CHEMOTACTIC FACTOR-INDUCED SUPEROXIDE RADICAL GENERATION BY HUMAN-NEUTROPHILS - REQUIREMENT FOR PROTEINASE (ESTERASE) ACTIVITY

  • 1 January 1979
    • journal article
    • research article
    • Vol. 94  (3) , 403-413
Abstract
The requirement for proteinase (esterase) activity in O2- generation by human peripheral neutrophils was investigated. Neutrophils were activated by exposure to the chemotactic peptide FMLP [formyl-methionyl-leucyl-phenylalanine] and superoxide generation was assessed by ferricytochrome C reduction. O2- generation inhibition was observed by pretreating cells with chloromethyl ketone derivatives of tosyl-phenylalanine (TPCK, ID50 [median inhibitory dose] 2.2 .times. 10-5 M) and tosyl-lysine (TLCK, ID50 1.9 .times. 10-4 M). Dose-dependent inhibition was also noted with synthetic proteinase substrates, especially those of chymotrypsin-like specificity, phenylalanine and tryptophan methyl esters [PheME and TryME] (ID50S .apprx. 1.5 .times. 10-4 M), as compared to derivatives of basic amino acids, arginine and acetyl-lysine methyl esters, which caused negligible inhibition at 10-3 M. O2- generation inhibition demonstrated by TryME was time- and temperature-dependent and reversible with cell washing, whereas inhibition seen with TPCK was irreversible. DFP at high concentrations, 10-3 M or greater, caused moderate O2- generation inhibition unaffected by exogenous serine and almost completely reversible by cell washing. TPCK and TryME, at concentrations that caused marked inhibition of O2- generation, had no effect on FMLP-induced 45Ca cell uptake. Intact proteinase function is apparently required for O2- generation and this step follows the Ca influx in the activation sequence induced by FMLP.