The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses alpha-ketoglutarate and O(2) as cosubstrates, and forms succinate and CO(2) HKI-272 in vivo as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing Substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into
(2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to SN-38 those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to Characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of alpha-ketoglutarate. Thus, the assay described herein Could facilitate biochemical analyses of this essential enzyme. (C) 2008 Elsevier Inc. All rights reserved.”
shock proteins (HSP) are induced during cellular stress. Their role is to chaperone cellular proteins giving protection from denaturation and ultimately preventing cell death. Monocytes are key cells involved in atherosclerosis and are highly responsive to HSP induction. Therefore, we wished to examine monocyte Hsp70 expression and induction in patients with peripheral arterial disease (PAD) and in healthy controls.\n\nWe measured cellular Hsp70 levels in freshly isolated monocytes and released Hsp70 levels in plasma and monocyte culture supernatants, obtained from patients
with PAD and from healthy controls. A-1155463 mouse We assessed the effect of statin therapy on Hsp70 levels and examined monocyte cell survival in culture with and without immunological stress.\n\nMonocyte cellular Hsp70 was lower in patients with PAD compared to healthy controls (11.3 +/- 7.4 ng/10(6) cells vs 20.7 +/- 16.0 ng/10(6) cells; p < 0.001). Individuals on statin therapy from both PAD and control groups had lower monocyte Hsp70 compared to those not treated with statins. Concentrations of Hsp70 released into culture supernatants were not dependent on PAD or statin therapy. Cell survival was inversely associated with Hsp70 concentrations in culture supernatants but had no association with cellular concentrations of Hsp70.\n\nCellular Hsp70 and released Hsp70 may play different roles in monocyte health.