Interaction of silkworm larval hemolymph antitrypsin and bovine trypsin.
Antitrypsin isolated from silkworm larval hemolymph can inhibit bovine trypsin. The apparent molar ratio of silkworm antitrypsin (sw-AT) to trypsin at extrapolated null trypsin activity was determined to be 1.3 by titration of trypsin with sw-AT. The undissociability of the complex between sw-AT and trypsin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by immunoblotting and fluorescence labeling techniques. Chemical analysis of the complex elucidated that it contained equimolar sw-AT and trypsin. Densitometric analysis of electrophoretic patterns obtained during the titration of trypsin by sw-AT suggested the presence of a suicide product formed from sw-AT. This was the reason why excess sw-AT was needed for complete inhibition of trypsin. In the complex, the sw-AT molecule was cut at one site but the fragments produced were still joined together. Trypsin in the complex was released by treatment at pH 10.0, and it was deduced that the complex formation involved acyl-bond formation between sw-AT and trypsin. The sw-AT component obtained from the alkali-treated complex possessed two kinds of NH2-terminal amino acid sequences. Non-covalent forces may bind the two fragments of sw-AT, which could be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequence analysis of the large fragment gave a sequence identical with that of intact sw-AT. This indicated that the reactive site of sw-AT with trypsin was located at the COOH-terminal region of the molecule. These characteristics resemble those of inhibitors belonging to the serpin family.
Sasaki
T
T
Department of Food Science and Technology, School of Agriculture, Nagoya University, Aichi.
Kobayashi
K
K
Ozeki
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D012831:Bombyx; D002417:Cattle; D006458:Hemolymph / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*
D000596:Amino Acids; D014361:Trypsin Inhibitors; D014357:Trypsin