1.9 nmol of this spin-labelled membrane probe per mg mitochondrial protein in a form which is not reducible by ascorbate. Upon energization with ATP the amount of the non-reducible CAT12 increases by about 37%. It is concluded that CAT12 which is not reducible by externally added ascorbate is bound to the inner surface of the inner mitochondrial membrane and/or accumulated in the matrix compartment. Therefore, CAT12 is not suitable to monitor the surface potential of mitochondria and other organelles which develop a transmembrane potential, negative inside.

Adenosine Triphosphate↗
PubMedDate not supplied

The intracellular potassium and chloride channels: properties, pharmacology and function (review).

Channels selective for potassium or chloride ions are present in membranes of intracellular organelles such as sarcoplasmic (endoplasmic) reticulum, mitochondria, nucleus, synaptic vesicles, and chromaffin, and zymogen granules. They probably play an important role in cellular events such as compensation of electrical charges during transport of Ca2+, delta pH formation in mitochondria or V-ATPase containing membrane granules, and regulation of volume changes, due to potassium and chloride transport into intracellular organelles. Intracellular potassium and chloride channels could also be the target for pharmacologically active compounds. This mini-review describes the basic properties, pharmacology, and current hypotheses concerning the functional role of intracellular potassium and chloride channels.

Animals↗
PubMedDate not supplied

Potassium channel openers induce mitochondrial matrix volume changes via activation of ATP-sensitive K+ channel.

Recently, using patch-clamp method, an ATP-sensitive potassium channel in rat liver mitochondrial inner membrane was identified. In this report we investigated the effects of different potassium channel openers on potassium ion permeability of rat liver mitochondria. For that purpose we employed light scattering technique, reflecting mitochondrial volume changes linked to solute transport. Among various potassium channel openers RP 66471, pinacidil, minoxidil sulfate and KRN 2391 were found to stimulate mitochondrial swelling with high efficiency. P1060, aprykalim and diazoxide had only a slight effect, while Ro 31-6930 and nicorandil were found ineffective. Activation by the K+ openers was abolished by the antidiabetic sulfonylurea, glibenclamide (the well known blocker of ATP-sensitive potassium channels). These findings may suggest that mitochondrial ATP-dependent K+ channel is involved in regulation of mitochondrial matrix volume.

Adenosine Triphosphate↗
PubMedDate not supplied

Guanidine derivative U-37883A, inhibits mitochondrial K+ uniport.

The activity of potassium uniport in rat liver mitochondria was measured using light scattering technique (mitochondrial swelling). It was shown, that guanidine derivative, U-37883A (4-morpholinecarboxamidine-N-1-adamantyl-N'-cyclohexylhyd rochloride), was able to inhibit A23187 induced potassium uniport activity. The inhibitory effect of U-37883A on mitochondrial swelling gave an IC50 an of 89 +/- microM. No inhibition was observed with U-37883A analog, U-42069D (4-morpholinecarboxamidine), which points to the specificity of U-37883A action. It was also shown, that U-37883A acted on a different binding site in rat liver mitochondria than glibenclamide, inhibitor of the ATP sensitive potassium channel.

Adamantane↗
PubMedDate not supplied

ATP-regulated potassium channel blocker, glibenclamide, uncouples mitochondria.

The purpose of the present study was to investigate the effect exerted by the antidiabetic sulfonylurea, glibenclamide, a well known blocker of ATP-regulated potassium channels, on membrane potential and proton gradient in rat liver mitochondria. Membrane potential of mitochondria was determined using a TPP+ selective electrode. Mitochondrial proton gradient value was measured with 2',7',bis(carboxyethyl)-5,6-carboxyfluorescein, the pH-sensitive fluorescent probe. Mitochondrial membrane potential and proton gradient were found to be dissipated by glibenclamide in a concentration-dependent manner, with IC50 of 70 +/- 7 microM. The present results provide evidence that the antidiabetic sulfonylurea, glibenclamide, uncouples rat liver mitochondria.

Adenosine Triphosphate↗