Partial purification, stabilization and characterization of adult honey bee midgut trehalase and a new trehalase specific disc gel stain method
1975; Elsevier BV; Volume: 5; Issue: 3 Linguagem: Inglês
10.1016/0020-1790(75)90070-0
ISSN1879-2928
Autores Tópico(s)Neurobiology and Insect Physiology Research
ResumoTrehalase was partially purified from the intestine of adult honey bees (Apis mellifera). It was shown to have only one band of activity on disc gel at both pH's 4.3 and 9.3 when stained by a new trehalase specific staining method and found to be quite different from the trehalases of the thorax. The intestinal trehalase was stable in the crude state but became very unstable after partial purification whereas the thoracic honey bee trehalases are quite stable even when purified. Of a series of compounds tried only Mn2+ and Mg2+ were able to slow down the rate of inactivation of the intestinal enzyme. Stability studies of the enzymes at temperature and pH extremes showed no significant differences between this enzyme and bee thorax trehalases but urea, even at low concentrations, caused rapid activity loss relative to the thorax trehalases. The Km of the intestinal trehalase was 1.11 mM and the activity was competitively inhibited by Tris and by p-nitrophenyl-β-d-glucoside. These properties are similar to those of the thorax trehalases. A pH optimum of 6.0 was found for the intestinal enzyme and a study of the variation of kinetic constants with pH showed a complex ionization relationship with groups dissociating at pH's 4.5 and 6.2. The energy of activation for the trehalase reaction was found to be 7.8 Kcal/mole which is lower than the energy of activation for the thorax trehalases of honey bees. The molecular weight of the intestinal trehalase was estimated to be 135,000 by Sephadex G-200 gel chromatography. This molecular weight value is about twice as great as the molecular weight found for thorax trehalases.
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