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Differential and sucrose gradient centrifugation of honey bee thoraces, disrupted by gentle methods and using mannitol-triethanolamine-EDTA buffer at pH 6.5, showed that in the honey bee thorax 92-94.8% of the trehalase was mitochondrial. Since only 92-95% of the cytochrome c oxidase, a known mitochondrial enzyme, was found in the mitochondrial fraction by these methods, it was concluded that honey bee trehalase is totally mitochondrial. Significant amounts of 'microsomal' or 'soluble' trehalase were formed only by harsh methods of thorax disruption and similar 'microsomal' or 'soluble' trehalases were also formed by harsh treatment of purified whole mitochondria. They thus seem to be artifacts of the isolation procedure. Studies (using marker enzymes) with purified intact mitochondria which were dispersed by various chemical, enzymatic, and physical methods showed that the trehalase in the mitochondria was membrane bound and that it was bound to either the outside of the inner membrane or to one of the sides of the outer membrane.

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.