Purification and characterization of soluble inositol 1,4,5-trisphosphate 5-phosphomonoesterase from rabbit peritoneal neutrophils.
A specific soluble inositol phosphate 5-phosphomonoesterase has been purified approximately 2,700-fold from a 120,000g supernatant of rabbit neutrophil homogenate. The specific enzyme represented 25-50% of the total hydrolytic activity toward inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3) with the remaining activity hydrolyzing both Ins-1,4,5-P3 as well as inositol 1,4-bisphosphate (Ins-1,4-P2). However, the enzyme could not be identified on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue, indicating that it represents a minor protein in the purified enzyme preparations. The purified enzyme has an apparent molecular mass of 43,000-47,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphomonoesterases. The enzyme hydrolyzes Ins-1,4,5-P3 with an apparent Km of 18 microM and a Vmax of 1.2 mumol/min/mg. The 5-phosphomonoesterase requires Mg2+ for activity and is not affected by physiological concentrations of Ca2+ or calmodulin. The pH optimum for activity is 7.5. Inositol 1,3,4,5-tetrakisphosphate is a potent competitive inhibitor of Ins-1,4,5-P3 hydrolysis (Ki = microM), whereas Ins-1,4-P2 is a weak inhibitor (Ki = 173 microM). Ins-1,4,5-P3 hydrolysis is relatively unaffected by monophosphorylated substrates, however, bisphosphorylated substrates are potent inhibitors. Comparisons of neutrophil 5-phosphomonoesterase characteristics with those of platelet and rat brain enzymes support the idea that each 5-phosphomonoesterase may be a unique enzyme and play a different role dependent upon the cell or tissue in which it acts.
Kennedy
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SP
Department of Pathology, University of Connecticut Health Center, Farmington 06032.
Sha'afi
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RI
Becker
E L
EL
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D010529:Peritoneal Cavity / Q000166:cytology; D010744:Phosphoric Monoester Hydrolases / Q000097:blood* / Q000502:physiology; D011817:Rabbits; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology
D002147:Calmodulin; C035215:inositol triphosphate phosphomonoesterase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D002118:Calcium