Effects of 1-chloro-2,4,6-trinitrobenzene on 5-lipoxygenase activity and cellular leukotriene synthesis.

5-Lipoxygenase (EC 1.13.11.34) is the key enzyme in the regulation of leukotriene synthesis. Here, the effects of various substituted nitrobenzene compounds on 5-lipoxygenase activity and the formation of leukotriene B4 (LTB4) were studied in polymorphonuclear leukocytes (PMNL), B lymphocytes, and human whole blood. 1-Chloro-2,4,6-trinitrobenzene (TNCB) was found to inhibit calcium ionophore A23187-induced leukotriene synthesis in PMNL in a biphasic manner. Thus, 1.0 microM TNCB caused 50% inhibition of LTB4 formation, but only 16% inhibition was found at 10 times higher concentration. In contrast, this higher concentration of TNCB activated the synthesis of LTB4 when PMNL were stimulated with arachidonic acid alone, demonstrating that TNCB can exert both stimulatory and inhibitory effects on leukotriene synthesis depending on the experimental conditions. The inhibitory effect of 1.0 microM TNCB on ionophore A23187-induced leukotriene synthesis could be circumvented by addition of exogenous arachidonic acid. At high concentrations of TNCB (25-100 microM), the drug blocked ionophore A23187-induced leukotriene synthesis. TNCB also inhibited LTB4 formation in B lymphocytes, as well as in human whole blood. The activity of recombinant 5-lipoxygenase was inhibited by TNCB, and reduced glutathione or beta-mercaptoethanol counteracted this inhibition. This suggests that TNCB might inhibit 5-lipoxygenase by alkylating thiol groups. TNCB possessed a high specificity for 5-lipoxygenase with only modest inhibitory effects on 12-lipoxygenase (EC 1.13.11.31), 15-lipoxygenase (EC 1.13.11.12), and phospholipase A2 (EC 3.1.1.4) activities. Taken together, these results show that TNCB can both specifically inhibit and stimulate leukotriene formation and might be useful in further studies on the regulation of 5-lipoxygenase.

Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity.

Recently, the cloning of a novel Ca2+-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA2 cDNA and its expression in B-cells and show that the iPLA2 gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA2 activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa. The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline-rich insertion in the last of the eight ankyrin repeats (residues 395-449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA2 but terminates just after the lipase active site, and two other isoforms contain only the iPLA2 ankyrin repeat sequence (ankyrin-iPLA2-1 and -2). Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA2 enzyme exists as a multimeric complex of 270-350 kDa, the expression of just the ankyrin-iPLA2 sequence suggested that these may also interact with the iPLA2 oligomeric complexes and perhaps modulate PLA2 activity. Transfection of the human iPLA2 cDNA into COS cells resulted in a substantial increase in calcium-independent PLA2 activity in cell lysate. No activity above background was observed following ankyrin-iPLA2-1 cDNA transfection. However, co-transfection of the ankyrin-iPLA2-1 and the iPLA2 cDNAs resulted in a 2-fold reduction in activity compared with iPLA2 alone. A similar co-transfection of ankyrin-iPLA2-1 cDNA with the cPLA2 cDNA had no effect on PLA2 activity. These results suggest that the ankyrin-iPLA2 sequence can function as a negative regulator of iPLA2 activity and that the alternative splicing of the iPLA2 gene can have a direct effect on the attenuation of enzyme activity.