Use of benzoyl-L-phenylalanyl-L-valyl-L-arginine (3H) methyl ester as a sensitive and selective substrate for the human lymphokine, leukocyte migration inhibitory factor (LIF).
Human leukocyte migration inhibitory factor (LIF) appears to be a serine esterase and protease exhibiting specific affinity towards arginine esters and amides. On the basis of indirect evidence that an amide of the oligopeptide benzoyl-phenylalanyl-valyl-arginine might have high and selective affinity for LIF, we prepared an ester of this oligopeptide, benzoyl-phenylalanyl-valyl-arginine (3H) methyl ester (3H-BPVAME) for the direct measurement of LIF activity in a double-phase radio-enzyme assay. The hydrolysis of 3H-BPVAME followed enzyme-substrate kinetics in that the reaction was time-, temperature-, pH-, and concentration-dependent. 3H-BPVAME rpoved to be more selective and approximately 20 times more sensitive as a substrate for LIF than previously used radiolabeled substrates such as 3H-BAEE and 3H-TAME. Moreover, hydrolysis of 3H-BPVAME by partially purified LIF preparations was significantly inhibited by 10(-8) to 10 (-5) M of guanosine 3',5'-cyclic monophosphate (cGMP), further supporting the hypothesis that cGMP acts as a regulator of LIF activity. Inhibition of LIF-induced esterolysis was also provided by dibutyryl cGMP, but only at concentrations 10(-7) to 10(-5) M; 8-bromo cGMP and adenosine 3',5'-cyclic monophosphate were both ineffective. These results provide support for the use of 3H-BPVAME as a more selective substrate to detect esterase activity in LIF preparations than heretofore described and the possible development of a biochemical assay for the measurement of this lymphokine.
D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004952:Esters; D006801:Humans; D007961:Leukocyte Migration-Inhibitory Factors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008222:Lymphokines / Q000378:metabolism*; D009842:Oligopeptides; D014106:Tosylarginine Methyl Ester / Q000494:pharmacology; D014316:Tritium
D004952:Esters; D007961:Leukocyte Migration-Inhibitory Factors; D008222:Lymphokines; D009842:Oligopeptides; D014316:Tritium; C026649:benzoyl-L-phenylalanyl-L-valyl-L-arginine-L-methyl ester; D014106:Tosylarginine Methyl Ester; D000242:Cyclic AMP; D006152:Cyclic GMP