PURIFICATION AND PARTIAL CHARACTERIZATION OF TRANSGLUTAMINASE FROM PHYSARUM-POLYCEPHALUM

被引:69
作者
KLEIN, JD [1 ]
GUZMAN, E [1 ]
KUEHN, GD [1 ]
机构
[1] NEW MEXICO STATE UNIV,GRAD PROGRAM MOLEC BIOL,BOX 3MLS,LAS CRUCES,NM 88003
关键词
D O I
10.1128/jb.174.8.2599-2605.1992
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 [C-14]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, [C-14]putrescine, [C-14]spermidine, and [C-14]spermine were all effective amine donor substrates with K(m) values of 49, 21.4, and 31.7-mu-M, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200-mu-M. 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.
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页码:2599 / 2605
页数:7
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