ALKALINE PHOSPHATASE IN AMOEBA PROTEUS
V. A. Sopina
Institute of Cytology, RAS, St. Petersburg, Russia;
e-mail: sopina@SS4363.spb.edu
In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 μg
of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic
mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases
were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+,
completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+,
Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1,
p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by
H2Î2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole,
L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents - p-(hydroxymercuri)benzoate and N-ethylmaleimide - had no
influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated
by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+,
phosphates and H2Î2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast"
phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+,
Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation
by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0,
only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain,
to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.
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