Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli.

To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate.

Activity of tricyclic nucleoside 5'-phosphate in model systems of human ovarian cancer.

Tricyclic nucleoside 5'-phosphate (TCN-P) was evaluated in two models of human ovarian cancer. TCN-P reduced both colony number and volume in clonogenic assays employing human ovarian cancer cell lines. TCN-P cytotoxicity depended on the concentration, exposure duration and cell line studied, but not on cell line plating efficiency or growth rate in soft agarose. Comparison of experimental IC50 concentrations for 1 hour or continuous TCN-P exposure with reported clinically relevant concentrations suggests that therapeutic TCN-P levels are more likely to be achieved by continuous infusions. Cell lines and sublines with resistance to several standard chemotherapeutic agents acquired both in vivo and in vitro were at most 2.6-fold cross-resistant to TCN-P with 1 hour drug exposure. Cross-resistance was not evident with continuous TCN-P exposure. Intermittent bolus TCN-P (100 mg/kg/d X 5) was ineffective in an in vivo xenograft model of human ovarian cancer. These data suggest that TCN-P is most likely to be clinically effective against ovarian cancer, and may be non-cross-resistant with several standard agents, if administered by continuous infusion. Preclinical evaluation of new agents, such as TCN-P, in these experimental models may provide information useful in subsequent clinical trials.