Biochemical and cytochemical studies on enzymes that dephosphorylate inositol (1,4,5)-trisphosphate in neutrophils.
Guinea pig neutrophils contain membrane-bound and soluble phosphatases that catalyze the dephosphorylation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. The activities were 5.1 +/- 0.2 and 1.3 +/- 0.2 (SD; n = 5) nmoles phosphate (Pi) released/min/10(7) cell equivalents, respectively. The membrane-bound enzyme dephosphorylated many substrates (e.g., beta-glycerophosphate), exhibited alkaline pH optima, and was inhibited by levamisole. In contrast, the soluble phosphatase was specific for Ins(1,4,5)P3, exhibited a neutral pH optimum, and was insensitive to levamisole. A cerium-based ultrastructural cytochemical procedure was employed to identify the subcellular sites of the membrane-bound activity. Staining was observed on the exterior of the plasmalemma and in a population of granules. Staining in the granules was observed only in permeabilized cells. Treatment of neutrophils with p-diazobenzenesulfonate (DBSA) (4.0 mM) for 20 min at 37 degrees C blocked the cytochemical reaction on the cell surface using beta-glycerophosphate as the substrate, but did not affect the staining of the granules on subsequent permeabilization. In biochemical studies, this treatment with DBSA inhibited the membrane-bound activity by c. 50% but did not affect the soluble phosphatase. Therefore, the membrane-bound phosphatase is, in fact, an alkaline phosphatase that resides in locales not accessible to Ins(1,4,5)P3 generated during cell stimulation. Breakdown of Ins(1,4,5)P3 generated during cell stimulation, therefore, would be catalyzed by the soluble enzyme.
Badwey
J A
JA
Department of Cell Physiology, Boston Biomedical Research Institute, Massachusetts 02114.
Robinson
J M
JM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D003979:Diazonium Compounds / Q000494:pharmacology; D005994:Glycerophosphates / Q000378:metabolism; D006168:Guinea Pigs; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D009504:Neutrophils / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D013425:Sulfanilic Acids / Q000494:pharmacology
D003979:Diazonium Compounds; D005994:Glycerophosphates; D010710:Phosphates; D013425:Sulfanilic Acids; D007978:Levamisole; D015544:Inositol 1,4,5-Trisphosphate; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; C031463:beta-glycerophosphoric acid; C000469:diazobenzenesulfonic acid