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J Storch

Publications and source records attributed to J Storch.

At least 73 records · Page 4Linked to original sources
PubMed1988

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase.

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.

Acridines↗
PubMed1988

Solubilization of the NADPH-oxidase from bone marrow derived macrophages by dialkylphospholipid and purification by HPLC-anion exchange chromatography.

The microsomal NADPH-oxidase of bone marrow-derived macrophages was partially purified by HPLC-anion exchange chromatography. Particulate preparations were solubilized by the Dialkylphospholipid 2-O-methyl-1-O-octadecyl-rac-glycero-3-phosphocholine. The enzyme was enriched up to 61-fold and proved to be stable under the assay conditions. The NADPH-oxidase depended on NADPH as electron donor with a Km of 2.1 microM, whereas no O2- -production was detected with up to 40 microM NADH. Ca2+ was found to be a strong inhibitor of the enzyme, whereas Mg2+ had no influence. O2- was determined by the very sensitive and selective method of superoxide dismutase-inhibitable chemiluminescence of lucigenin.

Animals↗
PubMed1987

Increased membrane permeability for an antitumoral alkyl lysophospholipid in sensitive tumor cells.

We have investigated cellular sensitivity to the antitumoral alkyl lysophospholipid (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) in vitro. The permeation of this lipid into the cell was not influenced by metabolic inhibitors of ATP biosynthesis. ET-18-OCH3 uptake was not saturable within sublytic concentrations, but could be inhibited in part by cytochalasin B (CB) and dipyridamole. The activation energy of the CB-sensitive uptake process was increased up to threefold compared to CB-insensitive uptake. ET-18-OCH3 influx and equilibrium binding of ET-18-OCH3 were decreased in a fibrosarcoma cell variant (MethA) selected for ET-18-OCH3 resistance. The resistant MethA cells were also less sensitive to cytolysis by lysophosphatidylcholine and other ALP. After 72 hr, the resistant MethA cells had metabolized only 11.8% more of the absorbed ET-18-OCH3 than sensitive MethA cells. However, they tolerated at least a 30-fold concentration of this ALP. The uptake mechanism, which could be inhibited by CB, was less active in resistant MethA cells and several other ALP-resistant cell lines. The concentration of CB, required for maximal uptake inhibition, was increased more than four times in the ALP-sensitive tumor cell lines. CB-specific ET-18-OCH3 uptake was also enhanced after virus transformation of 3T3 fibroblasts by SV 40. Dipyridamole retarded the ET-18-OCH3-mediated cell destruction.

Animals↗
PubMed1986

Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles.

Movement of free fatty acids (ffa) between small unilamellar vesicles (SUV) was studied by measuring the transfer of fluorescent n-(9-anthroyloxy)-labeled analogues (AOffa) between donor and acceptor vesicles. Donors were composed of egg phosphatidylcholine (PC) loaded with 1-2 mol % AOffa, and acceptors were egg PC containing 10-12 mol % N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE). The fluorescence of AO added directly to acceptor SUV was greater than 98% quenched by energy transfer to NBD. Thus, AOffa movement from donor to acceptor was monitored by the time-dependent decrease in AO fluorescence. The transfer of the short-chain AOffa, although too fast to be resolved by the methods used here, is consistent with studies that find transfer rates on the order of milliseconds and kinetics which are first order. In contrast, transfer rates for the long-chain AOffa are more than 2 orders of magnitude slower, and the kinetics of the transfer process are best described by the sum of two exponentials plus a constant. The ffa ionization state was also found to be an important determinant of transfer rate. The charged species transferred an average of 10-fold faster than the protonated ffa. The ffa pKa in the membrane is 9, as calculated from the pH dependence of transfer. Similar to results found for other lipids, long-chain AOffa are transferred via water rather than a collision-mediated process. The aqueous phase route of AOffa intermembrane transfer is indicated by the lack of effect on transfer of large alterations in the product of donor and acceptor phospholipid concentrations. Moreover, the transfer rate is decreased as [NaCl] is increased from 0.1 to 4 M. This effect of ionic strength is probably due not only to a decrease in the aqueous phase partition of the AOffa but also to an alteration in bilayer structure, as measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The observed kinetics are consistent with a model in which the transfer involves two steps: transbilayer movement between the inner and outer bilayer leaflets, followed by transfer from the outer leaflet to the aqueous phase (off rate). Within the framework of this model, the observed slow rate is primarily determined by the rate of transbilayer movement, and the observed fast rate is approximately equal to the off rate. The off rate is about 10-fold faster than the rate of transbilayer movement.

Fatty Acids, Nonesterified↗
PubMed1985

Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro.

Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.

Animals↗
PubMed1984

Dietary induction of acyl chain desaturases alters the lipid composition and fluidity of rat hepatocyte plasma membranes.

Rats were maintained on a regimen of intermittent starvation followed by refeeding a fat-free diet in order to induce hepatic acyl desaturase activities and other enzymes involved in lipid synthesis. The effects of the dietary regimen on the lipid composition and fluidity of isolated hepatocyte plasma membranes were compared to corresponding effects on microsomal preparations. The dietary regimen increased the content of monoenoic and polyenoic acyl chains and decreased the cholesterol/phospholipid molar ratio in the plasma membranes. Accordingly, the lipid fluidity of the plasma membranes was significantly increased as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 12-(9-anthroyloxy)stearate and the intramolecular excimer fluorescence of 1,3-di(1-pyrenyl)propane. In the microsomal membranes, substantial increases in the content of monoenoic acyl chains were offset by decreases in polyenoic acids, and no change in cholesterol/phospholipid ratio was observed. Correspondingly, the lipid fluidity of the microsomal membranes remained almost unchanged. The enhancement of lipid fluidity in the hepatocyte plasma membranes was accompanied by an increase of approximately 68% in the specific activity of the (Na+ + K+)-dependent adenosinetriphosphatase. The results demonstrate that a dietary regimen can modulate in vivo the lipid composition, fluidity, and enzyme function of the hepatocyte plasma membrane.

Animals↗
PubMed1983

Lipid fluidity of hepatocyte plasma membrane subfractions and their differential regulation by calcium.

Rat hepatocyte plasma membranes were subfractionated by several methods into canalicular, sinusoidal and mixed contiguous plus sinusoidal membranes. Assessment of lipid fluidity by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 12-(9-anthroyloxy)stearate indicates that the canalicular fraction is less fluid than the other membranes. Incubation with calcium decreases the fluidity of the sinusoidal and contiguous membranes by altering the lipid composition, an action which is not reversed by subsequent chelation of the cation. This effect of calcium is not observed in canalicular membranes.

Animals↗