Substrate specificity of the human lymphokine leucocyte migration-inhibitory factor (LIF): radioenzymic assay and inhibition by cGMP.
The human lymphokine, leucocyte 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 paper, three methods are described by which putative substrates for LIF may be investigated. Thus, molecules satisfying the substrate specificities of this lymphokine should (1) protect LIF against inactivation by PMSF, (2) reduce LIF activity in vitro on polymorphonuclear leucocytes, and (3) reduce the esterolytic activity of purified LIF-rich supernatants. The first two reactions were tested by means of the leucocyte migration agarose technique; the third reaction was tested by a sensitive enzyme assay using tritiated tosyl arginine methyl ester as substrate. Guanosine 3',5'-cyclic monophosphoric acid, 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, thombin and plasmin were investigated. Only one, the thrombin- and trypsin-specific benzoyl-phenylalanyl-valyl-agarine-p-nitroanilide, possessed high affinity for the LIF molecule and may therefore prove to be a potent substrate for this lymphokine.
D000813:Anilides / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology*; D004952:Esters / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008222:Lymphokines / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008722:Methods; D009504:Neutrophils / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D014106:Tosylarginine Methyl Ester / Q000494:pharmacology; D014316:Tritium
D000813:Anilides; D004952:Esters; D008222:Lymphokines; D009842:Oligopeptides; D014316:Tritium; D010664:Phenylmethylsulfonyl Fluoride; D014106:Tosylarginine Methyl Ester; D010447:Peptide Hydrolases; D006152:Cyclic GMP