Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax.
The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles.
D000818:Animals; D001516:Bees / Q000201:enzymology*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010879:Piperazines / Q000494:pharmacology; D013696:Temperature; D013909:Thorax / Q000201:enzymology; D014198:Trehalase / Q000302:isolation & purification / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D002729:Chloromercuribenzoates; D005960:Glucosides; D046911:Macromolecular Substances; D010879:Piperazines; D014508:Urea; D014198:Trehalase; D007460:Iodoacetamide