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Biomedical subjects

P Spangenberg

Publications and source records attributed to P Spangenberg.

69 records · Page 4Linked to original sources
PubMed1985

Localization of phosphatidylethanolamine in the plasma membrane of diamide-treated human blood platelets.

In human blood platelet plasma membranes phosphatidylethanolamine (PE) is asymmetrically distributed between the two leaflets. The main part of this phospholipid is localized at the inner half of the lipid bilayer. Upon stimulation of the cell a substantial transbilayer movement of PE as well as phosphatidylserine occurs and the outer leaflet then provides a procoagulant surface. The thrombin-induced PE flip-flop is inhibited by pretreatment of platelets with diamide, whereas pretreatment of platelets with diamide alone up to 5 mM did not change considerably the localization of PE in the platelet membrane. Thus, cytoskeletal proteins, which are modified by diamide, are not involved in the maintenance of the PE asymmetry but are important for the realization of the agonist-induced events in the platelets.

Adenosine Diphosphate↗
PubMed1984

Chemical modification of cytoskeletal proteins of human blood platelets by diamide.

Incubation of human blood platelets with diamide (azo-dicarboxylic acid-bis-dimethylamide, DIA) influences the aggregation behaviour considerably. Depending on concentration and incubation time DIA induces a reversible aggregation or brings about complete inhibition. The effect is reversible and may be due to the regeneration of reduced glutathione (GSH) which will be oxidized to GSSG by DIA. DIA causes disulfide-linked polymer formation of certain cytoskeletal proteins. At least three polymer families (Pa, Pb, Pc) with different molecular weights are formed depending on dosage and incubation time of DIA. The appearance of a double band in Pa correlates with reversible aggregation, the formation of Pc is always accompanied by a complete inhibition of aggregation. A disturbance of cytoskeleton-membrane interaction by polymer formation can be assumed. GSH serves as a reductant of disulfide-linked polymers whereby a direct link between the maintenance of SH/SS status of platelets and GSH can be established.

Azo Compounds↗
PubMed1984

Influence of diamide on aggregation, cytoskeletal proteins, and arachidonic acid metabolism in human platelets.

Simultaneous addition of diamide (azodicarboxylic acid-bis-dimethylamide, DIA), a SH-oxidizing agent, and collagen causes a deaggregation of otherwise irreversibly aggregating platelets. Thromboxane B2 (TXB2) and 12-HE-TE formation is inhibited depending on the concentration ratio between collagen and DIA. Thus, at 0.25 mM DIA and 20 micrograms/ml collagen neither TXB2 nor 12-HETE were measurable, but a full scale reversible aggregation is induced. Deaggregation is further attained by adding DIA to collagen-induced aggregates at a time, when maximum amplitude has been achieved. Investigation of arachidonic acid (AA) metabolites under these conditions revealed no influence of DIA on AA metabolism. Therefore, AA metabolization seems to play a minor role in collagen-induced aggregation and DIA-induced deaggregation. Polymerization of certain cytoskeletal proteins of the platelets, after addition of DIA, parallels DIA-induced deaggregation. DIA inhibits endogenous AA release, probably by interaction with platelet plasma membrane. DIA seems to inhibit the release of the alpha-granula protein thrombospondin.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1984

[Proteins and phospholipids of thrombocytes and erythrocytes in glucose-6-phosphate dehydrogenase deficient patients].

Proteins and phospholipids of platelets and red cell membranes of glucose-6-phosphate-dehydrogenase-deficient patients with chronic hemolytic disease were investigated. The enzyme deficiency is not connected with alteration in the protein and phospholipid pattern of platelets. However, marked differences in the diamide-induced protein polymerization between normal and deficient platelets were found. Whereas normal platelets show only a weak polymerization which is reversible during diamide incubation of the cells, the deficient platelets demonstrate a considerable and long lasting protein polymerization. The results point to a direct connection between the thiol-disulfide status of platelets and intracellular level of glutathione, which is able to repair oxidative damages. Two of the six patients showed already protein polymers in their erythrocyte membrane without addition of any SH-oxidizing agent. The phospholipid pattern of the deficient red-cell membranes were comparable to the controls.

Adolescent↗
PubMed1984

Dependence of arachidonic acid (AA) metabolization in human blood platelets on reduced coenzymes.

In human platelets the metabolization of AA is linked to a consumption of the reduced coenzymes NADPH and GSH which can be attributed to about 70% to the cyclooxygenase (CO) and to about 30% to the lipoxygenase (LO) pathway. In GSH depleted platelets the conversion of AA in the LO pathway to 12-HETE is strongly impaired, whereas the formation of the stable CO products from exogenous AA is not decreased, but accelerated. When platelets are deprived from GSH by oxidation to GSSG, the release of endogenous AA from phospholipids in activated platelets is inhibited.

Arachidonic Acid↗
PubMed1982

The effect of diamide (azodicarboxylic acid-bis-dimethylamide) on sulfhydryl group content, proteins, and the location of phosphatidylethanolamine in human blood platelets.

Treatment of human blood platelets with the thiol-oxidizing agent, diamide, causes rapid oxidation of glutathione and alterations in aggregation and release reaction. Moreover, cross-linking of proteins was observed. Three high molecular weight bands of 200 - 240 X 10(3) daltons and a band of 66 X 10(3) daltons were involved in this process. After reduction with dithiotreitol the normal pattern was received. In contrast to the erythrocyte, the cross-linking of platelet proteins was not accompanied by a reorientation of phosphatidylethanolamine in the membrane. Also a considerably smaller effect of diamide on platelet protein sulfhydryl group content was measured.

Azo Compounds↗
PubMed1982

Reconsideration of quantitative phospholipid analysis in human gel-filtered blood platelets.

In the literature the values for phospholipid content and the pattern of human gel-filtered platelets diverge considerably. In order to find the possible reasons for these discrepancies, the separation of plasma constituents from gel-filtered platelets and the influence of different extraction methods were reinvestigated. The critical point in phospholipid determination of platelets appeared to be the completeness of plasma lipid separation from platelets and not the extraction method. Avoiding contamination by plasma lipids the results with gel-filtered platelets correlated to that obtained with washed platelets.

Blood Platelets↗
PubMed1979

[Preparation of human platelets without the use of anticoagulants and study of the effect of Catt on aggregation].

A method for the preparation of human blood platelets is presented which substitutes the use of anticoagulants by a gelfiltration for the removal of plasma calcium from native blood. In a second step the platelets are separated from the gel filtered blood by a centrifugation on a Ficoll density gradient. The anticoagulant-free platelets reveal an intact morphological feature and a normal aggregation behaviour in response to different aggregation inducers. It was found that in ADP-induced aggregation monophasic aggregation is shifted to a biphasic one by increasing concentrations of extracellular calcium.

Blood Platelets↗
PubMed1976

[Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)].

The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.

Animals↗
PubMed1975

[Purification of an anionic trypsin from the gastric juice of the crayfish Cambarus affinis say by means of affinity chromatography].

Beside other digestion enzymes the crayfish Cambarus affinis SAY possess an anionic trypsin. This enzyme could be separated from all the other proteins of the crude gastric juice by a single affinity chromatographic step on immobilized natural inhibitor. The dissociation of the thus produced enzyme-inhibitor complex was achieved by specific elution with ben-amidine. The soluble benzamidine-trypsin-complex was dialyzed and separated by gelfiltration on Sephadex G-50. Of the ligands used Trasylol proved to be the most effective one. Trypsin isolated by this ligand was in a discelectrophoretic homogeneous state. On the other hand, trypsin purified by Soybean trypsin inhibitor and Contrykal contains impurities.

Animals↗