Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts.
Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.
Division of Applied Life Sciences, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea.
Soo Kwon
SK
Park
Jung Ro
JR
Lee
Seung Sik
SS
Lee
Hyo Jin
HJ
Son
Ji Young
JY
Yoo
Jeong Chan
JC
Moon
Heun Young
HY
Kwon
Chae Oh
CO
Lim
Jeong Dong
JD
Bahk
Moo Je
MJ
Cho
Sang Yeol
SY
Lee
D000906:Antibodies / Q000276:immunology; D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D003840:Deoxycholic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D012995:Solubility; D013025:Glycine max / Q000201:enzymology*; D010738:Type C Phospholipases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D000906:Antibodies; D019269:Phosphatidylinositol 4,5-Diphosphate; D003840:Deoxycholic Acid; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium