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Prevention of degradation of human polymorphonuclear leukocyte proteins by
diisopropylfluorophosphate
PC Amrein and TP Stossel
Proteases can complicate the characterization of proteins from cells,
especially human polymorphonuclear leukocytes (PMN), which contain abundant
neutral proteases. We tested the ability of agents to inhibit proteolysis,
with special reference to the subunit polypeptides of the contractile
proteins actin, myosin, and actin-binding protein (ABP).
Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N-
ethylmaleimide, alone or in combinations, failed to prevent extensive
proteolysis of the PMN proteins during solubilization of cells with dodecyl
sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin
inhibitor similarly could not prevent proteolysis during homogenization of
cells in cold isosomolar sucrose. Treatment of PMN with greater than or
equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or
homogenization markedly inhibited proteolysis. PMSF and DFP were equally
effective in inhibiting proteolysis in PMN extracts, suggesting that the
efficacy of DFP may result from its permeation of intact cells and granules
before barriers are disrupted by detergents or homogenization. Treatment of
PMN with DFP under conditions inhibiting proteolysis did not affect their
rate of phagocytosis. We recommend the use of DFP in future studies
correlating functions and protein structure of PMN.
Volume 56,
Issue 3,
pp. 442-447,
09/01/1980
Copyright © 1980 by The American Society of Hematology
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