Purification and properties of the soluble midgut trehalase from the gypsy moth, Lymantria dispar.
The midgut trehalase (THA) from fifth instar Lymantria dispar (gypsy moth) larvae was purified to homogeneity by two separate methods: gel filtration followed by Rotofor preparative IEF, and affinity chromatography on trehalose coupled to Sepharose 6B followed by preparative polyacrylamide gel electrophoresis. Midgut THA from the last stadium L. dispar larvae existed mainly in soluble form and displayed a single band of activity in nondenaturing polyacrylamide gels when stained by a THA-specific staining procedure. Analytical IEF of purified midgut THA revealed a single protein band with an apparent pI of 4.6. SDS-PAGE and gel permeation studies indicated that the smallest active form of THA in the late fifth instar larval midgut was a monomeric protein with an approximate size of 60 kDa. A specific activity of 67 units/mg of protein at 30 degrees C and at pH 6.4 was determined for the enzyme purified by affinity chromatography and preparative gel electrophoresis. The midgut enzyme exhibited a very high substrate specificity with a Km of 0.4 mM for trehalose. The enzyme was maximally active at pH 5.4-6.0 and was thermally stable at temperatures up to 65 degrees C. The midgut THA was insensitive to inhibition by a high concentration of Tris, sucrose, p-nitrophenyl-beta-D-glucoside or phloridzin. Divalent cations metal ions, hypertrehalosaemic hormone and octopamine had no significant effect on the activity of the purified enzyme in vitro. The purified enzyme was inactivated by modification with DEP and was competitively inhibited by castanospermine with an apparent Ki of 0.8 x 10(-6)M at pH 6.4.
Valaitis
A P
AP
USDA, Forest Service, Northeastern Forest Experiment Station, Delaware, OH 43015.
Bowers
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004064:Digestive System / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D007814:Larva / Q000737:chemistry; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification
D007527:Isoenzymes; D014198:Trehalase