thioacetamide and methionine-sulfoxide

Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Brain; Cattle; Cricetinae; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Hydrogen Peroxide; Methionine; Mice; Mice, Inbred C57BL; Models, Biological; Oxidation-Reduction; PrPC Proteins; PrPSc Proteins; Sheep; Tandem Mass Spectrometry; Thioacetamide

During the isolation of cholecystokinin from natural sources, as well as during its bioassay, inactivation by oxidation can cause problems. We have attempted to reactivate oxidized CCK by reduction at room temperature with N-methylmercaptoacetamide, recently stated to be the reducing agent of choice for the reduction of methionine sulfoxide to methionine [22]. We have not yet been unequivocally successful in these attempts, but the results seem promising. In the case of oxidized VIP and of oxidized tetragastrin, reduction with N-methylmercaptoacetamide does seem to result in reconversion of the peptides to their preoxidation states, as evidenced by thin layer chromatography on silica gel. We have, together with A. Holmgren and A. Ehrnberg, made observations suggesting the presence in rate liver cytosol of an enzyme which catalyzes the reductive reactivation of oxidized CCK with reduced thioredoxin as the immediate hydrogen donor. In collaboration with A. Light, Purdue University, we have found that enterokinase cleaves 39-CCK and 33-CCk with release of 8-CCK and the tetrapeptide immediately preceding it in the peptide chain. The conversion of 39-CCK to 33-CCK by the action of dipeptidyl amino-peptidase I has been confirmed.

Topics: Animals; Cats; Cholecystokinin; Enteropeptidase; Gastrointestinal Hormones; Guinea Pigs; Liver; Methionine; Methionine Sulfoxide Reductases; NADP; Oxidation-Reduction; Oxidoreductases; Peptide Fragments; Sincalide; Tetragastrin; Thioacetamide; Vasoactive Intestinal Peptide