Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties.
In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.
Zamolodchikova
T S
TS
Laboratory of Proteolytic Enzymes Chemistry, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Vorotyntseva
T I
TI
Antonov
V K
VK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D004386:Duodenum / Q000201:enzymology*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002241:Carbohydrates; D009842:Oligopeptides; D012697:Serine Endopeptidases; C078261:duodenase