Purification of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain.
The ATP-dependent, calmodulin-sensitive 3-kinase responsible for the conversion of D-myo-inositol 1,4,5-trisphosphate to D-myo-inositol 1,3,4,5-tetrakisphosphate has been purified 2,700-fold from rat brain to a specific activity of 2.3 mumol/min/mg protein. A method of purification is described involving chromatography on phosphocellulose, Orange A dye ligand, calmodulin agarose, and hydroxylapatite columns. Neither the highly purified enzyme nor enzyme eluting from the phosphocellulose column were activated by Ca2+. However, enzyme in the 100,000 x g supernatant from rat brain was activated by Ca2+ over the range from 10(-7) to 10(-6) M and Ca2+ sensitivity of the purified enzyme was restored by the addition of calmodulin. The enzyme has a catalytic subunit Mr of 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography of the purified enzyme on a Superose 12 column gave a Mr value of 70,000, indicating that the purified enzyme was present as a monomer. In contrast, the 100,000 x g supernatant and the purified enzyme after addition of calmodulin and 10(-6) M Ca2+ chromatographed on size exclusion chromatography with a Mr of 150,000-160,000. These results imply that the native enzyme is a dimeric structure of two catalytic subunits plus calmodulin. The purified enzyme showed a Km of 0.21 +/- 0.08 microM for D-myo-inositol 1,4,5-trisphosphate and had a pH optimum of 8.5. Addition of calmodulin increased both the Km and the Vmax of the purified enzyme about 2-fold. The high affinity of the 3-kinase for D-myo-inositol 1,4,5-trisphosphate together with its activation by Ca2+/calmodulin suggests that this enzyme may exert an important regulatory role in inositol phosphate signaling by promoting the formation of additional inositol polyphosphate isomers.
Johanson
R A
RA
Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Hansen
C A
CA
Williamson
J R
JR
D000818:Animals; D001391:Azo Compounds; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009281:Naphthalenes; D010770:Phosphotransferases / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011917:Rats, Inbred Lew; D011919:Rats, Inbred Strains; D014227:Triazines
D001391:Azo Compounds; D002147:Calmodulin; D004396:Coloring Agents; D009281:Naphthalenes; D014227:Triazines; C048536:matrex gel orange A; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium