Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase).
A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.
Yoshida
N
N
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Tsuruyama
S
S
Nagata
K
K
Hirayama
K
K
Noda
K
K
Makisumi
S
S
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis; D011402:Pronase / Q000032:analysis*; D011480:Protease Inhibitors / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis; D013305:Streptomyces griseus / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors; D011506:Proteins; C059145:tert-butoxycarbonyl-alanyl-alanyl-prolyl-glutamyl-4-nitroanilide; C059144:N-tert-butoxycarbonyl-alanyl-alanyl-prolyl-aspartyl-4-nitroanilide; D010450:Endopeptidases; D011402:Pronase