Detection and partial purification of inositol 1,4,5-trisphosphate 3-kinase from porcine skeletal muscle.
A myoplasmic 3-kinase was detected in porcine skeletal muscle that phosphorylated [3H]Ins(1,4,5)P3 [D-myo-inositol(1,4,5)trisphosphate] to [3H]Ins(1,3,4,5)P4 [D-myo-inositol(1,3,4,5)tetrakisphosphate]. The Ins(1,4,5)P3 3-kinase activity was ATP- and Mg(2+)-dependent, and was activated by Ca2+ and calmodulin. Ins(1,4,5)P3 3-kinase activity was purified 2632-fold from soluble extracts of skeletal muscle by a combination of DEAE-Sephacel, heparin-Agarose and Ins(1,4,5)P3 structural-analogue affinity chromatography. The highest specific activity obtained was 10.6 nmol of Ins(1,4,5)P3 phosphorylated/min/mg protein. The partially purified enzyme had a mean Km and Vmax of 0.46 microM and 3.15 nmol/min/mg protein for Ins(1,4,5)P3 metabolism, respectively. After analytical gel filtration two forms of soluble Ins(1,4,5)P3 3-kinase were observed with M(r) of 39,000 and 62,000. As in other cell types, muscle Ins(1,4,5)P3 3-kinase was soluble, and had a higher affinity but a lower capacity to metabolize Ins(1,4,5)P3 in comparison to Ins(1,4,5)P3 5-phosphatase.
Hogan
S P
SP
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Foster
P S
PS
Hansbro
P M
PM
Ozaki
S
S
Denborough
M A
MA
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000302:isolation & purification*; D013552:Swine
D002147:Calmodulin; D007295:Inositol Phosphates; C081655:inositol-1,3,4,5-tetrakisphosphate; D015544:Inositol 1,4,5-Trisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium