A myo-inositol D-3 hydroxykinase activity in Dictyostelium.
A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation.
Stephens
L R
LR
Biochemistry Department, A.F.R.C. Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Kay
R R
RR
Irvine
R F
RF
D002250:Carbon Radioisotopes; D002852:Chromatography, Ion Exchange; D004023:Dictyostelium / Q000201:enzymology* / Q000254:growth & development; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity; D014316:Tritium
D002250:Carbon Radioisotopes; D007295:Inositol Phosphates; D014316:Tritium; D007294:Inositol; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C066722:myo-inositol D-3 hydroxykinase