Physiological implications of trehalase from Phaseolus vulgaris root nodules: partial purification and characterization.
The purification and characterization of trehalase from common bean nodules as well as the role of this enzyme on growth, nodulation nitrogen fixation by examining the effects of the trehalase inhibitor validamycin A, was studied. Validamycin A did not affect plant and nodule mass, neither root trehalase and nitrogenase activity; however this treatment applied at the time of sowing increased nodule number about 16% and decreased nodule trehalase activity (16-fold) and the size of nodules. These results suggest that nodule trehalase activity of Phaseolus vulgaris could be involved in nodule formation and development. In addition, acid trehalase (EC 3.2.1.28) was purified from root nodules by fractionating ammonium sulfate, column chromatography on DEAE-sepharose and sephacryl S-300, and finally on native polyacrylamide gel electrophoresis. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 42 and 45 kDa on SDS-PAGE. The enzyme has the optimum pH 3.9, Km of 0.109 mM, Vmax of 3630 nkat mg-1 protein and is relatively heat stable. Besides trehalose, it shows maximal activity with sucrose and maltose and, to a lesser degree melibiose, cellobiose and raffinose, and it does not hydrolyze on lactose and turanose. Acid trehalase was activated by Na+, Mn2+, Mg2+, Li+, Co2+, K+ and inhibited by Fe3+, Hg+ and EDTA.
García
Noel A Tejera
NA
Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain. natejera@ugr.es
Iribarne
Carmen
C
López
Miguel
M
Herrera-Cervera
José A
JA
Lluch
Carmen
C
D002412:Cations / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009586:Nitrogen Fixation; D027805:Phaseolus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D018517:Plant Roots / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002412:Cations; D004791:Enzyme Inhibitors; C030790:validamycin A; D007294:Inositol; D014198:Trehalase