Characterization of 101-kDa transglutaminase from Physarum polycephalum and identification of LAV1-2 as substrate.

Plasmodial transglutaminase of Physarum polycephalum was purified by anion exchange and hydrophobic chromatography. Gel filtration and SDS-polyacrylamide gel electrophoresis indicate that it is a monomer of 96-101 kDa. It is Ca2+-dependent, with half-maximal activity at 0. 7 mM Ca2+. Optimal activity occurs at pH 7.5 and at 50 mM KCl. Inactivation by N-ethylmaleimide indicates that it is a thiol enzyme. With N,N-dimethylcasein as substrate, the Km for monodansylcadaverine is 33.9 +/- 1.8 microM. Damage of plasmodia by brief treatment with 15% ethanol activates the transglutaminase, with rapid accumulation of cross-linked proteins unable to enter gels during SDS-polyacrylamide gel electrophoresis. Added monodansylcadaverine is conjugated principally to LAV1-2, a plasmodia-specific 40-kDa protein with four EF-hand sequences believed to bind Ca2+. Actin is seen as an additional substrate only in plasmodial homogenates. Immunoblots show that upon ethanol treatment, a portion of LAV1-2 is modified quickly and shifts to 36 kDa; another portion is cross-linked to itself or other proteins. The modification of LAV1-2 may lead to localized release of Ca2+ and activation of transglutaminase for walling off damaged areas of plasmodia. No significant increase in amount of the transglutaminase occurs during starvation-induced differentiation of plasmodia to form spherules, but a 50% reduction in the amount of total protein leads to a doubling in the specific mass of the TGase. Neither the transglutaminase nor LAV1-2 is found in the ameboid form of the organism.