An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia¹

Abstract

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p‐nitrophenylphosphatase (pNPPase), glucose‐6‐phosphatase (G6Pase), and 5′‐nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose‐6‐phosphate (G6P) and adenosine 5′‐monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na+‐K+‐ATPase was observed. Since the hydrolysis of p‐nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β‐glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)‐tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β‐glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.

Occurrence of apara-Nitrophenyl Phosphate-Phosphatase with Calcineurin-like Characteristics inParamecium tetraurelia

“…Although other phosphatases, e.g., protein constitution experiments with fractions containing the CaN B subunit (data not shown). Immunoblots or 45 Ca Tyr phosphatases or alkaline or acid phosphatase occurring in Paramecium (61,62), also hydrolyze pNPP, overlay analysis unfortunately revealed that subunit B of Paramecium CaN coelutes with Peak III phosphaa possible interference for Peak I (and for Peak II) can be excluded by inhibitor responses. From further enzy-tase.…”