Biomedical subjects

S Rimon

Publications and source records attributed to S Rimon.

18 recordsLinked to original sources

PubMed1999

Purification, characterization and anticoagulant activity of a proteolytic enzyme from Vespa orientalis venom.

The anticoagulant effect of Vespa orientalis venom sac extract (VSE) was attributed to a proteolytic process, involving mainly coagulation factors VIII and IX [Joshua, H., Ishay, J., 1975. Toxicon 13, 11-20; Korenberg et al., 1988. Toxicon 26, 1169-1176]. Preliminary purification of the proteolytic activity showed the presence of three separate proteases. One of which, protease I, was purified. The purified enzyme migrated as a double band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The molecular weights of the bands, under reduced conditions were 42 and 44 kD. Both bands retained activity after the electrophoretic run. The enzyme hydrolyses bovine factor IX (BFIX), factor X (BFX) and prothrombin. The pH optimum for the degradation of BFIX was 7.0 and its isoelectric point is above pH 10. The amino acid composition of the protease was determined.

Amino Acids↗

PubMed1998

Association of myeloperoxidase with heparin: oxidative inactivation of proteins on the surface of endothelial cells by the bound enzyme.

Chromatography of human myeloperoxidase (MPO) on a heparin-agarose column demonstrated a tight association of the protein with the resin. The electrophoretic mobility of mixtures of MPO and heparin in polyacrylamide gels under nondenaturing conditions was consistent with a strong interaction of the cationic enzyme with the polyanionic polysaccharide. Purified MPO prebound to bovine aorta endothelial cells (BAEC) and supplemented with hydrogen peroxide dose- and time-dependently abrogated the interaction of coagulation factor IX (FIX) with factor IX-binding protein (FIXBP) on the surface of BAEC reflecting oxidative modification of the binding protein. This inactivation of FIXBP required the presence of chloride implicating hypochlorite in the reaction. Hypochlorite and activated neutrophils exerted a similar effect. The oxidative modification of FIXBP was only partially dependent on the addition of hydrogen peroxide and was abolished by exogenous heparin which displaced MPO from the cell surface, emphasizing the functional differences between cell-bound and free enzyme.

Animals↗

PubMed1995

Purification properties and specificity of cathepsin D from Cyprinus carpio.

Cathepsin D was purified 750-fold from a homogenate of Cyprinus carpio muscles. The purified enzyme has a molecular weight of 36,000, is inhibited by pepstatin and is active between pH 2.7 and 3.7 when tested on hemoglobin as the substrate. It consists of two isoenzymes with pIs of 5.65 and 6.1, respectively. The mode of cleavage of the beta chain of oxidized insulin was determined by analysis of the N-terminal amino acids of the cleaved peptides. The major points of cleavage of the beta chain of oxidized insulin are 56% at Tyr16-Leu17 and 40% at Phe25-Tyr26. The minor points of cleavage are at Leu15-Tyr16, Phe24-Phe25, Gly23-Phe24, Leu11-Val12, Ala14-Leu15 and Gln4-His5.

Amino Acid Sequence↗

PubMed1991

Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa.

Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.

Animals↗

PubMed1987

Purification and characterization of acetyl esterase from bull testes.

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from bull testes was purified 325-fold by ammonium sulphate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and finally, gel filtration on a Sephadex G-200 column. The purified enzyme appeared as a single protein band on native polyacrylamide gel electrophoresis and in isoelectric focusing (pI 5.25). In both methods, the activity coincided with the protein band. A single protein band corresponding to Mr 70,000 was obtained by SDS-polyacrylamide gel electrophoresis. The reported amino-acid composition indicates that the enzyme contains three half-cystine residues, of which only one could be detected, by titration, as a free -SH group. No free amino terminal was detected by dansylation.

Acetylesterase↗

PubMed1987

Identification of a factor IX/IXa binding protein on the endothelial cell surface.

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.

Animals↗

PubMed1986

Intraoperative identification of peripheral nerve fascicle. Use of a new rapid biochemical assay technique.

An original nerve-identification technique based on the enzymatic activity of choline acetylase enables precise differentiation between motor nerve fascicles and sensory nerve fascicles. Within 70 to 80 minutes, an accurate matching of cut nerve ends is possible, both in recent and in late nerve injuries. Preliminary results of the clinical intraoperative application of this method are encouraging.

Choline O-Acetyltransferase↗

PubMed1983

1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805.

The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.

17-Hydroxysteroid Dehydrogenases↗

PubMed1982

Intraoperative nerve fascicle identification using choline acetyltransferase: a preliminary report.

One reason for failure of nerve recovery after suture or nerve grafting is the inappropriate matching of motor and sensory fibers. Methods used in attempting to solve this problem have either been unprecise, too time consuming, or unpleasant to the patient. Both experimentally and on cadaver samples, the level of choline acetylase activity is eight times higher in motor nerve fascicles than in sensory fascicles. In experimentally cut nerves, the ChAC activity remained high even after a period of 45 days. Thus it was possible to identify both ends of the motor and sensory fascicles in fresh injuries, and the proximal parts in old injuries. We have since used this method of intraoperative nerve identification in three patients. It has proved to be a rapid (70-80 minutes), viable technique for the immediate differentiation between proximal parts of motor and sensory nerve fascicles in cases of old nerve injuries. In contrast to our animal studies we found that the ChAC activity in the distal segment was still different for the various fascicles five months after transection. Presumably the present need for distal exploration to identify the "target organ" can be avoided.

Adult↗

PubMed1981

Choline acetyltransferase nerve identification method in early and late nerve repair.

Choline acetyltransferase (ChAC) measurement after complete transection of the sciatic nerve in rats revealed a steady increase in ChAC in the proximal nerve stump and a rapid drop in ChAC in the distal nerve stump. Ninety-six hours following transection no appreciable ChAC activity could be detected in the distal segment. The increase in the proximal segments was followed by a slight decline and leveling off at about twice the normal level. End-to-end nerve anastomosis, using the enzymatic nerve differentiation technique, could therefore be performed up to the first 96 hours. Nerve grafts could be used, with markedly improved results, up to the forty-fifth day following transection.

Animals↗

PubMed1980

Choline acetyltransferase for differentiation between human motor and sensory nerve fibers.

A rapid and precise nerve fascicle identification method based on the enzymatic activity of choline acetyltransferase (choline acetylase, or ChAC) is described. Motor nerve facicles demonstrated significantly higher ChAC activity in comparison to sensory nerve facicles. Based on this difference in activity, matching of nerve fascicle cut ends is possible in cases of recent trauma.

Afferent Pathways↗

PubMed1977

Physico-chemical and immunological properties of allophycocyanins.

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure (alpha beta)3. Representative native allophycocyanins and their alpha and beta subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties. Renatured alpha and beta subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein. Trimeric allophycocyanin was readily reconstituted from the purified alpha and beta subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques. The results support the view that allophycocyanins are a highly conserved group of proteins.

Amino Acids↗

PubMed1976

Biliproteins of cyanobacteria and Rhodophyta: Homologous family of photosynthetic accessory pigments.

Amino-terminal sequence determinations are reported of the subunits of biliproteins of prokaryotic unicellular and filamentous cyanobacteria and of eukaryotic unicellular red algae. The biliproteins examined, allophycocyanin, C-phycocyanin, R-phycocyanin, b-phycoerythrin, and phycoerythrocyanin, vary with respect to the chemical nature and the number and distribution of the bilin chromophores between the two dissimilar subunits. The amino-terminal sequences fall into two classes, "alpha-type" and "beta-type", with a high degree of homology within each class.In those biliproteins where the number of bilin chromophores on the two subunits is unequal, the subunit with the greater number of chromophores has the beta-type amino-acid sequence.Extensive homology also exists between alpha- and beta-type sequences, strongly supporting the view that these arose by gene duplication to give rise to the ancestral alpha- and beta-type genes early in the evolution of the biliproteins. The subsequent generation of the various classes of biliproteins appears to be the result of further gene duplication of the alpha- and beta-type genes, ultimately to give rise to families of polypeptide chains of similar sequence, but varying in the number of chromophore attachment sites and the structure of the chromophores.

Journal Article↗

PubMed1972