BACKGROUNDTo establish a SAH hydrolase antiviral screening in vitro model for screening of broad spectrum antiviral agents.

METHODSSAH hydrolase was purified from rat livers by (NH4) 2SO4 fractionation, DEAE52,hydroxyapatite and Sephadex G-100 chromatography successively. The activity of SAH hydrolase was estimated by radio labeled substrate in synthesis direction by TLC.

RESULTSPurified SAH hydrolase showed a single band in SDS-PAGE electrophoresis with silver nitrate staining, the apparent molecular weight is 45 000. The Km for adenosine is (6.32 +- 0.17) micromol/L. The IC50 of S-DNPA, a known inhibitor of SAH hydrolase, was 7.6 micromol/L estimated in our system. The structure and activity relationships shown by racemic and regiosomer analogs of S-DHPA indicated that the structural specificity of SAH hydrolase was high. 42 compounds had been screened in the system and no compound showed more inhibitory activity against SAH hydrolase than S-DNPA.

CONCLUSIONSAn in vitro antiviral screening model has been established using SAH hydrolase. It can also be used to study kinetics of enzyme inhibition.

Adenine ; analogs & derivatives ; chemistry ; pharmacology ; Adenosylhomocysteinase ; Animals ; Antiviral Agents ; chemistry ; pharmacology ; Drug Evaluation, Preclinical ; Female ; Hydrolases ; antagonists & inhibitors ; isolation & purification ; metabolism ; In Vitro Techniques ; Liver ; enzymology ; Rats ; Rats, Wistar

3-Deazaadenosine (DZA), a potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential (DeltaPsim). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.
Adenosylhomocysteinase ; Amino Acid Chloromethyl Ketones ; Apoptosis ; bcl-2-Associated X Protein ; bcl-X Protein ; Cell Death ; Cytochromes c ; Cytosol ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; Mitochondria ; Tubercidin