Human Leukocyte Inhibitory Factor (LIF): Use of Benzoyl L-Arginine Ethyl Ester to Detect LIF Activity

Abstract

Abstract Human leukocyte inhibitory factor (LIF), a mediator elaborated by activated lymphocytes, has recently been shown to have the properties of an esterase. Further more, the LIF preparation contains an esteratic principle that hydrolyzes the amino acid ester, benzoyl arginine ethyl ester (3H-BAEE). In the present experiments, we sought to determine whether the esterase and biologic activities in the LIF preparation were due to the same or different molecules. This was accomplished by means of two series of experiments. With one approach, LIF was initially subjected to gel filtration and subsequently to polyacrylamide gel electrophoresis, sucrose density gradient electrophoresis, and affinity chromatography. The fractions obtained from each procedure were analyzed for biologic and esterase activities. The results indicate that both activities co-chromatographed with each technique. In addition, there are contaminating esterases present in the LIF preparation that can hydrolyze 3H-BAEE, some activity(ies) of which are eliminated by purification. The other approach directly demonstrated whether BAEE could serve as a substrate for LIF by substrate-competition experiments. BAEE and other structurally related amino acid esters were co-incubated with LIF and the ability of the former to protect the latter against inactivation by esterase inhibitors, or block biologic LIF activity on polymorphonuclear leukocytes was ascertained. It was shown that BAEE specifically prevented the destruction of LIF by diisopropylphosphofluoridate (DFP) and phenyl methyl sulfonyl fluoride (PMSF). Moreover, BAEE in a dose-dependent fashion, blocked LIF activity on PMN leukocytes in vitro when added to the culture supernatants. Taken together, these results strongly suggest that BAEE may serve as a substrate for LIF and point to the eventual development of a biochemical assay for this lymphocyte mediator.