Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases.
A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D004926:Escherichia coli / Q000032:analysis*; D029968:Escherichia coli Proteins; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010434:Pepsin A / Q000378:metabolism; D033903:Periplasmic Proteins; D011480:Protease Inhibitors / Q000302:isolation & purification*
D000596:Amino Acids; D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011480:Protease Inhibitors; D002918:Chymotrypsin; D010434:Pepsin A