Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.
Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.
D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007070:Immunoglobulin A; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors; D013298:Streptococcus sanguis / Q000201:enzymology*; D013379:Substrate Specificity; D013393:Sucrase / Q000378:metabolism
D007070:Immunoglobulin A; D010618:Phenanthrolines; D011480:Protease Inhibitors; D004492:Edetic Acid; D013393:Sucrase; D010447:Peptide Hydrolases