Purification and characterization of D-myo-inositol (1,4,5)/(1,3,4,5)- polyphosphate 5-phosphatase from skeletal muscle.
In this investigation we report the presence of two forms of inositol (1,4,5)P3/(1,3,4,5)P4-polyphosphate 5-phosphatase activity (types I and II) which were observed in soluble extracts of skeletal muscle after fractionation by DEAE-Sephacel chromatography. Hydrolysis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and D-myo-inositol 1,3,4,5-tetrakisphosphate by both phosphatases was 5-phosphate-specific, Mg2+ ion-dependent and inhibited by D-2,3-bisphosphoglycerate. Soluble type I 5-phosphatase activity was purified 27,300-fold to a specific activity of 2.54 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg protein after a combination of DEAE-Sephacel, Blue Sepharose, heparin-agarose and structural analogue affinity chromatography. Purified type I 5-phosphatase had an apparent mean Km of 8.9 and 1.1 microM and Vmax of 3.55 and 0.13 mumol of substrate hydrolyzed/min/mg protein for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. Investigations on soluble type II 5-phosphatase after DEAE-Sephacel chromatography indicated an apparent Km of 71.4 microM Ins(1,4,5)P3 and an apparent molecular mass of 81 kDa. Soluble type I phosphatase has an apparent molecular mass of 48 kDa and an isoelectric point of 5.8. Soluble type I 5-phosphatase has kinetic constants which suggest a role in the regulation of inositol polyphosphates at physiological concentrations. These results support a role for Ins(1,4,5)P3 in the regulation of Ca2+ homeostasis in skeletal muscle.
Hansbro
P M
PM
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Foster
P S
PS
Hogan
S P
SP
Ozaki
S
S
Denborough
M A
MA
D019794:2,3-Diphosphoglycerate; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D000072181:Inositol Polyphosphate 5-Phosphatases; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013552:Swine
D004163:Diphosphoglyceric Acids; D007295:Inositol Phosphates; C081655:inositol-1,3,4,5-tetrakisphosphate; D019794:2,3-Diphosphoglycerate; D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; D008274:Magnesium