- Email: [email protected]
Purification of phospholipase D from peanuts
BIOCHIMICA
290 BBA
RIOPHTSI(‘.~
ACTA
56099
PURIFICATION
K.
ET
TZIJK
AXD
Department
OF
hfay
D FROM
PEANUTS
SHAPIRO
B.
of Biochemistry,
(Received
PHOSPHOLIPASE
znd,
The Hebrew
Ulliversity
Hadassah
Medical
School, Jerusalenz
i Israel)
1972)
SUMMAR\
Phospholipase D (EC 3.1.4.4) was extracted from dry peanut seeds and purified to a specific activity of about zoo pmoles/min per mg protein. Some properties and optimal working conditions are described.
INTRODUCTION
Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of lecithin to form phosphatidic acid and cholinel-“, as well as the exchange of choline by alcohols, to form pl~ospl~atidylalcol~o1 and choline+*. The source of this enzyme, described in the hterature, has been mainly green leaves (cabbage) or carrotsl-S, and was initially located in the insoluble fraction (plastids). Later it was shown that it is also present in the soluble fraction, provided that Ca”i were addedg. An additional rich source of the enzyme was found in this laboratory in peanut seeds”). The enzyme in drv peanuts was soluble and showed properties similar to those of the cabbage enzyme, but was found to act also on cardiolipinlO and on phosphatidyl glycerol l1 in the absence of ether or anionic amphipathes. Purification procedures have been described for the enzyme from cabbagel”*‘” and for the peanut enzymelo. The present paper presents a procedure for advanced purification to yield preparations of very high specific activities. Some additional properties of the purified enzyme are tested and optimal assay conditions established. 1MATERIALS
Ah-I)
METHODS
Ovolecithin was obtained from egg yolk and purified according to the method of Pangbornl4. [3H]Choline-labeled lecithin was obtained from tissue cultures grown in the presence of F’Hjmethylcholine as described earlier-l0 or by inoculating a synthetic medium containing :3H]methylcholine with fresh yeastIs. DEAE-cellulose was obtained from Sigma Chemical Company. Sephadex G-zoo was obtained from Pharmacia Uppsala, Sweden. 7’hin-Zqer chromatogra@hy. Thin-layer chromatographic plates coated with silica gel G were activated at IIO “C. and developed in a solvent system consisting of Niochim
Biophys.
Acta,
280 (1972)
290.-296
R. TZCR, B. SHAPIRO
292
mercaptoethanol (1.5 g/l). Activity was eluted with the same solution immediately after elution of the void volume. Tubes with similar specific activities were pooled and concentrated either by lyophilization or by dipping a dialysis bag into solid sucrose. (Fig. I). Further purification was achieved by rechromatography on a DEAlScellulose column
and eluting
”
the activity
with 0.45-0.6
M KCl, as stated
before (Fig. 2).
10
20 30 LO 50 60 Fraction Number Fig. 2. Chromatography of phospholipase D on DEAE-cellulose, Step 1’. 2.4 ml of Sephadex G-zoo fraction containing 3 mg protein were applied on the column (I ,5 cm x 30 cm). Elution was performed with 150 ml of Tris-EDTA buffer, pH 7.4, as stated in Materials and Methods followed with 200 ml of a linear gradient of 0-1 M KC1 in the same buffer. l’ractions of 2.5 ml were collected.
0
RESCLTS
Table I presents
the results of the purification
purification relative to the particle free extract enzyme was about 30%. TABLE
and shows a rooo-fold
I
PURIFICATION
OF
PHOSPHOLIPASE
step
I. II. III. IV. Vl.
procedure
of the peanut seeds. The yield of active
Total pvotein (v)
“Post microsomes” 20% (NH&SO, DEAE-cellulose I Sephadex G-zoo DEAE-cellulose II
Experimental
conditions
6560 4’0 13 3 2
D Specific activity (fiumoleslmin PeJ, w) -TransphosphaHydrolase tidyZase 0.2
2.3 32.5 167.0 234.0
0.2
3.0 3X.8 2ocJ.o
N.D.
Yield of hydvolasc activity (“4) 100
71.8 32 37 35.6
Ratio tvans_fevase hydrolasr
I.2
I.3 I.19 I.2 -
as stated in Materials and Methods. N.D., not determined.
EfSEct of soda’uwtdodecyl sulphate on $hospholi@ase D activity Stimulation with sodium dodecyl sulphate of lecithin hydrolysis by phospholipase D exceeded stimulation with ether by about IO times. Furthermore, solubilizing lecithin in dodecyl sulphate had the advantage over ether application of providing for a single phase, with better defined substrate concentration, As a result, the surface Biochim.
Biophys.
Acta,
280 (1972) 290-296
D
PHOSPHOLIPASE
area between
293
phases of the shaking
velocity,
did not affect enzymatic
activity
as in
the case with etherll. Optimal enzymatic activity was obtained at a molar ratio of lecithin to dodecyl sulphate of about 2 : I as also found by Dawsons with the enzyme from cabbage. Deviation from this ratio by changing dodecyl sulphate concentration with constant lecithin or vice versa reduced activity considerably (Fig. 3). The same ratio was found to be optimal at all stages of enzyme purification.
I
0
2
3
4
pmoles
5
6
SDS
Fig. 3, Phospholipase D activity in the presence of different molar ratios of lecithin to sodium dodecyl sulphate. Enzyme preparations of Stage IV were incubated with 3 pmoles, 5 pmoles and 7 ILmoles of lecithin in the presence of different sodium dodecyl sulphate (SDS) concentrations. x--x , 3 pmoles lecithin; O-O, 5 ~moles lecithin; O-O, 7 [lmoles lecithin.
Phosphatidic acid had only a limited stimulatory effect on phospholipase D activity, when optimal molar ratios of lecithin to dodecyl sulphate were used. With suboptimal ratios, however, the stimulation was considerable (Table II). When phosphatidic acid was the sole activator (in the absence of dodecyl sulphate) very low activities were found. ThBLE
II
EPFECT OF PHOSPHATIDIC LECITHIN
TO SODIUM
Addition of phosphatidic acid (~wPVJlcs)
ACID
DODECYL
ON PHOSPHOLIPASE
Lecithin lzxpt
to dodecyl sulfihate
MOLAR
RATIOS
OF
molar ratio:
2 : I
I
Expt
100
182
0.5
320 7oo
117
900
IZj
I.5
2
100
0
0.2j
118 116
2
Lecithin
AT DIFFERENT
Activation relative to activity without phosphatidic acid 5 :I
I
D ACTIVITY
SULPHATE
concentration
was kept
at 5 m&T. For absolute
activities
Biochim.
see Fig.
Biophys.
3
Acta,
280 (1972) zgo--296
K.
294
TZGR,
R. SHAPIRO
Transphosphatidylic acid activity of phospholipase D, measured with methanol showed optimal activity with 0.2 ml methanol per I ml incubation mixture. Hydrolytic and transphosphatidylic activities of phospholipase D seem to be associated with the same protein. The ratio of their activities remained constant throughout purification (Table I). Transphosphatidylic acid activity was always higher and when suboptimal molar concentrations of lecithin to sodium dodecyl sulphate were used it exceeds hydrolysis several fold. In that case methanol probably substitutes for dodecyl sulphate as activating agent. When ;_14C]oleic acid labeled lecithin was incubated with methanol, analysis product
by thinlayer chromatography was phosphatidyl methanol.
revealed
that at least 95%
of the radioactive
Stability of phospholifase D preparations The lyophilyzed (NH&SO, fraction was stable for 4-5 months when stored at -20 “C. The concentrated enzyme solution obtained after gel filtration (Step IV) by dialysis against solid sucrose did not loose activity for 5-6 weeks at 4 “C whereas when frozen at -20 “C, 607; of the activity was lost after 4 weeks. When lyophilyzed, such an enzvme preparation could be stored at -20 “C for 2 months without loss of activity. Efect
of albwni~z Incubation of phospholipase D in the presence of serum albumin caused inactivation of the enzyme (Table III) contrary to what was found by Dawson and Hemington13
with the cabbage
enzyme.
Kinetic parameters Kinetic parameters were measured with the enzyme preparation after elution from Sephadex G-200. K, for hydrolytic activity was found to be 2.2 .IO@ M and for transphosphatidvlic activity 2.5. IO-” IN. Enzyme activity curves were linear up to at least 20-25~~ breakdown of the substrate, when enzyme concentration was changed (Fig. 4) or with different incubation times with the same enzyme concentration
(T;ig. 5). The efect ofphospholi$ase D on microsomes Purified phospholipase D preparations (Steps IV and 1’) act on microsomes to split phospholipids without addition of sodium dodecyl sulphate. As shown in Table IV, after 120 min of incubation 120 nmoles of phosphatidic acid were formed with a simultaneous reduction in the amount of phosphatidylcholine and phosphatidylethanolamine. Incubation for a longer period with the same amount of enzyme did not Biochiw.
Bio~hys.
.4&a, 280 (1972) 290+296
PHOSFHOLIPASE
k
0
D
295
1.0
0.5
2.0
1.5
10 15 20 minutes Fig. 4. l?~~yrne concentration curve. Hydrolase (O-O) and transphosphatidylascactiyity( X-X ) were measured with different enzyme (Sephadex G-zoo eluate) concentrations incubated for IO min. at 30 “C. pg
0
Fig. 5. Enzyme time curve. Hydrolase (0-e) measured with 0.5 yg and I pugof Sephadex
EFFECT
5
Protein
OF PHOSPHOLIPASE
D
ON
MICROSOMAL
and transphosphatidylase activity ( x -x eluate for the times indicated.
) were
G-200
PHOSPHOLIPIDS
and 3 mg protein of lyophilyzed rat liver microsomes were incubated with 50 ~~molcs CaCl, and 50 !Lrnoles acetate buffer, pH 5.7. in the presence of 33 and roe ~6g protein phospholipase D (PLD), Step V. Total lipid phosphate of the microsomes were determined as stated in Materials and Methods. Phosphate of the various phospholipids were determined after plating and scraping thin-layer chromatographic plates according to reference substrates. _____-. _______. ._ -__.. Fraction cluted from r mg miwosomal protein 3 mg microsomal protein thin-laycar chvomatographic 120 min incubatio+z 60 milt incubation ~__ plate -PLD - PLD 33 pg PLD 33 pg PLD IO0 pg PLD I
.____I__.
~-~
Phosphatidic acid Sphingomyelin Phosphatidylcholine Phos~~~tidylethanolamiilc -.-____ ~...
21 85
140.7 85
231.6
108.4
‘49
.__
I68
96
-.
2jZ
2j2
364 260
8jZ
780
576
74
.__
-_
__-._
_
give further hydrolysis, whereas with larger amounts of phospholipase D hydrolysis was increased. Similar results have also been shown with the crude preparationll.
The presently described purification procedure yields preparations of very high specific activities, exceeding markedly those described for the enzyme from cabbage’“. The starting material, the aqueous extract of peanut seeds, in spite of somewhat lower specific activity has a much higher total activity due to the high protein content of peanut extracts, compared to those of cabbage. Biochim.
Biophys.
Acta,
280
(1972)
zgo-296
Ii. TZUR, B. SHAPIRO
296
Contrary to the finding with the cabbage enzymeIs, no considerable loss was encountered by absorption of the enzyme to glass and linear activity curves were obtained, although the amount of enzyme employed in the assay was of the order of I ,~g. In spite of the high degree of purification, the enzyme is not yet homogenous as seen on polyacrylamide gel electrophoresis. Scanning of the stained gel revealed that about 80% of the protein appeared in bands devoid of activity. Polyacrylamide gel electrophoresis could not be used for purification since only a small part of the activity was eluted from thegel. Further attempts at purification were not successful due to the small amounts of protein obtained. This will require a scale-up to almost industrial levels. REFERENCES I D. J, Hanahan and I. L. Chaikoff, J. Biol. Chem., 168 (1947) 233. 2 D. J. Hanahan and I. L. Chaikoff, J. Riol. Chem., 169 (1947) 699. 3 I>. J. Hanahan and I. L. Chaikoff, J. Biol. Chem., 172 (1948) 191. 4 M. Kates, Can. J. R&hem. Physiol., 32 (1954) 571. 5 M. Kates, Can. J. B&hem. Physiol., 34 (1956) 967. 6 :I. A. Benson, S. Freer and S. F. Yan g, 9th Int. Conf. oy1 Biochemistry Netherlands, 7 8 9 IO II 12
13 14
15 16
of Lipids,
Noovdwijk,
1965.
R. Douce, M. Faux-e and J. Marechal, C. R. Sot. Biol. (Paris), 262 (1966) 1549. K. hl. C. Damson, Biochrm. J., 102 (1967) 205. F. M. Davidson and C. Long, Biochem. J., 69 (1958) 45X. ill. Heller, E. Aladjem and B. Shapiro, Bull. Sot. Chem. Biol., 50 (x968) 1x95. M. Heller and R. Arad, Biochim. Biophys. Acta, 210 (1970) 276. S. I;‘. Yang, S. Freer and A. A. Benson, J, Biol. Ckem., 24~ (1967) 477. K. M. C. Damson and N. Hemington, Biochem. J., 102 (1967) 76. bl. C. Pangborn, /. Biol. Chem., 188 (1951) 471. A. D. Bangham and K. XI. C. Dawson, Biochem. J,, 75 (1960) ‘33. G. R. Barlett, J. Riol. Chem., 234 (1959) 466.
Biochim.
Biophys.
Acta,
280 (1972)
290-296
The
290 BBA
RIOPHTSI(‘.~
ACTA
56099
PURIFICATION
K.
ET
TZIJK
AXD
Department
OF
hfay
D FROM
PEANUTS
SHAPIRO
B.
of Biochemistry,
(Received
PHOSPHOLIPASE
znd,
The Hebrew
Ulliversity
Hadassah
Medical
School, Jerusalenz
i Israel)
1972)
SUMMAR\
Phospholipase D (EC 3.1.4.4) was extracted from dry peanut seeds and purified to a specific activity of about zoo pmoles/min per mg protein. Some properties and optimal working conditions are described.
INTRODUCTION
Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of lecithin to form phosphatidic acid and cholinel-“, as well as the exchange of choline by alcohols, to form pl~ospl~atidylalcol~o1 and choline+*. The source of this enzyme, described in the hterature, has been mainly green leaves (cabbage) or carrotsl-S, and was initially located in the insoluble fraction (plastids). Later it was shown that it is also present in the soluble fraction, provided that Ca”i were addedg. An additional rich source of the enzyme was found in this laboratory in peanut seeds”). The enzyme in drv peanuts was soluble and showed properties similar to those of the cabbage enzyme, but was found to act also on cardiolipinlO and on phosphatidyl glycerol l1 in the absence of ether or anionic amphipathes. Purification procedures have been described for the enzyme from cabbagel”*‘” and for the peanut enzymelo. The present paper presents a procedure for advanced purification to yield preparations of very high specific activities. Some additional properties of the purified enzyme are tested and optimal assay conditions established. 1MATERIALS
Ah-I)
METHODS
Ovolecithin was obtained from egg yolk and purified according to the method of Pangbornl4. [3H]Choline-labeled lecithin was obtained from tissue cultures grown in the presence of F’Hjmethylcholine as described earlier-l0 or by inoculating a synthetic medium containing :3H]methylcholine with fresh yeastIs. DEAE-cellulose was obtained from Sigma Chemical Company. Sephadex G-zoo was obtained from Pharmacia Uppsala, Sweden. 7’hin-Zqer chromatogra@hy. Thin-layer chromatographic plates coated with silica gel G were activated at IIO “C. and developed in a solvent system consisting of Niochim
Biophys.
Acta,
280 (1972)
290.-296
R. TZCR, B. SHAPIRO
292
mercaptoethanol (1.5 g/l). Activity was eluted with the same solution immediately after elution of the void volume. Tubes with similar specific activities were pooled and concentrated either by lyophilization or by dipping a dialysis bag into solid sucrose. (Fig. I). Further purification was achieved by rechromatography on a DEAlScellulose column
and eluting
”
the activity
with 0.45-0.6
M KCl, as stated
before (Fig. 2).
10
20 30 LO 50 60 Fraction Number Fig. 2. Chromatography of phospholipase D on DEAE-cellulose, Step 1’. 2.4 ml of Sephadex G-zoo fraction containing 3 mg protein were applied on the column (I ,5 cm x 30 cm). Elution was performed with 150 ml of Tris-EDTA buffer, pH 7.4, as stated in Materials and Methods followed with 200 ml of a linear gradient of 0-1 M KC1 in the same buffer. l’ractions of 2.5 ml were collected.
0
RESCLTS
Table I presents
the results of the purification
purification relative to the particle free extract enzyme was about 30%. TABLE
and shows a rooo-fold
I
PURIFICATION
OF
PHOSPHOLIPASE
step
I. II. III. IV. Vl.
procedure
of the peanut seeds. The yield of active
Total pvotein (v)
“Post microsomes” 20% (NH&SO, DEAE-cellulose I Sephadex G-zoo DEAE-cellulose II
Experimental
conditions
6560 4’0 13 3 2
D Specific activity (fiumoleslmin PeJ, w) -TransphosphaHydrolase tidyZase 0.2
2.3 32.5 167.0 234.0
0.2
3.0 3X.8 2ocJ.o
N.D.
Yield of hydvolasc activity (“4) 100
71.8 32 37 35.6
Ratio tvans_fevase hydrolasr
I.2
I.3 I.19 I.2 -
as stated in Materials and Methods. N.D., not determined.
EfSEct of soda’uwtdodecyl sulphate on $hospholi@ase D activity Stimulation with sodium dodecyl sulphate of lecithin hydrolysis by phospholipase D exceeded stimulation with ether by about IO times. Furthermore, solubilizing lecithin in dodecyl sulphate had the advantage over ether application of providing for a single phase, with better defined substrate concentration, As a result, the surface Biochim.
Biophys.
Acta,
280 (1972) 290-296
D
PHOSPHOLIPASE
area between
293
phases of the shaking
velocity,
did not affect enzymatic
activity
as in
the case with etherll. Optimal enzymatic activity was obtained at a molar ratio of lecithin to dodecyl sulphate of about 2 : I as also found by Dawsons with the enzyme from cabbage. Deviation from this ratio by changing dodecyl sulphate concentration with constant lecithin or vice versa reduced activity considerably (Fig. 3). The same ratio was found to be optimal at all stages of enzyme purification.
I
0
2
3
4
pmoles
5
6
SDS
Fig. 3, Phospholipase D activity in the presence of different molar ratios of lecithin to sodium dodecyl sulphate. Enzyme preparations of Stage IV were incubated with 3 pmoles, 5 pmoles and 7 ILmoles of lecithin in the presence of different sodium dodecyl sulphate (SDS) concentrations. x--x , 3 pmoles lecithin; O-O, 5 ~moles lecithin; O-O, 7 [lmoles lecithin.
Phosphatidic acid had only a limited stimulatory effect on phospholipase D activity, when optimal molar ratios of lecithin to dodecyl sulphate were used. With suboptimal ratios, however, the stimulation was considerable (Table II). When phosphatidic acid was the sole activator (in the absence of dodecyl sulphate) very low activities were found. ThBLE
II
EPFECT OF PHOSPHATIDIC LECITHIN
TO SODIUM
Addition of phosphatidic acid (~wPVJlcs)
ACID
DODECYL
ON PHOSPHOLIPASE
Lecithin lzxpt
to dodecyl sulfihate
MOLAR
RATIOS
OF
molar ratio:
2 : I
I
Expt
100
182
0.5
320 7oo
117
900
IZj
I.5
2
100
0
0.2j
118 116
2
Lecithin
AT DIFFERENT
Activation relative to activity without phosphatidic acid 5 :I
I
D ACTIVITY
SULPHATE
concentration
was kept
at 5 m&T. For absolute
activities
Biochim.
see Fig.
Biophys.
3
Acta,
280 (1972) zgo--296
K.
294
TZGR,
R. SHAPIRO
Transphosphatidylic acid activity of phospholipase D, measured with methanol showed optimal activity with 0.2 ml methanol per I ml incubation mixture. Hydrolytic and transphosphatidylic activities of phospholipase D seem to be associated with the same protein. The ratio of their activities remained constant throughout purification (Table I). Transphosphatidylic acid activity was always higher and when suboptimal molar concentrations of lecithin to sodium dodecyl sulphate were used it exceeds hydrolysis several fold. In that case methanol probably substitutes for dodecyl sulphate as activating agent. When ;_14C]oleic acid labeled lecithin was incubated with methanol, analysis product
by thinlayer chromatography was phosphatidyl methanol.
revealed
that at least 95%
of the radioactive
Stability of phospholifase D preparations The lyophilyzed (NH&SO, fraction was stable for 4-5 months when stored at -20 “C. The concentrated enzyme solution obtained after gel filtration (Step IV) by dialysis against solid sucrose did not loose activity for 5-6 weeks at 4 “C whereas when frozen at -20 “C, 607; of the activity was lost after 4 weeks. When lyophilyzed, such an enzvme preparation could be stored at -20 “C for 2 months without loss of activity. Efect
of albwni~z Incubation of phospholipase D in the presence of serum albumin caused inactivation of the enzyme (Table III) contrary to what was found by Dawson and Hemington13
with the cabbage
enzyme.
Kinetic parameters Kinetic parameters were measured with the enzyme preparation after elution from Sephadex G-200. K, for hydrolytic activity was found to be 2.2 .IO@ M and for transphosphatidvlic activity 2.5. IO-” IN. Enzyme activity curves were linear up to at least 20-25~~ breakdown of the substrate, when enzyme concentration was changed (Fig. 4) or with different incubation times with the same enzyme concentration
(T;ig. 5). The efect ofphospholi$ase D on microsomes Purified phospholipase D preparations (Steps IV and 1’) act on microsomes to split phospholipids without addition of sodium dodecyl sulphate. As shown in Table IV, after 120 min of incubation 120 nmoles of phosphatidic acid were formed with a simultaneous reduction in the amount of phosphatidylcholine and phosphatidylethanolamine. Incubation for a longer period with the same amount of enzyme did not Biochiw.
Bio~hys.
.4&a, 280 (1972) 290+296
PHOSFHOLIPASE
k
0
D
295
1.0
0.5
2.0
1.5
10 15 20 minutes Fig. 4. l?~~yrne concentration curve. Hydrolase (O-O) and transphosphatidylascactiyity( X-X ) were measured with different enzyme (Sephadex G-zoo eluate) concentrations incubated for IO min. at 30 “C. pg
0
Fig. 5. Enzyme time curve. Hydrolase (0-e) measured with 0.5 yg and I pugof Sephadex
EFFECT
5
Protein
OF PHOSPHOLIPASE
D
ON
MICROSOMAL
and transphosphatidylase activity ( x -x eluate for the times indicated.
) were
G-200
PHOSPHOLIPIDS
and 3 mg protein of lyophilyzed rat liver microsomes were incubated with 50 ~~molcs CaCl, and 50 !Lrnoles acetate buffer, pH 5.7. in the presence of 33 and roe ~6g protein phospholipase D (PLD), Step V. Total lipid phosphate of the microsomes were determined as stated in Materials and Methods. Phosphate of the various phospholipids were determined after plating and scraping thin-layer chromatographic plates according to reference substrates. _____-. _______. ._ -__.. Fraction cluted from r mg miwosomal protein 3 mg microsomal protein thin-laycar chvomatographic 120 min incubatio+z 60 milt incubation ~__ plate -PLD - PLD 33 pg PLD 33 pg PLD IO0 pg PLD I
.____I__.
~-~
Phosphatidic acid Sphingomyelin Phosphatidylcholine Phos~~~tidylethanolamiilc -.-____ ~...
21 85
140.7 85
231.6
108.4
‘49
.__
I68
96
-.
2jZ
2j2
364 260
8jZ
780
576
74
.__
-_
__-._
_
give further hydrolysis, whereas with larger amounts of phospholipase D hydrolysis was increased. Similar results have also been shown with the crude preparationll.
The presently described purification procedure yields preparations of very high specific activities, exceeding markedly those described for the enzyme from cabbage’“. The starting material, the aqueous extract of peanut seeds, in spite of somewhat lower specific activity has a much higher total activity due to the high protein content of peanut extracts, compared to those of cabbage. Biochim.
Biophys.
Acta,
280
(1972)
zgo-296
Ii. TZUR, B. SHAPIRO
296
Contrary to the finding with the cabbage enzymeIs, no considerable loss was encountered by absorption of the enzyme to glass and linear activity curves were obtained, although the amount of enzyme employed in the assay was of the order of I ,~g. In spite of the high degree of purification, the enzyme is not yet homogenous as seen on polyacrylamide gel electrophoresis. Scanning of the stained gel revealed that about 80% of the protein appeared in bands devoid of activity. Polyacrylamide gel electrophoresis could not be used for purification since only a small part of the activity was eluted from thegel. Further attempts at purification were not successful due to the small amounts of protein obtained. This will require a scale-up to almost industrial levels. REFERENCES I D. J, Hanahan and I. L. Chaikoff, J. Biol. Chem., 168 (1947) 233. 2 D. J. Hanahan and I. L. Chaikoff, J. Riol. Chem., 169 (1947) 699. 3 I>. J. Hanahan and I. L. Chaikoff, J. Biol. Chem., 172 (1948) 191. 4 M. Kates, Can. J. R&hem. Physiol., 32 (1954) 571. 5 M. Kates, Can. J. B&hem. Physiol., 34 (1956) 967. 6 :I. A. Benson, S. Freer and S. F. Yan g, 9th Int. Conf. oy1 Biochemistry Netherlands, 7 8 9 IO II 12
13 14
15 16
of Lipids,
Noovdwijk,
1965.
R. Douce, M. Faux-e and J. Marechal, C. R. Sot. Biol. (Paris), 262 (1966) 1549. K. hl. C. Damson, Biochrm. J., 102 (1967) 205. F. M. Davidson and C. Long, Biochem. J., 69 (1958) 45X. ill. Heller, E. Aladjem and B. Shapiro, Bull. Sot. Chem. Biol., 50 (x968) 1x95. M. Heller and R. Arad, Biochim. Biophys. Acta, 210 (1970) 276. S. I;‘. Yang, S. Freer and A. A. Benson, J, Biol. Ckem., 24~ (1967) 477. K. M. C. Damson and N. Hemington, Biochem. J., 102 (1967) 76. bl. C. Pangborn, /. Biol. Chem., 188 (1951) 471. A. D. Bangham and K. XI. C. Dawson, Biochem. J,, 75 (1960) ‘33. G. R. Barlett, J. Riol. Chem., 234 (1959) 466.
Biochim.
Biophys.
Acta,
280 (1972)
290-296
The