Purification and partial characterization of transglutaminase from Physarum polycephalum.
An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.
Graduate Program in Molecular Biology, New Mexico State University, Las Cruces 88003-0001.
J D
JD
Klein
E
E
Guzman
G D
GD
Kuehn
D000818:Animals; D002454:Cell Differentiation; D003619:Dansyl Compounds / Q000378:metabolism; D008970:Molecular Weight; D010804:Physarum / Q000166:cytology / Q000201:enzymology*; D011700:Putrescine / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D003619:Dansyl Compounds; D011503:Transglutaminases; D011700:Putrescine