The extent and distribution of N-glycosylation and the nature of most of the disulfide bond linkages were determined for bovine lactoperoxidase through proteolytic and glycolytic digestions combined with matrix-assisted laser desorption/ionization mass spectrometric analysis. In addition, 98% of the primary sequence of the protein was confirmed. All five of the asparagines present in sequons were found to be glycosylated, predominantly by high mannose and complex structures. Six disulfide bonds were assigned, including Cys 32-Cys 45, Cys 146-Cys 156, Cys 150-Cys 174, Cys 254-Cys 265, Cys 473-Cys 530 and Cys 571-Cys 596.

BACKGROUND:The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however.RESULTS:Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation.CONCLUSIONS:Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.

The high-energy collision-induced dissociation (CID) spectra of a number of peptides containing S-β-(4-pyridylethyl)-, S-carbamidomethyl- and S-carboxymethylcysteine have been generated and several hitherto undescribed fragmentation pathways identified. For peptides containing pyridylethylcysteine at the nth position, these include xn − 105, an+1 + 1 − 106, an+1 + 1 − 138, zn+1 + 1 − 106, zn+1 + 1 − 138, and combined side chain loss ions. The analogous fragment ions were also observed in the spectra of peptides containing other S-alkylcysteines. Also, two fragmentations unique to pyridylethylcysteine-containing peptides, resulting in the formation of bn+ ≥ 1 - RC + H and CO + peC ions, were studied. Precursor scanning, exact mass measurement and CID of the appropriate source-generated fragment ions have been employed to identify the origin of these ions and to facilitate the proposal of mechanisms for their formation.