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J Römisch

Publications and source records attributed to J Römisch.

At least 37 records · Page 2Linked to original sources
PubMed1992

Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Amino Acid Sequence↗
PubMed1992

Detection of human anti-annexin autoantibodies by enzyme immunoassays.

Annexins belong to a family of proteins characterized by calcium-dependent binding to the cytoskeleton and phospholipid surfaces. Basing on these properties annexins are discussed to be involved in the regulation of cytodynamic, anticoagulatory and antiinflammatory processes. Since autoantibodies against annexin I had been detected in patients suffering from inflammatory or autoimmune diseases, an impact on the pathophysiological outcome was assumed. Therefore we developed solid phase, enzyme-linked immunoassays for the quantitative determination of autoantibodies directed against six members of the annexin family. Some preliminary results obtained from sera of patients with malignant melanoma show a quite frequent presence of such autoantibodies. These data suggest that autoantibodies are generated against all annexins. Furthermore, in the individual patient autoantibodies of the IgG-type are monospecific, while about 1/4 of the IgM-type are directed against several annexins. These observations imply that for investigation of anti-annexin autoantibodies in inflammatory and autoimmune diseases as well as cancer all members of the annexin family have to be taken into consideration.

Autoantibodies↗
PubMed1992

Coagulation and fibrinolysis in cancer.

Haemostasis is a system of finely adjusted interactions between cells, enzymatic reaction cascades and inhibitors. Disturbances of this balance occur in many disorders, especially in inflammatory processes, septicaemia and cancer. In such cases malignant cells and infectious organisms activate the plasmatic enzyme cascades, especially of the coagulation and fibrinolysis cascades. The resulting consumption and proteolytic degradation of the regulatory proteins contribute to hypercoagulability and secondarily to reactive fibrinolysis, and these may then lead to local thromboses and haemorrhages. These pathogenic events culminate in disseminated intravascular coagulation (DIC), frequently with organ failure and death. Factors of both plasmatic systems are also "misused" by malignant cells for the purposes of growth and metastasis. Prominent examples of this misuse are the formation of a protective fibrin shield against the endogenous defence mechanisms and the local degradation of tissues for tumor proliferation as well as for cell permeation and invasion. In the search for a potential therapy a number of protease inhibitors, predominantly of enzymes of coagulation and fibrinolysis, have been tested in vivo with regard to their efficacy. So far, however, it has not been possible to find a new uniform treatment principle to inhibit the growth and/or metastasis of different types of tumor. The haemorrhagic diathesis and thromboses frequently associated with tumors are generally treated by substitution with plasma components, especially concentrates of coagulation factors and inhibitors.

Blood Coagulation↗
PubMed1992

Annexins I to VI: quantitative determination in different human cell types and in plasma after myocardial infarction.

Concentrations of annexins I to VI were quantitatively determined in extracts of placenta and different human cell types. They were detectable in all extracts studied, but lymphocytes/monocytes, endothelial cells and fibroblasts had very high annexin contents. The results indicate cell type specific annexin-repertoires. Annexins are intracellular proteins lacking signal sequences but which are detectable in trace amounts in plasma of healthy humans. The majority of plasma samples drawn from 14 patients suffering from myocardial infarction had elevated annexin III, IV and V concentrations. Shortly after infarction increased annexin levels were detected, reaching maximal values 24 to 48 h later. In the course of the following days annexin concentrations returned towards normal plasma levels.

Calcium-Binding Proteins↗
PubMed1991

Comparative study on the in vitro effectiveness of antithrombotic agents.

The synthetic low molecular weight inhibitors MD 805, FUT-175 and FOY as well as heparin and r-Hirudin were compared for their in vitro antithrombotic potencies and protease specificities. The amidolytic activity of thrombin and the plasma coagulation were effectively inhibited by MD 805 and, in particular, by r-Hirudin. FUT-175 and FOY revealed only weak inhibition. None of the synthetic substances discriminated between alpha-, beta- and gamma-thrombin. Beside r-Hirudin only MD 805 revealed a relatively good specificity for thrombin. On the contrary, FUT-175 and FOY are unspecific and can not be classified as thrombin inhibitors.

Amino Acid Sequence↗
PubMed1991

In-vivo antithrombotic potency of placenta protein 4 (annexin V).

The antithrombotic properties of Placenta Protein 4 (PP4) were investigated in laser or photochemically induced thrombus formation models in rats. In both in-vivo test-systems PP4 displayed a significant antithrombotic effect at dose levels as low as 0.3 and 1.0 mg/kg body weight. Bleeding times, surprisingly, were not prolonged significantly at these dose regimens. Maximal inhibition of thrombus formation in the laser-model was observed 15 min after intravenous administration of PP4, but was not recognizable in a clear-cut reaction in the second model. Determination of PP4 plasma levels in two monkeys revealed a half-life of 11.5 and 14.9 min, respectively. The maximal anticoagulant effect was observed between 15 and 30 min after administration of PP4 as determined functionally by means of thrombelastography.

Animals↗
PubMed1991

Thrombin-hirudin complex stability: a comparison with the thrombin-antithrombin III complex.

In the present in vitro study the stabilities of the thrombin-hirudin and the thrombin-antithrombin III complexes were investigated. After incubation of the complexes with free inhibitors the thrombin-antithrombin III levels were determined by ELISA. The thrombin-hirudin complex proved to be stable in the presence of antithrombin III or heparin. However, in the presence of heparin and plasma equivalent concentrations of antithrombin III, the thrombin-hirudin complex dissociated and hirudin was displaced. In contrast, both thrombin-antithrombin III and thrombin-antithrombin III/heparin complexes are very stable even in the presence of a large excess of hirudin.

Antithrombin III↗
PubMed1991

[Lipocortins and phospholipases: new aspects in the physiology of glucocorticosteroid effect].

Glucocorticosteroids are among the most useful and most widely prescribed anti-inflammatory drugs. Despite their wide use little is yet known about their mode of action. In the last 10 years a group of proteins called lipocortins or annexins has been characterized. Those proteins exert an inhibitory effect on the synthesis of lipid mediators by way of an important proinflammatory enzyme, phospholipase A2. Phospholipases are known to be involved in cell-signal transduction and generation of inflammatory mediators like prostaglandins, leukotrienes and platelet-activating-factor. The cellular expression of lipocortins is induced by glucocorticosteroids. The inhibition of cellular phospholipases via lipocortins may account for some aspects of the action of glucocorticosteroids.

Annexins↗
PubMed1990

Anticoagulant properties of placenta protein 4 (annexin V).

A placenta protein, originally termed PP4, was found to inhibit the aPTT in a concentration-dependent manner. PP4 which turned out to be identical with a vascular anticoagulant of the annexin type, inhibits the blood clotting process by binding of the essential lipids in a reaction which is dependent on calcium ions. Also in the presence of calcium PP4 combines with platelet membranes neutralizing their procoagulant effect. By fluorescence-microscopy binding of PP4 to stimulated macrophages is shown. The antithrombotic effect of PP4 is demonstrated by means of thrombelastography of human blood. Coagulation triggered by the addition of thromboplastin/lipid-mixtures is extinguished by PP4.

Anticoagulants↗
PubMed1990

The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.

Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specific calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.

Amino Acid Sequence↗
PubMed1990

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield. The proteins were purified to homogeneity. The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts. Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria. Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test.

Annexin A5↗
PubMed1990

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker.

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of six annexins from human placenta.

Isolation of six calcium-binding proteins from human placenta is described by means of hydrophobic chromatography, calcium-dependent adsorption to heparin-Sepharose and ion-exchange chromatography. These proteins were characterized and identified as PP4, PP4-X, PAP III, p68 and lipocortins I and II belonging to the family of annexins. Antibodies raised against PP4, PAP III and p68 revealed to be highly specific, while those raised against PP4-X reacted with all investigated annexins, except PP4. Cross-reactivity was also observed between lipocortins I and II. All annexins inhibited in a concentration-dependent manner blood coagulation but with different potencies as was determined by means of a modified thromboplastin time test. The most potent inhibitors turned out to be PP4 and PAP III, followed by PP4-X, lipocortin I, p68 and lipocortin II.

Annexin A5↗
PubMed1990

[Hemostasis, fibrinolysis, proteolysis: interaction with inflammatory reactions].

Many physiological processes are based on the finely regulated interaction between cells and enzymatic reaction cascades. Mainly proteinases are involved in these processes, which are regulated by inhibitors, principally proteins. If this sensitive balance is disturbed, uncontrolled pathophysiological events can be induced, which are often associated with inflammatory reactions. Characteristic for inflammation are events like contact activation of hemostasis, increasing permeability of blood vessels caused by activation of the Kallikrein-Kinin- and the Complement-system and Plasmin-release induced by activation of fibrinolysis. The following uncontrolled proteolysis, leading to tissue destruction, is mainly associated with the degree of illness. Inflammatory cells excrete besides proteinases also mediators maintaining and increasing these processes. Only when the balance between proteinases and inhibitors is restored, inflammation subsides. Afterwards the controlled course of physiological reactions is possible again.

Endopeptidases↗
PubMed1987

The primary structure of cytochrome c1 from Neurospora crassa.

The primary structure of the cytochrome c1 subunit of ubiquinol-cytochrome-c reductase from mitochondria of Neurospora crassa was determined by sequencing the cDNA of a bank cloned in Escherichia coli. From the coding region the sequence of 332 amino acids, corresponding to the molecular mass of 36,496 Da, was derived for the precursor protein. The mature protein, the N terminus of which was previously sequenced [Tsugita et al. (1979) in Cytochrome oxidase (King, T. E. et al., eds) pp. 67-77, Elsevier, New York], consists of 262 amino acids and has the molecular mass of 29,908 Da including the heme. The sequence contains an N-terminal hydrophilic part of 211 residues, which carries the heme, a hydrophobic stretch of 15 residues, which is assumed to anchor the protein to the membrane, and a C-terminal hydrophilic part of 36 residues. The N-terminal presequence of 70 amino acids contains 9 positive charges but only 1 negative charge and is characterized by a stretch of 20 uncharged residues.

Amino Acid Sequence↗