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Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg²⁺) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca²⁺/CaM). In the presence of Ca²⁺ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca²⁺ dependent mobility shift on denaturing gels. Its Ca²⁺ binding was confirmed by Chelex-100 assay and gel overlays using ⁴⁵CaCl₂. Glyoxalase I was activated by over 7-fold in the presence of Ca²⁺ (25 μM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 μM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in …