myo-inositol monophosphatase from rat testes: purification and properties.
myo-Inositol monophosphatase (EC 3.1.3.25) has been purified to homogeneity from the high-speed supernatant of rat testes and its properties were investigated. By means of ammonium sulfate precipitation, followed by heating, anion exchange, and gel filtration high-pressure liquid chromatographic techniques, polylysine agarose and phenyl-Sepharose column chromatographic methods, this phosphatase was purified 2563-fold to a specific activity of 7972 mU/mg protein. It showed an apparent native molecular weight of 58,000 as determined by gel filtration chromatography and was composed of two identical subunits of molecular weight of 29,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Among several divalent cations tested for activation of the enzyme, Mg2+ was most effective and optimally active at pH 7.8. The Km values for D- and L-myo-inositol 1-phosphate (which were equal) and 2'-AMP were 0.12 +/- 0.02 and 0.17 +/- 0.03 mM, respectively. Lithium ions inhibited this phosphatase specifically and kinetic studies demonstrated uncompetitive inhibition. Preparations of polyclonal antibodies against the homogeneous enzyme in rabbits cross-reacted with the partially purified enzyme preparations from liver, kidney, heart, and brain show immunological identity. Western blot analysis after SDS-polyacrylamide gel electrophoresis confirmed a major band corresponding to a subunit molecular weight of 29,000. A sensitive enzyme staining method was also developed to localize the site of myo-inositol monophosphatase activity on polyacrylamide gels which helped to differentiate this phosphatase from nonspecific contaminating phosphatases. To explain the unusual stereospecificity of this enzyme on its isomeric substrates, a working model was suggested involving the production of a myo-inositol 1,3-cyclic phosphate intermediate during the course of its reaction.
Department of Psychiatry, University of Tennessee, Memphis 38104.
L
L
Parthasarathy
R E
RE
Vadnal
T G
TG
Ramesh
C S
CS
Shyamaladevi
R
R
Parthasarathy
D000818:Animals; D002412:Cations; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D002412:Cations; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase