Inactivation of thromboxane synthase with hydroperoxy-fatty acids.

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The structure-activity relationship of several hydroperoxy fatty acids and their ability to inactivate thromboxane synthase was investigated. Millimolar quantities of hydrogen peroxide and tert-butylperoxide were required to inactivate the enzyme: whereas micromolar quantities of C18 and C20 hydroperoxy fatty acids inactivated the enzyme. Pretreatment of the enzyme with long chain hydroperoxy-fatty acids resulted in a decreased synthesis of both TXB2 and HHT.

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production.

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.

Measurement of PGH synthase activity using a pan-specific monoclonal antibody.

A pan-specific monoclonal antibody that recognizes a variety of prostaglandin moieties and does not recognize arachidonic acid or the hydroxy-eicosatetraenoic acids was used to assess the general ability of a tissue to produce prostaglandins. Although this assay does not give a quantitative measure of PGH synthase activity, it does provide a sensitive and convenient means of screening a large number of samples for prostaglandin production.

Alterations in human circulating and bone marrow mononuclear cell polyamine levels in hematologic malignancies as a consequence of difluoromethylornithine administration.

The effect of administering increasing intravenous doses of difluoromethylornithine on human tumor cell polyamine levels was determined in patients with hematologic malignancies. Difluoromethylornithine from 5.5. to 64 gm/m2 per day was administered to nine patients with refractory acute leukemia or multiple myeloma. Putrescine, spermidine, and spermine levels were determined on a daily basis in the circulating mononuclear cells and on a weekly basis in the mononuclear cells of the bone marrow. Tumor cell putrescine levels declined in 5 patients, spermidine levels declined in 4 patients, and spermine levels declined in 3 patients. Alterations in the polyamine levels of the bone marrow mononuclear cells paralleled those occurring in the peripheral blood mononuclear cells in the patients with leukemia. Seven to ten days of DFMO treatment were required for mononuclear cell polyamine levels to decrease. The higher drug doses were not significantly more effective than the lower doses in bringing about a decline in tumor cell polyamine levels, either with respect to treatment time required for onset of response or with respect to the ultimate extent of response.