title: Determination of the human lymphokine leukocyte migration inhibitory factor (LIF) by a sensitive radioenzymatic assay. Inhibitory effect of cGMP on the esterolytic activity of highly purified LIF.
abstract: Indirect experiments using irreversible enzyme inhibitors have shown that the human lymphokine leukocyte migration inhibitory factor (LIF) is a serine esterase and protease exhibiting specific affinity towards arginine esters and amides. A sensitive assay for direct measurement of esterase activity using p-tosyl-L-arginine (3H) methyl ester (3H-TAME) as substrate is described. Esterolytic activities are demonstrated in crude supernatants of human lymphocytes stimulated with concanavalin A (LIF-rich) and, more pronounced, in supernatants of unstimulated cells (control). To follow the effects of purification procedures, the serine esterases of LIF-rich and control preparations were specifically labeled with the irreversible, active site directed agent (1,3-3H)di-isopropylphosphorofluoridate. Most of these enzymes, visualized by Sephadex chromatography, were removed by a gentle three-step procedure, allowing at least 50% of the initial LIF activity to be recovered. The resulting LIF-rich preparation, purified to contain serine esterases at a concentration corresponding to less than 1 ng per ml original supernatant, still showed estrolytic activity towards 3H-TAME. The optimal conditions for the radioenzymatic assay of purified LIF and the inhibitory effect of 10(-4) M cGMP, which on the basis of indirect experiments has been implicated as a specific regulator of LIF activity, are described.
mesh_terms: D000345:Affinity Labels; D002464:Cell Migration Inhibition; D003208:Concanavalin A; D003470:Culture Media; D006152:Cyclic GMP / Q000494:pharmacology*; D004950:Esterases / Q000037:antagonists & inhibitors*; D006801:Humans; D007531:Isoflurophate; D007961:Leukocyte Migration-Inhibitory Factors / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects; D013379:Substrate Specificity; D014106:Tosylarginine Methyl Ester; D014316:Tritium
chemical_list: D000345:Affinity Labels; D003470:Culture Media; D007961:Leukocyte Migration-Inhibitory Factors; D014316:Tritium; D003208:Concanavalin A; D007531:Isoflurophate; D014106:Tosylarginine Methyl Ester; D004950:Esterases; C022635:serine esterase; D006152:Cyclic GMP