Serine protease from midgut of Bombus terrestris males

0391467 - ÚOCHB 2014 RIV US eng J - Journal Article
Brabcová, Jana - Kindl, Jiří - Valterová, Irena - Pichová, Iva - Zarevúcka, Marie - Brabcová, J. - Jágr, Michal - Mikšík, Ivan
Serine protease from midgut of Bombus terrestris males.
Archives of Insect Biochemistry and Physiology. Roč. 82, č. 3 (2013), s. 117-128. ISSN 0739-4462. E-ISSN 1520-6327
R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969
Institutional support: RVO:61388963 ; RVO:67985823
Keywords : Bombus terrestris * midgut * serine protease * bumblebee
Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C)
Impact factor: 1.160, year: 2013

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 degrees 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 mol p-nitroalanine produced min1 mg protein1, 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.
Permanent Link: http://hdl.handle.net/11104/0220513