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.
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.
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
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