title: Serine protease from midgut of Bombus terrestris males.
abstract: A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine 4-nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55±0.042 mM and 0.714±0.056 μmol p-nitroalanine produced min(-1) mg protein(-1) , respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, which indicated the trypsin-like but not chymotrypsin-like specificity. The identity of the serine protease was confirmed by nanoliquid-tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Brabcová
authors.forename: Jiří
authors.initials: J
authors.lastname: Kindl
authors.forename: Irena
authors.initials: I
authors.lastname: Valterová
authors.forename: Iva
authors.initials: I
authors.lastname: Pichová
authors.forename: Marie
authors.initials: M
authors.lastname: Zarevúcka
authors.forename: Jana
authors.initials: J
authors.lastname: Brabcová
authors.forename: Michal
authors.initials: M
authors.lastname: Jágr
authors.forename: Ivan
authors.initials: I
authors.lastname: Mikšík
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000201:enzymology*; D041981:Gastrointestinal Tract / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D057057:Serine Proteases