CHARACTERIZATION OF 101-KDA TRANSGLUTAMINASE FROM PHYSARUM-POLYCEPHALUM AND IDENTIFICATION OF LAV1-2 AS SUBSTRATE

Plasmodial transglutaminase of Physarum polycephalum was purified by a nion exchange and hydrophobic chromatography. Gel filtration and SDS-p olyacrylamide gel electrophoresis indicate that it is a monomer of 96- 101 kDa. It is Ca2+-dependent, with half-maximal activity at 0.7 mM Ca 2+. Optimal activity occurs at pH 7.5 and at 50 mM KCl. Inactivation b y N-ethylmaleimide indicates that it is a thiol enzyme. With N,N-dimet hylcasein as substrate, the K-m for monodansylcadaverine is 33.9 +/- 1 .8 mu M. Damage of plasmodia by brief treatment with 15% ethanol activ ates the transglutaminase, with rapid accumulation of cross-linked pro teins unable to enter gels during SDS-polyacrylamide gel electrophores is. 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 plasmo dial homogenates. Immunoblots show that upon ethanol treatment, a port ion of LAV1-2 is modified quickly and shifts to 36 kDa; another portio n is cross-linked to itself or other proteins. The modification of LAV 1-2 may lead to localized release of Ca2+ and activation of transgluta minase for walling off damaged areas of plasmodia. No significant incr ease in amount of the transglutaminase occurs during starvation-induce d differentiation of plasmodia to form spherules, but a 50% reduction in the amount of total protein leads to a doubling in the specific mas s of the TGase. Neither the transglutaminase nor LAV1-2 is found in th e ameboid form of the organism.