1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.
We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types.
Rubiera
C
C
Departamento de Biologia Funcional, Universidad de Oviedo, Spain.
Velasco
G
G
Michell
R H
RH
Lazo
P S
PS
Shears
S B
SB
D019794:2,3-Diphosphoglycerate; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology*; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000097:blood / Q000378:metabolism*; D051381:Rats; D013403:Sugar Phosphates / Q000378:metabolism*; D014018:Tissue Distribution
D004163:Diphosphoglyceric Acids; D005958:Glucosephosphates; D007295:Inositol Phosphates; D013403:Sugar Phosphates; D019794:2,3-Diphosphoglycerate; D019298:Glucose-6-Phosphate; D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; C043422:myo-inositol 1,4,5-triphosphate phosphatase