Purification and properties of a specific proteolytic enzyme present in spores of Bacillus magaterium.
A proteolytic enzyme with high activity on the specific, low molecular weight dormant spore proteins (termed A and B proteins) degraded during spore germination has been purified approximately 1000-fold from Bacillus megaterium spores. This enzyme accounts for greater than 85% of the proteolytic activity toward the A and B proteins in crude spore extracts. The protease has a pH optimum of approximately 7.5, is inactivated by phenylmethylsulfonyl fluoride and EDTA (10 mM), and is inhibited approximately 70% by NaCl (1 M). The enzyme is unstable and requires glycerol, divalent cations, and high enzyme concentrations for maximum stability. The protease shows a high degree of specificity for the A and B proteins, since high levels of enzyme catalyze no detectable bond cleavage on a variety of amide, ester, peptide, or other protein substrates. The enzyme is an endoprotease and digestion of the A and B proteins in vitro generates a number of peptide fragments at least one of which appears identical with a fragment isolated from lysates of spores carrying out hydrolysis of endogenous A and B proteins. In vitro, the peptide products can be rapidly degraded to amino acids by an aminopeptidase which has also been partially purified from spores. Although high levels of the protease are present in extracts of dormant spores, and of spores germinated for a few minutes, the enzyme is undetectable in log phase and early stationary phase cells. Furthermore, the protease disappears rapidly (t1/2 approximately 30 min) and completely (greater than 90%) as the process of germination proceeds.
D000626:Aminopeptidases / Q000302:isolation & purification; D001410:Bacillus megaterium / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D013171:Spores, Bacterial / Q000201:enzymology; D013329:Structure-Activity Relationship
D010447:Peptide Hydrolases; D000626:Aminopeptidases; D005990:Glycerol; D002118:Calcium