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.
Mottahedeh
J
J
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688, USA.
Marsh
R
R
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000737:chemistry; D005260:Female; D007700:Kinetics; D051379:Mice; D016839:Physarum polycephalum / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D011503:Transglutaminases / Q000302:isolation & purification / Q000378:metabolism*
D000199:Actins; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine; D005033:Ethylmaleimide