Purification and characterization of Bombyx cysteine proteinase specific inhibitors from the hemolymph of Bombyx mori.
Protein inhibitors capable of inhibiting BCP (Bombyx cysteine proteinase) were found in the larval-pupal hemolymph of Bombyx mori. Two forms of the inhibitors, named BCPI (BCP inhibitor) alpha and BCPI beta, were purified from the pupal hemolymph by heat treatment and column chromatographies on CM-cellulose, Toyopearl HW-50, Phenyl-Sepharose, and Mono Q. Purified BCPI beta gave a single protein band with a molecular mass of 10,500 daltons on SDS-PAGE. BCPI alpha is mostly composed of the same molecular mass protein as BCPI beta. Both forms were inhibitory towards other cysteine proteinases such as cathepsins L,B and papain but had no effects on trypsin and pepsin. Both forms inhibited the processing of the enzymatically inactive proform of BCP (pro-BCP) to the activated mature BCP. BCPI alpha and BCPI beta shared many other features such as molecular mass determined by gel filtration, antigenicity, and HPLC profiles. NH(2)-terminal amino acid sequencing of the purified inhibitors revealed that three amino acid residues were different in the BCPI alpha and BCPI beta sequences, all others being identical. The hemolymph BCP inhibitor increased activity approximately four- to fivefold at the time of spinning and maintained this level of activity during pupation.
Yamamoto
Y
Y
Department of Biochemistry and Radiation Biology, College of Agriculture, Yamaguchi University, Yamaguchi, Japan. yamataka@po.yb.cc.yamaguchi-u.ac.jp
Watabe
S
S
Kageyama
T
T
Takahashi
S Y
SY
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D012831:Bombyx / Q000378:metabolism*; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015853:Cysteine Proteinase Inhibitors / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence
D015853:Cysteine Proteinase Inhibitors; D019476:Insect Proteins; C401887:cysteine proteinase inhibitor, Bombyx