Introduction Materials and Methods Materials Nunc Immuno plates II were obtained from Nunc (Roskilde, Denmark). Horseradish peroxidase1) was obtained from Boehringer Mannheim (Mannheim, Germany) and 2,2'-azino-bis(3ethyl-benzthiazoline-6-sulphonic acid (ABTS) from Sigma (Deisenhofen, Germany). Human recombinant angiogenin was kindly donated by Behringwerke, Marburg. Blood samples were supplied by Prof. Kleesiek, Herzzentrum Bad Oeynhausen-(Bad Oeynhausen, Germany). 5-[2,3-bis(palmitoyloxy)propyl]-Npalmitoyl-cysteinyl-serinyl-glycine-succinimidyl-ester (Pam3 CysSerGlyOSu) and S-[(2J?S)-2,3-bis(palmiloyloxy)-propyl]-Npalmitoyl-cysteinyl-lysyl-lysyl-lysyl-lysine (Pam3Cys-Ser-Lys4) were kindly donated by Prof. Jung (Tübingen, Germany). Methods Preparation of the S-[2t3,bis(palmitoyloxy)propyl]-N-palmitoyl-cysfeinyl-sennyl-glycine/angiogenin (Pam3CysSerGIy/angiogenin) conjugate Recombinant angiogenin (0.5 mg) was mixed with 5mg Pam3CysSerGlyOSu in 500 freshly distilled dimethylformamide and incubated under continuous stirring for 15 h at room temperature. After removal of the dimethylformamide, the product was dissolved in /-butanol/water (3 + 1, by vol.) and lyophilised (10). Angiogenin is a single chain Afr 14100 protein, first isolated and characterised from a HT-29 human adenocarcinoma cell line (1). It has been shown to be an inducer of vascular growth. The protein has 35% identity with pancreatic ribonuclease (2) and has been shown to inhibit protein synthesis in vitro (3). Saxena et al. described angiogenin as a cytotoxic, t-RNAspecific ribonuclease iii the RNase A superfamily (4). Angiogenic and ribonucleolytic activities are blocked by a tight-binding placental ribonuclease inhibitor (5).
Clinical Chemistry and Laboratory Medicine – de Gruyter
Published: Jan 1, 1993
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