Partial Purification and Properties of a Phosphatidylinositol 4,5-Bisphosphate Hydrolyzing Phospholipase C from the Soluble Fraction of Soybean Sprouts

다국어 초록 (Multilingual Abstract)

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 M 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.