L-myo-inositol-1-phosphate synthase expressed in developing organ: isolation and characterisation of the enzyme from human fetal liver.
L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0-60 % ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46 % of MIPS could be recovered. The human fetal liver MIPS specifically utilised D-glucose-6-phosphte and NAD(+) as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40 °C. The enzyme activity was remarkably stimulated by NH (4) (+) , slightly stimulated by K(+) and Ca(2+) and highly inhibited by Zn(2+), Cu(2+) and Hg(2+). The K (m) values of MIPS for D-glucose-6-phosphate and NAD(+) were found to be as 1.15 and 0.12 mM respectively while the V (max) values were 280 nM and 252 nM for D-glucose-6-phosphate and NAD(+) correspondingly. The apparent molecular weight of the native enzyme was determined to be 170 kDa.
Biochemistry and Molecular Biology Laboratory, Darjeeling Government College, Darjeeling, West Bengal, India. dhanirajc@gmail.com
Dhani Raj
DR
Chhetri
Seema
S
Gupta
Asok Kumar
AK
Mukherjee
Jukta
J
Adhikari
D004795:Enzyme Stability; D005260:Female; D005333:Fetus / Q000737:chemistry / Q000196:embryology / Q000201:enzymology*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000196:embryology* / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D011247:Pregnancy; D013379:Substrate Specificity
D009243:NAD; D019298:Glucose-6-Phosphate; D007296:Myo-Inositol-1-Phosphate Synthase