PubMed Health⌕ Search

Biomedical subjects

R Brugger

Publications and source records attributed to R Brugger.

23 records · Page 2Linked to original sources
PubMed1994

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.

Animals↗
PubMedDate not supplied

Isolation and pharmacological characterization of microsomal human liver flumazenil carboxylesterase.

PURPOSE: In vivo the biotransformation of the imidazobenzodiazepine antagonist flumazenil leads to the formation of two metabolites, flumazenil acid and N-demethylated flumazenil. In the present study we investigated the role of carboxylesterases for the metabolism of flumazenil. METHODS: We purified a non-specific carboxylesterase (EC 3.1.1.1) from human liver microsomes that catalyzes the hydrolysis of flumazenil to flumazenil acid and, in presence of methanol the formation of flumazenil methyl ester an in vivo unknown metabolite. The purification procedure included solubilization of the microsomes obtained from human livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, DEAE-sepharose, hydroxyapatite and final chromatofocusing. RESULTS: The purified esterase isozyme exhibited an apparent subunit molecular weight of 59 kDa as estimated by SDS gelelectrophoresis, a native molecular weight of 170 kDa determined by a calibrated gel filtration column suggesting that the active enzyme is a trimer. The isoelectric point of the enzyme was approximately 5.4. The specific activities of the purified enzyme were 5.8 nmol/(min*mg protein) protein for the formation of flumazenil acid and 31 nmol/(min*mg protein) for the synthesis of the flumazenil methylester. The purified enzyme obeys simple Michaelis-Menten kinetics with K(M) values of 665 microM for flumazenil acid, 1011 mM for methanol and 900 microM for the flumazenil methylester. PMSF, a specific inhibitor for serine proteases and mammalian acetylcholinesterase, completely inhibited the formation of flumazenil -acid and the flumazenil methylester at a concentration of 100 microM. No synthesis of the flumazenil -methylester could be observed by incubation of the purified esterase with flumazenil acid in the presence of methanol leading to the conclusion that the enzymatically catalyzed reaction is a transesterification. The purified esterase was digested with endoproteinase LysC. A 15 amino acid long peptide was isolated and showed identical matches to carboxylesterase cDNAs from human liver and lung. CONCLUSION: Our results show that carboxylesterase isozymes play an important role in the detoxification and metabolism of flumazenil. Because of enzymatic, catalytic and structural properties a similarity of the characterized flumazenil carboxylesterase with human liver cocaine carboxylesterase is possible.

Animals↗