New subtilisin-like collagenase from leaves of common plantain.
A new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain leaves. The protease named plantagolisin was isolated by ammonium sulfate precipitation of the leaves' extract followed by affinity chromatography on bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime. Its molecular mass is 19000 Da and pI 5.0. pH-stability range is 7-10 in the presence of 2 mM Ca(2+), temperature optimum is 40 degrees C. The substrate specificity of subtilase towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases: proteinase from leaves of the sunflower and taraxalisin. Besides, the proteinase is able to hydrolyze substrates with Pro in P(1) position. The enzyme hydrolyzes collagen. alpha and beta chains are hydrolyzed simultaneously in parallel; there are only low-molecular-mass hydrolysis products in the sample after 2 h of incubation. Pure serine proteinase was inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and Hg(2+). The plantagolisin N-terminal sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology with that of subtilisin from yeast Schizosaccharomyces pombe.
Bogacheva
A M
AM
Moscow State University, Chemistry Department, Vorobevy Gory, B-234, 119899, Moscow, Russia. annamb@genebee.msu.su
Rudenskaya
G N
GN
Dunaevsky
Y E
YE
Chestuhina
G G
GG
Golovkin
B N
BN
D000595:Amino Acid Sequence; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology*; D010943:Plantago / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry*; D013696:Temperature
D061965:Matrix Metalloproteinase Inhibitors; D020860:Subtilisin; D017364:Collagenases; C438651:plantagolisin