FoodSci. Technol. Res., 6 (1), 29-33, 2000
Purification and
Characterization of Phospholipase D from
Cabbage Leaves
Hiroaki SAT0,1 Toshihiro WATANABE,1 Yoshimasa SAGANE,1 Yozo NAKAZAWA2 and Katsunu TAKANO IDepartment oJ' Food Science and Technolog_v, Faculty of Bioindustry Toky'o University ofAgriculture, 1 96, Yasaka, Abashiri, Hokhaido, 099-
2493, Japan 2Department ofApplied Biology and Chemistry, Faculty ofApplied Bio-Science, Tokyo University ofAgriculture, 1-1-], Sakuragaoka, Setagayaku, Tokyo, 156L8502, Japan Received April 30, 1 999; Accepted December 7, 1 999
Phospholipase D (PLD) was purified from cabbage leaves. The molecular weight of purified PLD was estimated as approximately 73 and 87 kDa by gel filtration using Superdex 200 HR column and, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively! The transphosphatidylation capacity for phosphatidylcholine of this enzyme reached over 90 % , and the enzyme's hydrolysis efficiency for phosphatidylcholine was five-fold higher than that for phosphatidylglycerol. These findings indicated that the cabbage PLD efficiently transferred phos-
phatidylcholine to phosphatidylglycerol. On N-terminal amino acid sequence analysis of the band separated by SDS-
PAGE, two sequences with differing N-terminus were detected. This N-terminal difference may have been generated by processing during maturation of PLD. Keywords : phospholipase D, cabbage, N-terminal amino acid sequence
In recent years, the oil and fat industries have been attempting
Chemical Co. (St. Louis, MO). Horseradish peroxidase was from
to develop novel phospholipid. In this regard, phosphatidylcho-
Wako Pure Chemical Industries (Osaka). Lecithin (PC-98S, con-
line (PC) obtained as a by-product during purification of oil and
taining 99.1% phosphatidylcholine, 0.4% Iysophosphatidylcho-
fat or from yolk lecithin is particularly widely used.
line by TLC/FID) from egg yolk was obtained frorn Q.P. Co.
To obtain a highly functional phospholipid, PC is modified by
(Tokyo). L-oe-Phosphatidyl-DL-glycerol (PG), prepared by reac-
replacing the choline residue by specific alcohols (Nagao, 1995;
tion of cabbage PLD with egg yolk L-(x-phosphatidylcholine in
Hidano et al., 1995; D'Arrigo & Servi, 1997). In addition to hydrolysis of phospholipids releasing alcohol and phosphatidic
the presence of glycerol, was obtained from Sigma Chemical Co.
acid, phospholipase D (PLD, EC 3. I .4.4.) was shown to catalyze
Kanto Chemical Co. (Tokyo). Determiner L TG was from
transfer of a phosphatidyl group to compounds with a hydroxyl
Kyowa Medex Co. (Tokyo). Protein assay dye reagent concentrate was from Bio-Rad Laboratories (Hercules, CA). Octyl Sepharose CL-4B , Mono Q HR 5/5 column, Superdex 200 HR 10/30 column, gel filtration molecular weight standards and SDS-PAGE molecular weight standards (low range) were obtained from Pharmacia Biotech (Uppsala, Sweden). Coomassie Brilliant Blue (CBB) R-250 was from Fluka Chemie AG (Buchs, Switzerland) . Mini ProBlottTM membranes were from Applied
group (Yang et al., 1967; Dawson, 1967). PLD catalyzes hydrolysis by transferring a phosphatidyl group to a water molecule, Therefore, these two transfer reactions compete in the presence
of water and alcohol. The order of precedence between hydrolysis and transphosphatidylation as well as transfer capacity varies
among PLD from different origins (Nagao, 1 995).
We previously investigated the primary structure of cabbage
Sodium deoxycholate (SDC) and 4-aminoantipyrine were from
PLD at the protein level and showed it to be similar to other plant
Biosystems (Foster City, CA). All other reagents used were of
PLD (Sato et al., 1999). In this study, we purified and partially
the highest grade commercially available.
characterized cabbage PLD and analyzed its N-tenninal amino
Enzyme preparation and punfication PLD was prepared
Materials and Methods Cabbage samples Fresh spring cabbage (Brassica olera-
by heat coagulation and acetone precipitation from 400 g of inner yellowish-white leaves of cabbage (Davidson & Long, 1958). The crude lyophilized acetone powder suspended in 20 mM PIPES buffer (pH 6.2) containing 50 mM CaC12 Was centri-
cea var. capitata cv. Ishii) produced in Hyogo was obtained from
fuged to remove insoluble materials. The supernatant containing
a vegetable market.
PLD activity was applied to an Octyl Sepharose CL-4B column (Lambrecht & Ulbrich-Hofmann, 1992). The active fractions
acid sequence.
Enzymes and chemicals Choline oxidase from Alcaligenes species and phosphatidylglycerol were purchased from Sigma
were dialyzed, and then applied to a Mono Q HR5/5 column (Abousalham et al. , 1 993). The pooled fractions containing PLD
E-mail:
[email protected],jp Abbreviations: PLD, phospholipase D; PAGE, polyacrylamide gel electro-
phoresis; SDS, sodium dodecyl sulfate; PC, phosphatidylcholine; SDC, sodium deoxycholate; CBB, Coomassie brilliant blue; EDTA.E2Na, ethylendiamine-N,N,NINLtetraacetic acid disodium salt; PG, phosphatidylglycerol; TLC, thin layer chromatography; FID, flame ionization detector; PA, phosphatidic acid; PVDF, polyvinylidene difluoride.
activity were dialyzed and recovered.
Protein determination Protein content was determined using the microassay procedure with a dye-binding assay (Bradford, 1976) according to the manufacturer's instructions (Bio-
Rad Laboratories). Bovine serum albumin was used as the stan-
30
H. SATO e/ a/.
dard .
albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30
Enzyme assay PLD activity with lecithin (PC) as a substrate was assayed spectrophotometrically. The amount of free
kDa), trypsin inhibitor (20.1 kDa) and c(-lactalbumin ( 14.4 kDa).
choline generated was estimated colorimetrically, after oxidation
to betaine by choline oxidase with simultaneous production of
H.O,, which was oxidatively coupled with 4-aminoantipyrine and phenol by peroxidase to give a chromophore with maximal absorbance at 500 nm (Imamura & Horiuti, 1 978). Egg yolk lecithin emulsion ( I % (w/v) concentration, 100 mg of PC-98S in lO
ml of 40 mM SDC solution) was prepared with a sonicator
N-termina/ sequence ana!ysis Proteins separated by SDSPAGE on 7.5% (w/v) acrylamide gels were electroblotted onto polyvinylidene difluoride (PVDF) membrane (Mini ProBlottTM)
with a semi-dry blotting apparatus (Nippon Eido, Tokyo). The transfer was completed in 60 min at a constant I OO mA. The membranes were briefiy stained with O. I % (w/v) CBB R-250 to visualize protein bands and then destained (Hirano & Watanabe, l 990). Each excised protein band was sequenced with a pulse-
(model; Sonifier 450, Branson Co.. Danbury, CT) under ice-cold
liquid phase protein sequencer (model; 477All20A, Applied
conditions. A mixture (0.9 ml) composed of 0.1 ml of 20 mM Tris-HCI (pH 7.0) containing 40 mM CaC12' 0.7 ml of distllled
Biosystems, Foster City. CA).
water and 0.1 ml of enzyme solution was preincubated at 37'C.
pholipids Mixtures (0.9 ml) composed of 0.1 ml of 20 mM Tris-HCI (pH7.0) containing 40 mM CaC12' 0.1 ml of enzyme
After 5 min, the reaction was started by adding 0.1 ml of I % (w/ v) Iecithin emulsion as a substrate, and then the reaction mixture
( I .O ml) was incubated at 37'C. After 30 min, the reaction was
Transphosphatidy'lation reaction and extraction of phos-
solution and desired concentrations of glycerol were preincubated at 37'C. After 5 min, the reaction was started by adding
stopped by adding 0.2 ml of 0.5 M Tris-HCI (pH 8.0) containing
0.1 ml of I % (w/v) Iecithin emulsion as a substrate, followed by
50 mM ethylendiamine-N,N,NINLtetraacetic acid disodium salt (EDTA.2Na). To this I .2 ml of mixture was added, 0.4 ml of dis-
incubation at 37'C. The reaction was stopped by adding 0.2 ml of I N HCl. To this I .2 ml of mixture was then added 5.0 ml of
tilled water containing I .5 units of chollne oxidase. 0.2 units of
Folch solution (chloroform-methanol, 2: I ) and 0.9 ml of distilled
peroxidase, I .5 umol of 4-aminoantipyrine and 2. I umol of phe-
water. After mixing well, the solution was centrifuged for 5 min
nol. The mixture was incubated at 37'C for 30 min, then the
at 2500 r.p.m. and the lower layer was collected and evaporated
reaction was stopped by adding 2 ml of I % (w/v) Triton X- I OO,
with a vacuum rotary evaporator. The evaporated samples were
and the optical density was read at 500 nm. As a blank test, 0.l
dissolved in an appropriate amount of chloroforrn.
ml of distilled water was used instead of the enzyme solution,
Phospholipids analysis Quantitative phospholipid analysis was performed by thin layer chromatography with a fiame ionization detector (TLC/FID) (model; Iatroscan Analyzer new MK-5, Iatron Laboratories Co., Tokyo) connected to an integra-
and treated in the same way as described above. The amount of choline liberated was calculated from the absorbance obtained by subtracting the absorbance of the blank from that of the sample,
using the standard curve obtained with known amounts of choline chloride. One unit of PLD activity was defined as the amount of enzyme that produced I umol of choline/min under
tor (model; Chromatopac C-R6A, Shimadzu Co., Kyoto). The
the conditions described above.
with silica gel (Iatron Laboratories). Just before use, the blank
PLD activity with PG as a substrate was assayed spectrophoto-
metrically. The amount of free glycerol generated was estimated
evaporated samples dissolved in an appropriate quantity of chlo-
roform were spotted onto Chromarod SIII quartz rods coated rods were activated by passing them through the flame of the TLC/FID attached to the integrator. Disposable micropipettes
colorimetrically to give a chromophore with a maximal absorbance at 550 nm (Uwajima & Terada, 1 980). A suitable portion
were used for spotting. Aliquots ( 1-3 u1) of each sample together
of PG solution (in chloroform-methanol (98:2), approx. 99%) was evaporated to dryness under nitrogen gas. The dried PG was dispersed in 40 mM SDC to a final concentration of I % (w/v)
The spotted rods enclosed in a frame were put into a glass tank
under ice-cold conditions. The mixture (0.9 ml) composed of O. 1
mm from the bottom and developed again with the second developing solvent (chloroform-acetone-methanol-acetic acid-distilled
ml of 20 mM Tris-HCI (pH 7.0) containing 40 mM CaC12' 0.7 ml of distilled water and 0.1 ml of enzyme solution was preincu-
with phospholipid standards were spotted onto the chromarods . containing the first developing solvent (acetone-acetic acid, 70:
0.25). After development, the rods were burned again leaving 20
water, 6.5:2:1:1:0.3). After development, the solvent was
bated at 37'C. After 5 min, the reaction was started by adding O. 1
removed by heating the rods with a hot dryer; the rods were then
ml of I % (w/v) PG dispersion as a substrate, followed by incuba-
transferred to the instrument and scanned. TLC/FID was con-
tion of a I .O ml aliquot at 37'C. After 30 min, the reaction was
ducted at a hydrogen gas flow rate of 160 mVmin, air flow rate of
stopped by adding 0.1 ml of 0.5 M Tris-HCI (pH 8.0) containing
200 mUmin and scanning speed of 40 s/scan. The concentrations
50 mM EDTA.2Na. To this I . I ml of mixture was added, 0.3 ml
of PC, PG and PA were estimated from their peak areas with the
of Determiner L TG (R- I :R-2, 2:1) and incubation followed at
integrator (Juneja et al. , 1 987).
37'C for 30 min. The reaction was stopped by adding 2 ml of l % (w/v) Triton X- I OO, and the optical density was read at 550
Results and Discussion
Punfication of cabbage PLD by chromatography PLD
nm.
SDS-PAGE analysis SDS-PAGE was performed with a
was extracted from cabbage leaves. PLD in the extract was pre-
7.5% (w/v) acrylamide gel as described by Laemrnli (1 970). Pro-
cipitated with acetone. The precipitate was resuspended and
tein samples containing 10 mM Tris-HCI (pH 6.8), I % (w/v) SDS, I % (v/v) 2-mercaptoethanol and 20% (v/v) glycerol were boiled for 5 min. After electrophoresis the gels were stained with
loaded onto an Octyl Sepharose CL-4B hydrophobic chromatography column equilibrated with 30 mM PIPES buffer (pH 6.2) containing 50 mM CaC12' then eluted with stepwise CaC12 gradi-
0.25% (w/v) CBB R-250 and then destained. The molecular
ent in the buffer changing the CaCl, concentration from 50 to 30,
weight standards were phosphorylase b (94 kDa), bovine serum
then to O mM. The elution profile is shown in Fig. IA.
31
Puriflcation and Characterization of Phospholipase D f~rom Cabbage Leaves
After dialysis and concentration, active fractions obtained by
hydrophobic chromatography were loaded onto a Mono Q HR 51 5 column equilibrated with 20 mM Tris-HCI buffer (pH 7.5) and
the molecular weight of cabbage PLD was 89.000 and 87,000 by SDS-PAGE analysis, respectively. These values approximate that calculated in this paper. Furthermore, the molecular weights of
is shown in Fig. I B. PLD activity yield was 0.67% at 1 1 .6-fold
PLDS from other plants were reported as approximately 90 kDa (for example, rice: 82 kDa, soybean: 92 kDa, castor bean: 92
purification (Table I ).
kDa). The molecular weight of cabbage PLD was similar to that
eluted with a linear gradient of O to 0.5 NaCl. The elution profile
of PLDS from other plants.
Molecular weight oj'cabbage PLD To estimate the molecular weight of native cabbage PLD, purified PLD was applied to
a Superdex 200 HR I 0/30 equilibrated with 30 mM PIPES buffer
(pH 6.7) containing 50 mM CaCl, and 0.15 M NaCl. The PLD
o.05
was eluted with a retention volume of 1 3.38 ml as shown in Fig.
~
2. The molecular weight of native cabbage PLD was estimated as
O OO
approximately 73 kDa by retention volume relative to protein
C~I
standards. On SDS-PAGE using 7.5% gel in contrast, the cabbage PLD demonstrated as a single band with molecular weight
0.03
~, C:$ Os~
of approximately 87 kDa on (Fig. 3). Since the value of molecu-
0.04
0.02
C:l
~H
lar weight estimated by gel filtration and by SDS-PAGE were similar the cabbage PLD was believed to be a monomer. Lee et
O 0.01 ~) ,~
~1
~c~
Q) 20 ~ O~
O
~'~
.;::
o 40 q)
5
~' >1 $:)~c:l
o ,~
~L ,~'
80
,J Ft
'~ ,~
~O ~
1 OO
1 OO
20 ~::O ~~ ~~
-5
lO
5
O
c:lh
strates. Reaction mixtures were composed of 0.1 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 40 mM CaCl., 0.1 ml of purified PLD (1.4xl0-3 and 3.3xl0-3 mg for PC and PG, respectively). 0.7 ml of distilled water and various concentrations of substrate. The reaction was initiated by addition of
PLD and carried out at 37'C. Free polar head groups of choline and glycerol were measured by the method described by Imamura and Horiuti, and Uwajima, and Terada, respectively. Each line is fitted to the data points by the least-squares method.
H
O1234 () 5
l/[S] (mM)-l Fig. 4. Kinetic analysis of hydrolysis activity of purified PLD with different phospholipids as substrates. Double reciprocal plots of the velocity (v) of hydrolysis versus the concentrations ([S]) of PC (o) and PG ([]) as sub-
o
O
Reaction time h
Fig. 5. Time course of transphosphatidylation activity by purified PLD from cabbage in the presence of lO% (w/v) glycerol, o, PC;JL, PA;A, PG; [],
transphosphatidylation capacity. Reaction mixtures were composed of 0,l ml of 20 mM Tris-HCI buffer (pH 7.0) containing 40 mM CaCl,, O.1 ml of purified PLD (5.5xl0-3 units), 0.7 ml of distilled water and O.1 ml of l% (w/v) PC containing 40 mM SDC. The reaction was carried out at 3_ 7'C. PC,
PA and PG were measured by TLC/FID. The measurement conditions were described in Materials and Methods.
Table 3. N-termlnal amlno acld sequences of punfied PLD from cabbage leaves. seq. 1
Substrate
Parameter
K~ (mM) Vm" (umol/min/mg) V~**/K~
l O 20 15
Table 2. Kinetics parameters of PLD from cabbage leaves.
seq. 2
PC
PG
o.380 0.907 2.385
0.422
NVEET I GFGK GEXQL YAXXD KI I SNVEET I GFGKG XTQXY
The purified PLD showed a single protein band estimated at 87 kDa on
O. 1 74
SDS-PAGE (Pig. 2). The sequences were directly determined by protein sequencer. Seq. I and 2 indicate sequences detected peaks of phenylthiohy-
O.41 l
dantoin derevatives on a chromatogram.
33
Purification and Characterization of Phospholipase D from Cabbage Leaves
nus. Pannenberg et al. (1998) recently reported the entire amino
acid sequences deduced from the nucleotide sequences of mRNA and gene encoding cabbage PLD. Further, they demon-
466. Hidano, N.. Kamata. M., Hara. S. and Totani, Y. (1995). Specificity of
scriptional initiation codon. Sequence I and 2 were homologous
phospholipase D for alcohol substrate in transphosphatidylation. Yukagaku, 44, 49-55 (in Japanese). Hirano, H. and Watanabe, T. ( 1990). Microsequencing of proteins electrotransferred onto immobilllzing matrices from polyacrylamide gel electrophoresis: Application to an insoluble protein. Electrophoresis, Il, 573-580. Imamura, S. and Horiuti, Y. ( 1978). Enzymatic determination of phospholipase D activity with choline oxidase. J. Biochem., 83, 677680.
to the sequence starting at positions 37 and 33 from the N-termi-
Juneja, L.R., Hibi, N., Inagaki, N., Yamane, T. and Shimizu, S. (1987).
nus of the deduced PLD2 sequence, respectively. These findings
Comparative study on conversion of phosphatidylcholine to phosphatidylglycerol by cabbage phospholipase D in micelle and emulsion systems. Enzyme Microb. Technol., 9, 350-354. Laemmli, U.K. ( 1 970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Lambrecht, R. and Ulbrich-Hofmann, R. (1992). A facile purification procedure of phospholipase D from cabbage and its characterization. Biol. Chem. Hoppe-Seyer, 373, 81-88. Lee, H., Choi, M. and Koh, E. (1989). Purification and characteriza-
strated two cabbage PLD isozymes termed PLD I and PLD2 and deduced their amino acid sequences.
We previously confirmed that the PLD purified from cabbage leaves is PLD2 (Watanabe et al., 1 999). The N-tenninal amino acid of the deduced PLD2 sequence is Met, encoded by the tran-
indicated that mature PLD2 is generated by removal of 36 or 32
N-terminal amino acid residues from the translation product of
the gene encoding PLD2. Formation of mature PLD by removing the N-terminal amino acid sequences has also been confirmed in PLD derived from other plants; 30 and 46 N-terminal residues are removed in castor
bean PLD and rice PLD, respectively (Wang et al., 1994; Ueki et al., 1995).
Generally, functional proteins (for example, enzyme, peptide
tion of the active site of phospholipase D. Korean Biochem. J., 22,
487~}93. Nagao, A. (1995). Synthesis of new phospholipids using lipolytic
processing.
enzyme. Syokuryou sono Kagaku to Gljutsu, 34, 1-13 (in Japanese). Nakajima, J.. Nakashima, T., Shima, Y., Fukuda, H, and Yamane, T. ( 1994). A facile transphoshatidylation reaction using a culture
Although the details of the processing mechanism of cabbage PLD have not yet been elucidated, the stepwise processing of PLD may give two proteins having a different N-terminus during
suqernatant of actinomycetes directly as a phospholipase D catalyst with a chelating agent. Biotechnol. Bioeng., 44, I 193-1 198. Pannenberg, I., Mansfeld, J. and Ulbrich-Hofmann, R. (1998). Identifi-
hoamones and serum proteins) are thought to mature by stepwise
membrane penetration and protein transport.
cation of two isoenzymes (Accession Nos. AF090444 and AF090445) of two phospholipase D from cabbage. Plant Physiol., 118, I 102.
References Abousalham, A., Riviere, M., Teissere, M. and Verger, R. (1993). Improved purification and biochemical characterization of phospholipase D from cabbage. Biochim. Biophys. Acta, I158, 1-7. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana/. Biochem., 72, 248-254. Comfurius, P., Bevers, E.M, and Zwaal. R.F.A. (1990). Enzymatic synthesis of phosphatidylserine on small scale by use of a one-phase system. J. Lipid. Res., 31, 1719-1721 .
Dawson, R.M.C. (1967). The formation of phosphatidylglycerol and other phospholipids by the transferase activity of phospholipase D.
Biochem. J., 102, 205-210. D'Arrigo, P. and Servi, S. (1997). Using phospholipases for phospholipid modification. Trends Biotechnol., 15, 90-96. Davidson, F.M. and Long, C. (1958). The structure of the naturally occurring phosphoglycerides-4. Action of cabbage-leaf phospholipase D on ovolecithin and related substances. Bioche,n. J., 69, 458-
Sato, H., Watanabe, T.. Sagane, Y. and Takano, K. (1999). Purification
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tion and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of CDNA for PLD from rice and maize (Zae mays L.). Plant Cell Physiol., 36, 903-9 14. Uwajima, T. and Terada, O. ( 1980). Properties of new enzyme glycerol oxidase from Aspergillus japonicus AT008. Agric. Biol. Chem., 44, 203 9-2045 .
Wang, X., Xu, L. and Zheng, L. (1994). Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L. J. Biol. Chem., 269, 203 1 2-203 17.
Watanabe, T., Sato. H., Sagane, Y., Nakazawa, Y. and Takano, K. ( 1999). Primary structure of phospholipase D purified from cabbage leaves. Seibutsu Butsuri Kagaku, 43, 159-164 (in Japanese). Yang, S.F., Freer. S. and Benson, A.A. (1967). Transphosphatidylation by phospholipase D. J. Biol. Chem., 242, 477~84.
