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Biomedical subjects

L Fesus

Publications and source records attributed to L Fesus.

44 records · Page 3Linked to original sources
PubMed1987

Lipids associated with tissue transglutaminase.

A substantial amount of lipids (cholesterol and its esters, mono-, di- and triacylglycerols, free fatty acids and the phospholipids phosphatidylethanolamine and phosphatidylinositol) was found associated with tissue transglutaminase purified to apparent homogeneity from guinea pig liver. Removal of lipids results in an increased tendency of the enzyme for self-association and a decreased stability. Lauric acid was detected following hydroxylamine treatment of the enzyme, suggesting the occurrence of a fatty acid-type, covalent, posttranslational modification of transglutaminase. The results provide support for the idea that part of tissue transglutaminase may be localized in the cell membrane.

Animals↗
PubMed1986

Quantitation of tissue transglutaminase by a sandwich ELISA system.

A sandwich ELISA system has been developed to quantitate transglutaminase in human cell extracts. It utilizes affinity-purified rabbit anti-human transglutaminase as the capture antibody and a mouse monoclonal anti-transglutaminase (Birckbichler et al., 1985) as the indicator antibody (together with peroxidase-labeled anti-mouse immunoglobulin). The sensitivity of the assay was less than 1.0 ng/mg cellular protein. Significantly higher concentrations of the enzyme were found in resting versus proliferating or transformed fibroblasts in good agreement with previous activity measurements. The levels of transglutaminase in normal T and B lymphocytes, malignant lymphoid cells and monocytes were also determined.

Animals↗
PubMed1986

Transglutaminase-sensitive glutamine residues of human plasma fibronectin revealed by studying its proteolytic fragments.

The sites of transglutamination of fibronectin and fibronectin fragments, by coagulation factor XIIIa and tissue transglutaminase, were studied. It was shown that the intact fibronectin molecule has two sites sensitive to coagulation factor XIIIa and four sites sensitive to tissue transglutaminase: 180--190-kDa gelatin/heparin-binding fragments, 2 and 5--6 sites; 29-kDa heparin-I/fibrin-I-binding N-terminal fragments, 1 and 2 sites; 70-kDa gelatin-binding fragments, 0 and 1 site; 60-kDa cell-binding central fragments, 1 and 3--4 sites; 60-kDa, 45-kDa, 30-kDa heparin-II-binding C-terminal fragments, 1 and 2 sites. Thus, we have found a new coagulation-factor-XIIIa-sensitive site localized in the cell-binding central fragment, inaccessible to enzyme in the intact fibronectin molecule. Tissue transglutaminase appeared to interact with all of the three coagulation-factor-XIIIa-sensitive sites and, in addition, some others which are either available on the intact molecule or can be revealed only in proteolytic fragments of the fibronectin. We suggest that interdomain and intersubunit interactions in the intact fibronectin molecule account for the masking of glutamine residues potentially accessible to transglutaminases.

Binding Sites↗
PubMed1986

Purification of human erythrocyte transglutaminase by immunoaffinity chromatography.

Human erythrocyte transglutaminase was purified using a reusable immunoaffinity column prepared from a monoclonal antibody described previously (Birckbichler et al., Hybridoma, 4, 179-186, 1985). The purified TGase was catalytically active and exhibited a single band of apparent Mr = 85,000 on SDS-PAGE and Western blotting. The amino acid composition of the enzyme was determined. The amino terminus was blocked, and the carboxy-terminal residue appeared to be isoleucine.

Amino Acids↗
PubMed1985

Development of selective inhibitors of transglutaminase. Phenylthiourea derivatives.

For the purpose of developing a transglutaminase inhibitor which could be effective in physiological and pharmacological studies, a series of phenylthiourea derivatives of alpha, omega-diaminoalkanes were designed, synthesized, and evaluated kinetically as inhibitors of transglutaminases. A homologous series of compounds of the structure phenylthiourea-(CH2)n-NH2, where n = 2, 3, 4, 5, and 6, were tested for the inhibition of both guinea pig liver transglutaminase-catalyzed amine incorporation into various glutamine-containing substrates and plasma transglutaminase (factor XIIIa)-catalyzed amine incorporation into fibrin and fibrin cross-linking. It was found that the inhibitory activity of the compounds increases with increasing number of methylene groups in the side chain up to a maximum of n = 5. A further increase in the length of the methylene side chain to n = 6 results in decreased activity. The Ki value (4.9 X 10(-5) M) of 1-(5-aminopentyl)-3-phenylthiourea (PPTU) (n = 5) for the inhibition of guinea pig transglutaminase-catalyzed amine incorporation into the B chain of oxidized insulin is in close agreement to its Km(app) value (7.1 X 10(-5) M) obtained using 14C-labeled PPTU. PPTU was also found to be a potent inhibitor of plasma transglutaminase-catalyzed fibrin cross-linking. The finding that the specificity of the alkylamines for inhibition is correlated with the length of their methyl side chains is compatible with those reported for aliphatic amines and monodansylcadaverine analogues (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl). The phenylthiourea derivatives, however, are far less toxic in mice than monodansylcadaverine as indicated by their LD50 values: PPTU, 400 +/- 25 mg/kg; and monodansylcadaverine, 160 +/- 20 mg/kg.

Animals↗
PubMed1985

Activation of transglutaminase and production of protein-bound gamma-glutamylhistamine in stimulated mouse mast cells.

The identification of transglutaminase in the growth-factor-dependent mouse mast cell line PT18 was accomplished through its characteristic catalytic properties (specificity, calcium dependency, and inhibition by iodoacetamide); and by both immunoprecipitation and Western blot analysis using affinity purified antibody. The enzymatic activity in these cells increased in association with the release of histamine from the cells induced by an IgE-dependent mechanism or by exposure to the ionophores A23187 or Br-x537A. The increase in transglutaminase activity was paralleled by a marked increase in the level of protein-bound gamma-glutamylhistamine, determined in radiolabeled form in mast cells that were either metabolically labeled with [3H]histidine or incubated with [3H]histamine before degranulation. The highest level of bound gamma-glutamylhistamine was found in the immunologically stimulated cells. Enzymatic activity and the gamma-glutamyl derivative were associated primarily with the cells, both before and after stimulation. Separation of gamma-glutamylhistamine in a proteolytic digest of these cells was carried out using a combination of ion exchange chromatography and high performance liquid chromatography. The gamma-glutamyl compound was identified and quantitated through the enzymatic production of histamine with the use of gamma-glutamylamine cyclotransferase, an enzyme specific for the disassembly of gamma-glutamylamines.

Animals↗