PHOSPHATASES SUBSTRATE SPECIFITY IN AMOEBA PROTEUS
V. A. Sopina
Institute of Cytology RAS, St. Petersburg
e-mail: vsop39@mail.ru
Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and
a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of
these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All
phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a
preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl
phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a
greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast"
and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow"
phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2-naphthyl phosphate to a lesser degree than 1-naphthyl
phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower
intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast"
phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow"
phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this
phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase
is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture
medium where its activity is low. The enzyme displays both phosphomonoand phosphodiesterase activities, in addition
to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the
amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.
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