SUBSTRATE-SPECIFICITY OF THE HUMAN LYMPHOKINE LEUKOCYTE MIGRATION-INHIBITORY FACTOR (LIF) - RADIOENZYMIC ASSAY AND INHIBITION BY CGMP

The human lymphokine, leukocyte migration-inhibitory factor (LIF), appears to be a serine esterase and protease by virtue of its susceptibility to the irreversible enzyme inhibitor, phenylmethylsulfonyl fluoride (PMSF), and by the ability of arginine esters and amides to protect LIF against PMSF-induced inactivation. In this study, 3 methods were described where putative substrates for LIF could be investigated. Molecules satisfying the substrate specificities of this lymphokine should protect LIF against inactivation by PMSF, reduce LIF activity in vitro on polymorphonuclear leukocytes, and reduce the esterolytic activity of purified LIF-rich supernatants. The first 2 reactions were tested by the leukocyte migration agarose technique; the 3rd reaction was tested by a sensitive enzyme using tritiated tosyl arginine methyl ester as substrate. Cyclic GMP, which is capable of protecting LIF against PMSF-induced inhibition, also inhibited the esterolytic activity of the purified LIF preparation. Four synthetic oligopeptide substrates for trypsin, thrombin and plasmin were investigated. Only 1, the thrombin- and trypsin-specific benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, possessed high affinity for the LIF molecule and may prove to be a potent substrate for this lymphokine.