Purification and characterization of an acidic amino acid-specific endopeptidase of Bacillus subtilis obtained from a commercial preparation (Protease Type XVI, Sigma).
An acidic amino acid-specific endopeptidase was purified from Protease Type XVI (Sigma), a commercial product from culture filtrate of Bacillus subtilis, by a series of column chromatographies on CM-Toyopearl (Fractogel) and Mono-S, guided by activity assay using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase HPLC. The molecular weight of the protease was estimated to be 18,000 by gel filtration on TSK gel G3000SWXL column using 6 M guanidine hydrochloride as an eluent, and 17,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the protease was 7.7. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that the protease hydrolyzes the peptide bonds on the carboxyl-terminal side of acidic amino acids, especially of glutamic acid. The protease was completely inactivated by DFP, indicating the serine protease nature of the protease. The activity of the protease was also inhibited by EDTA and GEDTA, and reactivated by Ca2+. The protease contained 1.3 +/- 0.2 mol/mol protein of Ca2+. These results suggest that Ca2+ plays a vital role in the protease activity.
Niidome
T
T
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Yoshida
N
N
Ogata
F
F
Ito
A
A
Noda
K
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D006146:Guanidines / Q000494:pharmacology; D006868:Hydrolysis; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D006146:Guanidines; D008623:Mercaptoethanol; D004492:Edetic Acid; D010450:Endopeptidases; D002118:Calcium