title: Evidence for control of mast cell granule protease in situ by low pH.
abstract: The second order rate constant, k2, for the inhibition of mast cell protease I by phenylmethanesulfonyl fluoride (PMSF) is lower for intact mast cells and isolated granules with intact membranes than for granules stripped of their membranes and suspended in medium at pH 7.1. In order to test the hypothesis that the decreased activity of the protease in intact granules is attributable to low pH, two agents capable of lowering pH in intracellular compartments similar to mast cell granules were tested. Ammonium chloride increased k2 of the protease in isolated granules with intact membranes and mast cells and wash out of the salt partially reversed this effect. Treatment of cells with nigericin also substantially increased the rate of protease inactivation by PMSF. These results are consistent with the proposal that the observed k2 is determined in whole or part by a low pH of the granule in situ or isolated with intact membranes. If the low k2 in situ is solely dependent on low pH, then the rate of protease inhibition can be utilized as an endogenous probe of granule pH. On this basis we have estimated the pH of the intracellular granule as 5.2 and that of the isolated granule with its membrane intact as 6.0. The value for the pH of granules in situ is lower than that previously estimated, and we have considered possible bases for this discrepancy.
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008407:Mast Cells / Q000201:enzymology*; D009550:Nigericin / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D051381:Rats
chemical_list: D000643:Ammonium Chloride; D010664:Phenylmethylsulfonyl Fluoride; D010447:Peptide Hydrolases; D009550:Nigericin