Trehalase from the cellular slime mold Dictyostelium discoideum: purification and characterization of the homogeneous enzyme from myxamoebae.

Trehalase (alpha-alpha'-trehalose 1-D-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a Km of 1.2 mM. The enzyme was maximally active at 50 degrees C and had an energy of activation of 12-13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45-50 degrees C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mM, respectively. Of five heavy metals tested, HgCl2 was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mM. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10(5) +/- 5 X 10(3). Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95-100 X 10(3). The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3-10 ampholytes was 7.2-7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 M) and was eluted with alpha-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.