ARCHIVES
OF BIOCHEMISTRY
Partial
AND
BIOPHYSICS
Purification
and Properties Spinach
BRUCE Department
of Biochemistry
177, 24-30
A. MACHER’
(1976)
of Ethanolamine Leaf AND
J. B. MUDD
and Statewide Air Pollution Research Center, Riverside, Riverside, California 92502 Received
February
Kinase from
University
of California-
20, 1976
Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,OOOg supernatant from spinach leaf. The 100,OOOg supernatant preparation was stable for weeks’, but the partially purified preparation lost half of the ethanolamine kinase activity in lo-14 days at 0-4°C or -20°C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (K,, 42 PM), MgATP (K,, 63 PM), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparentKi of 6.7 mM. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and ophenanthroline. EDTA above 0.9 mM inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 mM) and inhibited at 1.8 mM. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 mM). The ethanolamine kinase and choline kinase activities of the 100,OOOg supernatant preparation could be separated by gel electrophoresis.
al. (2) have characterized the enzyme responsible for the synthesis of CDP-diglyceride in a number of tissues. Marshall and Kates (14, 15) have demonstrated both the CDP-diglyceride dependent synthesis of PS and its decarboxylation in spinach leaf preparations. From these studies, it is apparent that plants are capable of synthesizing PE through the bacterial pathway. Regarding the enzyme catalyzing reactions of the CDP-ethanolamine pathway, a number of studies are also available. Moore et al. (16) have demonstrated phosphatidic acid phosphatase in castor bean. Borkenhagen and Kennedy (3) have reported that preparations from carrot root are capable of catalyzing the synthesis of CDP-ethanolamine from CTP and phosphorylethanolamine. We have previously characterized an enzyme from spinach that catalyzes the formation of PE from DG and CDP-ethanolamine (13). This activity is also found in other plant tissues (13). Marshall and Kates (15) have shown that phosphorylethanolamine can be
Our basic knowledge of the major pathways for phospholipid synthesis in animal tissue comes from the studies of Kennedy and co-workers (3,4,7, 11). Their work has also been important in the elucidation of the major pathways of phospholipid synthesis in bacterial systems (9, 19). There are unique pathways for PE2 synthesis. In bacteria the final steps are formation of PS by way of CDP diglyceride, and then decarboxylation of PS (19): in animals the major pathway for PE synthesis appears to be by way of CDP ethanolanine (11). The enzymes which catalyze a number of these reactions have been studied in plants. Sumida and Mudd (22) and Bahl et 1 Present address: Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824.
* Abbreviations used: DG, 1,2 diacyl-sn-glyceroh EGTA, ethyleneglycol-bis-N,N’-tetraacetic acid; HEPES, N-2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic acid; PA, phosphatidic acid; PE, phosphatidylethanolamine; PS, phosphatidylserine; buffer A, 100 mM Tris-HCl, pH 7.6, 1 rnM Mg Cl,. 24 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
ETHANOLAMINE
KINASE
formed from ethanolamine and ATP in the spinach leaf. However, the data so far reported for ethanolamine kinase activity in spinach do not characterize the enzyme and cannot eliminate the possibility that phosphorylethanolamine formation is catalyzed by choline kinase (24). This report is the outcome of our desire to characterize ethanolamine kinase from spinach and to determine whether the activity is separable from choline kinase. EXPERIMENTAL
PROCEDURE
Materials. Spinach (Spinacia oleracea) was purchased at local stores. [‘4ClEthanolamine and l’4Clcholine were purchased from ICN, Irvine, California. [‘4C]Phosphorylethanolamine and [‘4C1phosphorylcholine were obtained from New England Nuclear, Boston, Massachusetts. Sephadex G-ZOO was a product of Pharmacia, Uppsala, Sweden. DEAE-filter disks were purchased from Whatman, Maidstone, England. Enzyme assays. The ethanolamine kinase assay contained in a volume of 0.55 ml: Tris-chloride, pH 8.5 (110 mM), MgCl, (1.8 mM), ATP (0.9 mM), [“Clethanolamine (0.11 mM, the amount of radioactivity varied with experiment), and enzyme. In assays for choline kinase, [‘Y!]choline replaced [‘“Clethanolamine. Reactions were started by addition of the enzyme, run 15 min at 3o”C, and stopped by placing in a boiling water bath for 5 min. An aliquot (0.1 ml) of the reaction mixture was assayed by a modification of the method of Preiss et al. (181, by applying it to a DEAE-filter disk. The assay method depends on the binding of the phosphorylated product to the filter disk, while the substrate is washed free. One unit of enzyme activity represents the formation of 1 nmol of phosphorylethanolamine per minute per milligram of protein. The products of the assay systems were characterized by paper chromatography in the solvent system, butanol-acetic acid--water, 5:3:2 (21. Other methods. Polyacrylamide gel electrophoresis was carried out in a discontinuous buffer system of Tris-chloride/Tris-glycine (8). Glycerol (final concentration, 8%) was added to the protein sample. The sample was layered over a stacking gel and electrophoresed at a constant current of 3 mA. The gels were sliced into 1.5-mm sections and each section was cut into two pieces. One piece was assayed for ethanolamine kinase activity. The other piece was assayed for choline kinase activity. Protein was determined by the procedure of Lowryet al. (12). Bovine serum albumin was used as a protein standard. All steps during the purification of ethanolamine kinase were carried out at 0-4°C. Data processing. All calculations and fits of data
FROM
SPINACH
25
LEAF
were carried out with programs written for the Wang 720 C programmable calculator. Saturation curves for MgATP, varying MgATP at several ethanolamine concentrations, and varying MgATP at several total magnesium concentrations were fitted to an equation which describes substrate inhibition assuming that substrate combines twice with the enzyme (5). The fit is by the least-squares method to a multiple linear regression analysis to the equation, Ko -1 = vA v
f gK-
+ +,
IA
PI
Saturation curves for ethanolamine at fixed varying MgATP concentrations were analyzed by the weighted fit method to a rectangular hyperbola (61. K,‘s for ethanolamine and MgATP were determined by reporting the slopes vs MgATP concentration and the intercepts vs MgATP concentrations from Fig. 2 (171. RESULTS
Purification
of the Enzyme
Spinach extracts were prepared by homogenizing a given weight of material with twice as much homogenizing medium containing sucrose (250 mM), Trischloride, pH 7.6 (10 mM), and EDTA (1 mM). The leaf tissue was shredded with a razor blade and then homogenized in a Waring Blendor (3-5 s burst). The homogenate was pressed through two layers of cheesecloth and centrifuged for 20 min at 20,OOOg. The resulting supernatant was centrifuged for 60 min at 100,OOOg. The majority of the ethanolamine kinase remained in the 100,OOOg supernatant. The yellow-green supernatant was adjusted to 45% saturation of ammonium sulfate by addition from a saturated solution (pH 7). Precipitated material was removed by centrifugation (20,OOOg for 10 min), and the supernatant was adjusted to 75% ammonium sulfate saturation. After 75 min without stirring, the precipitate then formed was removed by centrifugation and taken up in Buffer A. The dissolved precipitate was thoroughly dialyzed overnight against Buffer A (three changes of 100 vol). The sample was stored at -20°C at least 3 days and not more than 4 days. If the sample was stored frozen for 2 weeks, half the enzyme activity was lost. After the sample was thawed, it was spun briefly in a clinical centrifuge to remove
26
MACHER
AND
the large brown precipitate which had formed. The clarified sample was applied to a Sephadex G-200 column (2 x 100 cm) and eluted with Buffer A. Two major protein areas were separated by the column. Ethanolamine kinase eluted midway between two major protein peaks. Recovery was 50 to 60% of the initial activity. About 50% of the protein remaining after gel chromatography could be removed by adjusting the combined column fractions to 50% saturation with solid ground ammonium sulfate. The enzymatic activity was retained in the supernatant. The enzyme had been purified 60-fold at this stage. Polyacrylamide gel electrophoresis indicated that the preparation was not homogeneous. When the enzyme solution was adjusted to 90% saturation with solid ammonium sulfate and dialyzed, only 20% of the initial activity precipitated. Table I summarizes the steps used in the purification procedure. The molecular weight of ethanolamine kinase was estimated through the use of a standardized Sephadex G-200 column according to the method of Andrews (1). The relationship between the molecular weights of standard proteins (aldolase, ovalbumin, chymotrypsinogen A, and cytochrome c) and K,, was plotted. The molecular weight of ethanolamine kinase was estimated to be 110,000. The 100,OOOg preparation (Table I) was stable to repeated freezing and to prolonged storage at 0-4°C and room temperTABLE PURIFICATION
100,OOOg supernatant First ammonium sulfate tionation Freeze-thaw Chromatography Sephadex 200 Second ammonium sulfate tionation
frac-
Gfrac-
MUDD
ature (90% recovery of initial activity after 20 h at room temperature). The partially purified enzyme lost 50% of its activity during lo-14 days storage at -20°C. The 100,OOOg supernatant preparation lost 60% of its initial activity when it was heated at 45°C for 3 min. Characteristics Enzyme
of the Partially
Purified
Results of a time course experiment showed that with the enzyme at a concentration of 50 pug/ml in the reaction mixture, the rate of phosphorylethanolamine formation was linear for 35 min. The effect of changing protein concentration showed that product formation increased linearly with added protein to a concentration of 115 pg/ml. The partially purified ethanolamine kinase had maximal activity at the highest pH tested for each buffer (HEPES, pH 8.5; Tris-chloride, pH 9.0; and Tris-glycine, pH 9.2; Fig. 1). There appears to be a definite buffer effect. This pH profile is similar to that reported for ethanolamine kinase from Ehrlich Ascites cells (23) and rat liver (ethanolamine kinase II) (25). Although the crude and purified preparations of ethanolamine kinase from spinach were similar in most respects, the two preparations showed different responses to pH changes with Tris-glycine. While the purified preparation had maximal activity with Tris-glycine, the crude preparation showed less activity with Tris-glyI
OF ETHANOLAMINE
KINASE”
Total volume (ml)
Total activity (units)
Specific activity (unitslmg)
Puriti-
protein (mn)
50.0 13.0
66.7 74.7
262.0 65.0
0.25 1.15
1.0 4.6
100 112
12.8 34.0
72.6 40.6
30.5 4.4
2.38 9.23
9.5 36.9
109 61
59.0
35.6
2.4
14.80
59.2
53
Total
Yield
cation (%I
u One hundred grams of spinach leaf were homogenized with 200 ml of homogenizing medium. The homogenate was centrifuged and the enzyme was further purified as described in Experimental Procedure. Specific activity of each fraction was determined from the linear portion of a protein concentration curve.
ETHANOLAMINE
7.5
I
1
I
I 8.0
I 8.5 PH
I 9.0
KINASE
9.5
FIG. 1. The effect of pH on the activity of the partially purified preparation of ethanolamine kinase (0, 110 mM HEPES; A, 110 mM Tris-chloride; 0, 110 mM Tris-glycine). Enzyme activity was determined using standard assay conditions given under Experimental Procedure (40 pg protein assay).
tine than in either HEPES or Tris-chloride.:’ Kinetic Properties The effect of varying the concentration of ethanolamine (7.3 to 164 PM) at several concentrations of MgATP is shown in Fig. 2. When the data were presented in the Lineweaver-Burk form, a set of lines w.hich intersect to the left of the vertical axis was obtained (Fig. 2). This pattern is expected for an enzyme which catalyzes a two substrate reaction via a sequential mechanism (5). The K,, for ethanolamine was 42 PM and for MgATP, 63 PM as computed by the method cited in Experimental Procedure. Substrate inhibition was observed at the highest MgATP concentration (1.36 mM). Ethanolamine was not inhibitory at the concentrations tested. The effect of varying the concentration of MgATP at several concentrations of ethanolamine is shown in Fig. 3a. Double reciprocal plots of initial velocity against various concentrations of MgATP (0.05 to 1.7 mM) at several concentrations of ethanolamine are shown in Fig. 3b. Substrate 3 Data
not presented
FROM
SPINACH
LEAF
27
inhibition was again apparent at high concentrations of MgATP. When MgATP was the variable substrate (0.1 to 3.5 mM), at several levels of total magnesium, an unusual pattern was obtained (Figs. 4a and b). This plot shows that even at low levels of free ATP (line 41, substrate inhibition occurred at high concentrations of MgATP, suggesting that the substrate inhibition observed in Figs. 2 and 3 was due to high MgATP concentrations and not to high levels of free ATP. Therefore, from this plot both free magnesium and MgATP appeared to produce inhibition. The apparent Ki for MgATP was 6.7 mM. Free magnesium was required for maximum activity. Other Properties of Ethanolamine
Kinase
Table II shows the effect of metal-ion chelators and calcium on the ethanolamine kinase activity of dialyzed preparations of the 100,OOOg supernatant. EDTA inhibits the formation of phosphorylethanolamine at concentrations above 0.9 mM. EGTA activated ethanolamine kinase at low concentrations (0.4 and 0.9 mM) and inhibited it at a concentration of 1.8 mM. Ethanolamine kinase was inhibited to the same degree at all concentrations of o-phenanthroline tested. The
FIG. 2. Initial velocity pattern versus ethanolamine concentrations at several concentrations of MgATP. Enzyme activity was determined using standard assay conditions given in Experimental Procedure, except that the initial concentration of magnesium was 1.8 mM, and the concentrations of ATP were: 0.23 mM, 0; 0.43 mM, 0; 0.90 mM, A; 1.80 mM, 0 (30 pg purified enzyme protein assay). Reciprocal plots of the data are presented.
28
MACHER
AND
MUDD
i / /
z z 0’ aI E g Y/ E
I : : :’ /--A AA--. \:9.20 t \- * : : 730 -------a---- .--___CL-.. dimethylethanolamine > choline. ADP also inhibited ethanolamine kinase. This inhibition was most likely produced by MgADP. Since the partially purified ethanolamine kinase retained choline kinase activity, another method was used to separate these enzymes. Ethanolamine kinase and choline kinase were separated by polyacrylamide electrophoresis (Fig. 5). Ethanolamine kinase moved to a position in the gel between two areas of choline kinase activity.
ETHANOLAMINE
KINASE
FROM
From studies with animal cells (23) and tissue (251, it is clear that the protein responsible for ethanolamine phosphorylaTABLE
II
OF METAL-ION ETHANOLAMINE
-~ Additions
Concentration
CHELATORS KINASE”
ON
Nanomoles per minute
Percentage of control
(IIIM)
Pione
-
0.12
100
EDTA
0.4 0.9 1.8
0.13 0.11
108 92 42
0.4 0.9
0.20
166
0.18
1.8
0.09
150 75
0.4
0.10 0.10 0.10
83
0.5 0.9
0.10
83
0.07
1.8 9.1
0.06 0.02
58 50 17
EGTA
o-Phenanthroline
0.9 1.8
c92+
0.05
--
83 83
FIG. 5. Polyacrylamide gel electrophoresis separation of ethanolamine kinase and choline kinase (8% resolving gel, 630 pg of the postmicrosomal supernatant preparation). Preparation of gel slices and assay conditions are given under Experimental Procedure (ethanolamine kinase, 0; choline kinase,
(i Each reaction mixture contained 1.80 mM ATP, and 520 1.80 mM Mg**, 0.11 mM [‘“Clethanolamine, pg of protein of the 100,OOOg supernatant.
l ).
TABLE EFFECT
OF N-METHYL
Additions
DERIVATIVES
0.03 0.13 0.21
0.10 1.00 5.00
Dimethylethanolamine
0.10 1.00
Choline
102 74
reaction mixture of the 100,OOOg
contained supernatant.
72 44
30
0.080 0.032 0.026
64 26
89
0.108 0.076
87 61 31
62 32
20
1.80 mM ATP.
100
0.186
..--____ ’ Each of protein
0.120 0.135
31 65 26 21
101 100 97 59 49
3.64 18.20
KINASE”
Percentage control
0.189
0.10 1.00 1.80
ON ETHANOLAMINE
Ethanolamine (nmolesimin)
45
5.00
5.00
ADP
AND ADP
Disintegrations per minute (x10-:‘) ._______--.
0.05
Monomethylethanolamine
III
OF ETHANOLAMINE
Concentration (mM)
None Ethanolamine
29
LEAF
tion is not the same protein which phosphorylates choline. Sung and Johnstone (23) differentiated ethanolamine kinase and choline kinase, in Ehrlich Ascites cell extracts, on the basis of a number of kinetic and physical properties. For example, ethanolamine kinase was less stable during storage than choline kinase; ethanolamine kinase is more sensitive to thio reagents and calcium than choline kinase; and choline analogs, which stimulate choline kinase, inhibit ethanolamine kinase. Weinhold and Rethy (25) demonstrated two forms of ethanolamine kinase. Etha-
DISCUSSION
EFFECT
SPINACH
1.80 mM Mg”,
0.038 0.122
0.121 0.117
0.072 0.059 0.024
-.-____ 0.03 mM 1°C lethanolamine,
of
21
99 98 95 58 48 20
and 520 kg
-
30
MACHER
nolamine kinase I was physically separated from choline kinase by DEAE-cellulose column chromatography. Ethanolamine kinase II could be differentiated from choline kinase by its stability during storage and its kinetic properties. Comparable studies with plant tissues have not been reported previously. However, some comparisons of our results can be made to the available literature on choline kinase. Ramasarma and Wetter (20) have purified choline kinase from rapeseed. The purified enzyme did not phosphorylate ethanolamine, but no data were presented on ethanolamine phosphorylation by crude enzyme preparations. Choline kinase has also been purified from spinach (24), but no data were presented for ethanolamine phosphorylation. Choline kinase from spinach and rapeseed had pH curves similar to that found in this study for ethanolamine kinase. However, the choline kinases were stable to freezing and required equal amounts of magnesium and ATP for maximum activity. This is not true for ethanolamine kinase from spinach. The partially purified preparation of ethanolamine kinase is unstable to freezing and requires an excess of magnesium for maximum activity, suggesting that the enzyme requires both MgATP and free magnesium. The fact that ethanolamine kinase is inhibited by monomethylethanolamine and dimethylethanolamine but not choline also suggests that ethanolamine kinase and choline kinase are different proteins. The separation of the two activities by disc gel polyacrylamide electrophoresis confirms the theory that the two activities are due to separate proteins. It is interesting to point out that Weinhold and Rethy (25) demonstrated two ethanolamine kinase activities in rat liver, while we have found two choline kinase activities in spinach. With the data presented in this report, it would be premature to propose a reaction mechanism for ethanolamine kinase . However, it is clear that ethanolamine and MgATP produce a sequential initial velocity pattern. It is also important to consider the fact that free magnesium is essential for maximum velocity, suggesting that magnesium plays two roles.
AND
MUDD ACKNOWLEDGMENTS
The authors are indebted to Dr. Paul Cook for his assistance with some of the data analyses and interpretation.
REFERENCES 1. ANDREWS, P. (1964) Biochemistry 91, 222-233. 2. BAHL, J., GUILLOT-SALOMON, T., AND DOUCE, R. (1970) Physiol. Veg. 8, 55-74. 3. BORKENHAGEN, L. F., AND KENNEDY, E. P. (1957) J. Biol. Chem. 227, 951-962. 4. BORKENHAGEN, L. F., KENNEDY, E. P., AND FIELDING, A. (1961)J. Biol. Chem. 236, 28-30. W. W. (1970) in The Enzymes (Boyer, 5. CLELAND, P. D., ed.), Vol. 2, pp. l-65, Academic Press, New York. 6. CLELAND, W. W. (1967)Adu. Enzymol. 29, l-32. 7. DENNIS, E. A., AND KENNEDY, E. P. (1972) J. Lipid Res. 13, 263-267. 8. FURLONG, C. E., CIRAKOGLU, C., WILLIS, R. C., AND SANTY, P. A. (1973) Anal. Biochem. 5, 297-311. 9. KANFER, J., AND KENNEDY, E. P. (1964)J. Biol. Chem. 239, 1720-1726. E. P. (1956) J. Biol. Chem. 222, 185 10. KENNEDY, 192. 11. KENNEDY, E. P., AND WEISS, S. B. (1956)5. Biol. Chem. 222, 193-214. 12. LOWRY, 0. M., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. MACHER, B. A., AND MUDD, J. B. (1974) Plant Physiol. 53, 171-175. 14. MARSHALL, M. O., AND KATES, M. (1974) Canad. J. Biochem. 52, 469-482. 15. MARSHALL, M. O., AND KATES, M. (1973) FEBS Lett. 31, 199-202. 16. MOORE, T. S., LORD, J. M., KAGAWA, T., AND BEEVERS, H. (1973) Plant Physiol. 52,50-53. 17. PLOWMAN, K. M. (1972) Enzyme Kinetics, pp. 40-55, McGraw-Hill, New York. 18. PREISS, J., SHEN, L., GREENBERG, E., AND GENTNER, N. (1966) Biochemistry 5, 1833-1845. 19. RAETZ, C. R. M., AND KENNEDY, E. P. (1972) J. Biol. Chem. 247, 2008-2014. 20. RAMASAMRA, T., AND WETTER, L. (1957) Canod. J. Biochem. Physiol. 35, 853-863. W. C. (1969) in Methods in Enzy21. SCHNEIDER, mology, Vol. 14, pp. 684-690, Academic Press, New York. 22. SUMIDA, S., AND MUDD, J. B. (1970) Plant Physiol. 45, 719-722. 23. SUNG, C., AND JOHNSTONE, R. M. (1967) Biothem. J. 105, 497-503. 24. TANAKA, K., TOLBERT, N. E., AND GOHLKE, A. F. (1966) Plant Physiol. 41, 307-312. 25. WEINHOLD, P. A., AND RETHY, V. B. (1974) Biochemistry 13, 5135-5141.
OF BIOCHEMISTRY
Partial
AND
BIOPHYSICS
Purification
and Properties Spinach
BRUCE Department
of Biochemistry
177, 24-30
A. MACHER’
(1976)
of Ethanolamine Leaf AND
J. B. MUDD
and Statewide Air Pollution Research Center, Riverside, Riverside, California 92502 Received
February
Kinase from
University
of California-
20, 1976
Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,OOOg supernatant from spinach leaf. The 100,OOOg supernatant preparation was stable for weeks’, but the partially purified preparation lost half of the ethanolamine kinase activity in lo-14 days at 0-4°C or -20°C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (K,, 42 PM), MgATP (K,, 63 PM), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparentKi of 6.7 mM. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and ophenanthroline. EDTA above 0.9 mM inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 mM) and inhibited at 1.8 mM. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 mM). The ethanolamine kinase and choline kinase activities of the 100,OOOg supernatant preparation could be separated by gel electrophoresis.
al. (2) have characterized the enzyme responsible for the synthesis of CDP-diglyceride in a number of tissues. Marshall and Kates (14, 15) have demonstrated both the CDP-diglyceride dependent synthesis of PS and its decarboxylation in spinach leaf preparations. From these studies, it is apparent that plants are capable of synthesizing PE through the bacterial pathway. Regarding the enzyme catalyzing reactions of the CDP-ethanolamine pathway, a number of studies are also available. Moore et al. (16) have demonstrated phosphatidic acid phosphatase in castor bean. Borkenhagen and Kennedy (3) have reported that preparations from carrot root are capable of catalyzing the synthesis of CDP-ethanolamine from CTP and phosphorylethanolamine. We have previously characterized an enzyme from spinach that catalyzes the formation of PE from DG and CDP-ethanolamine (13). This activity is also found in other plant tissues (13). Marshall and Kates (15) have shown that phosphorylethanolamine can be
Our basic knowledge of the major pathways for phospholipid synthesis in animal tissue comes from the studies of Kennedy and co-workers (3,4,7, 11). Their work has also been important in the elucidation of the major pathways of phospholipid synthesis in bacterial systems (9, 19). There are unique pathways for PE2 synthesis. In bacteria the final steps are formation of PS by way of CDP diglyceride, and then decarboxylation of PS (19): in animals the major pathway for PE synthesis appears to be by way of CDP ethanolanine (11). The enzymes which catalyze a number of these reactions have been studied in plants. Sumida and Mudd (22) and Bahl et 1 Present address: Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824.
* Abbreviations used: DG, 1,2 diacyl-sn-glyceroh EGTA, ethyleneglycol-bis-N,N’-tetraacetic acid; HEPES, N-2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic acid; PA, phosphatidic acid; PE, phosphatidylethanolamine; PS, phosphatidylserine; buffer A, 100 mM Tris-HCl, pH 7.6, 1 rnM Mg Cl,. 24 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
ETHANOLAMINE
KINASE
formed from ethanolamine and ATP in the spinach leaf. However, the data so far reported for ethanolamine kinase activity in spinach do not characterize the enzyme and cannot eliminate the possibility that phosphorylethanolamine formation is catalyzed by choline kinase (24). This report is the outcome of our desire to characterize ethanolamine kinase from spinach and to determine whether the activity is separable from choline kinase. EXPERIMENTAL
PROCEDURE
Materials. Spinach (Spinacia oleracea) was purchased at local stores. [‘4ClEthanolamine and l’4Clcholine were purchased from ICN, Irvine, California. [‘4C]Phosphorylethanolamine and [‘4C1phosphorylcholine were obtained from New England Nuclear, Boston, Massachusetts. Sephadex G-ZOO was a product of Pharmacia, Uppsala, Sweden. DEAE-filter disks were purchased from Whatman, Maidstone, England. Enzyme assays. The ethanolamine kinase assay contained in a volume of 0.55 ml: Tris-chloride, pH 8.5 (110 mM), MgCl, (1.8 mM), ATP (0.9 mM), [“Clethanolamine (0.11 mM, the amount of radioactivity varied with experiment), and enzyme. In assays for choline kinase, [‘Y!]choline replaced [‘“Clethanolamine. Reactions were started by addition of the enzyme, run 15 min at 3o”C, and stopped by placing in a boiling water bath for 5 min. An aliquot (0.1 ml) of the reaction mixture was assayed by a modification of the method of Preiss et al. (181, by applying it to a DEAE-filter disk. The assay method depends on the binding of the phosphorylated product to the filter disk, while the substrate is washed free. One unit of enzyme activity represents the formation of 1 nmol of phosphorylethanolamine per minute per milligram of protein. The products of the assay systems were characterized by paper chromatography in the solvent system, butanol-acetic acid--water, 5:3:2 (21. Other methods. Polyacrylamide gel electrophoresis was carried out in a discontinuous buffer system of Tris-chloride/Tris-glycine (8). Glycerol (final concentration, 8%) was added to the protein sample. The sample was layered over a stacking gel and electrophoresed at a constant current of 3 mA. The gels were sliced into 1.5-mm sections and each section was cut into two pieces. One piece was assayed for ethanolamine kinase activity. The other piece was assayed for choline kinase activity. Protein was determined by the procedure of Lowryet al. (12). Bovine serum albumin was used as a protein standard. All steps during the purification of ethanolamine kinase were carried out at 0-4°C. Data processing. All calculations and fits of data
FROM
SPINACH
25
LEAF
were carried out with programs written for the Wang 720 C programmable calculator. Saturation curves for MgATP, varying MgATP at several ethanolamine concentrations, and varying MgATP at several total magnesium concentrations were fitted to an equation which describes substrate inhibition assuming that substrate combines twice with the enzyme (5). The fit is by the least-squares method to a multiple linear regression analysis to the equation, Ko -1 = vA v
f gK-
+ +,
IA
PI
Saturation curves for ethanolamine at fixed varying MgATP concentrations were analyzed by the weighted fit method to a rectangular hyperbola (61. K,‘s for ethanolamine and MgATP were determined by reporting the slopes vs MgATP concentration and the intercepts vs MgATP concentrations from Fig. 2 (171. RESULTS
Purification
of the Enzyme
Spinach extracts were prepared by homogenizing a given weight of material with twice as much homogenizing medium containing sucrose (250 mM), Trischloride, pH 7.6 (10 mM), and EDTA (1 mM). The leaf tissue was shredded with a razor blade and then homogenized in a Waring Blendor (3-5 s burst). The homogenate was pressed through two layers of cheesecloth and centrifuged for 20 min at 20,OOOg. The resulting supernatant was centrifuged for 60 min at 100,OOOg. The majority of the ethanolamine kinase remained in the 100,OOOg supernatant. The yellow-green supernatant was adjusted to 45% saturation of ammonium sulfate by addition from a saturated solution (pH 7). Precipitated material was removed by centrifugation (20,OOOg for 10 min), and the supernatant was adjusted to 75% ammonium sulfate saturation. After 75 min without stirring, the precipitate then formed was removed by centrifugation and taken up in Buffer A. The dissolved precipitate was thoroughly dialyzed overnight against Buffer A (three changes of 100 vol). The sample was stored at -20°C at least 3 days and not more than 4 days. If the sample was stored frozen for 2 weeks, half the enzyme activity was lost. After the sample was thawed, it was spun briefly in a clinical centrifuge to remove
26
MACHER
AND
the large brown precipitate which had formed. The clarified sample was applied to a Sephadex G-200 column (2 x 100 cm) and eluted with Buffer A. Two major protein areas were separated by the column. Ethanolamine kinase eluted midway between two major protein peaks. Recovery was 50 to 60% of the initial activity. About 50% of the protein remaining after gel chromatography could be removed by adjusting the combined column fractions to 50% saturation with solid ground ammonium sulfate. The enzymatic activity was retained in the supernatant. The enzyme had been purified 60-fold at this stage. Polyacrylamide gel electrophoresis indicated that the preparation was not homogeneous. When the enzyme solution was adjusted to 90% saturation with solid ammonium sulfate and dialyzed, only 20% of the initial activity precipitated. Table I summarizes the steps used in the purification procedure. The molecular weight of ethanolamine kinase was estimated through the use of a standardized Sephadex G-200 column according to the method of Andrews (1). The relationship between the molecular weights of standard proteins (aldolase, ovalbumin, chymotrypsinogen A, and cytochrome c) and K,, was plotted. The molecular weight of ethanolamine kinase was estimated to be 110,000. The 100,OOOg preparation (Table I) was stable to repeated freezing and to prolonged storage at 0-4°C and room temperTABLE PURIFICATION
100,OOOg supernatant First ammonium sulfate tionation Freeze-thaw Chromatography Sephadex 200 Second ammonium sulfate tionation
frac-
Gfrac-
MUDD
ature (90% recovery of initial activity after 20 h at room temperature). The partially purified enzyme lost 50% of its activity during lo-14 days storage at -20°C. The 100,OOOg supernatant preparation lost 60% of its initial activity when it was heated at 45°C for 3 min. Characteristics Enzyme
of the Partially
Purified
Results of a time course experiment showed that with the enzyme at a concentration of 50 pug/ml in the reaction mixture, the rate of phosphorylethanolamine formation was linear for 35 min. The effect of changing protein concentration showed that product formation increased linearly with added protein to a concentration of 115 pg/ml. The partially purified ethanolamine kinase had maximal activity at the highest pH tested for each buffer (HEPES, pH 8.5; Tris-chloride, pH 9.0; and Tris-glycine, pH 9.2; Fig. 1). There appears to be a definite buffer effect. This pH profile is similar to that reported for ethanolamine kinase from Ehrlich Ascites cells (23) and rat liver (ethanolamine kinase II) (25). Although the crude and purified preparations of ethanolamine kinase from spinach were similar in most respects, the two preparations showed different responses to pH changes with Tris-glycine. While the purified preparation had maximal activity with Tris-glycine, the crude preparation showed less activity with Tris-glyI
OF ETHANOLAMINE
KINASE”
Total volume (ml)
Total activity (units)
Specific activity (unitslmg)
Puriti-
protein (mn)
50.0 13.0
66.7 74.7
262.0 65.0
0.25 1.15
1.0 4.6
100 112
12.8 34.0
72.6 40.6
30.5 4.4
2.38 9.23
9.5 36.9
109 61
59.0
35.6
2.4
14.80
59.2
53
Total
Yield
cation (%I
u One hundred grams of spinach leaf were homogenized with 200 ml of homogenizing medium. The homogenate was centrifuged and the enzyme was further purified as described in Experimental Procedure. Specific activity of each fraction was determined from the linear portion of a protein concentration curve.
ETHANOLAMINE
7.5
I
1
I
I 8.0
I 8.5 PH
I 9.0
KINASE
9.5
FIG. 1. The effect of pH on the activity of the partially purified preparation of ethanolamine kinase (0, 110 mM HEPES; A, 110 mM Tris-chloride; 0, 110 mM Tris-glycine). Enzyme activity was determined using standard assay conditions given under Experimental Procedure (40 pg protein assay).
tine than in either HEPES or Tris-chloride.:’ Kinetic Properties The effect of varying the concentration of ethanolamine (7.3 to 164 PM) at several concentrations of MgATP is shown in Fig. 2. When the data were presented in the Lineweaver-Burk form, a set of lines w.hich intersect to the left of the vertical axis was obtained (Fig. 2). This pattern is expected for an enzyme which catalyzes a two substrate reaction via a sequential mechanism (5). The K,, for ethanolamine was 42 PM and for MgATP, 63 PM as computed by the method cited in Experimental Procedure. Substrate inhibition was observed at the highest MgATP concentration (1.36 mM). Ethanolamine was not inhibitory at the concentrations tested. The effect of varying the concentration of MgATP at several concentrations of ethanolamine is shown in Fig. 3a. Double reciprocal plots of initial velocity against various concentrations of MgATP (0.05 to 1.7 mM) at several concentrations of ethanolamine are shown in Fig. 3b. Substrate 3 Data
not presented
FROM
SPINACH
LEAF
27
inhibition was again apparent at high concentrations of MgATP. When MgATP was the variable substrate (0.1 to 3.5 mM), at several levels of total magnesium, an unusual pattern was obtained (Figs. 4a and b). This plot shows that even at low levels of free ATP (line 41, substrate inhibition occurred at high concentrations of MgATP, suggesting that the substrate inhibition observed in Figs. 2 and 3 was due to high MgATP concentrations and not to high levels of free ATP. Therefore, from this plot both free magnesium and MgATP appeared to produce inhibition. The apparent Ki for MgATP was 6.7 mM. Free magnesium was required for maximum activity. Other Properties of Ethanolamine
Kinase
Table II shows the effect of metal-ion chelators and calcium on the ethanolamine kinase activity of dialyzed preparations of the 100,OOOg supernatant. EDTA inhibits the formation of phosphorylethanolamine at concentrations above 0.9 mM. EGTA activated ethanolamine kinase at low concentrations (0.4 and 0.9 mM) and inhibited it at a concentration of 1.8 mM. Ethanolamine kinase was inhibited to the same degree at all concentrations of o-phenanthroline tested. The
FIG. 2. Initial velocity pattern versus ethanolamine concentrations at several concentrations of MgATP. Enzyme activity was determined using standard assay conditions given in Experimental Procedure, except that the initial concentration of magnesium was 1.8 mM, and the concentrations of ATP were: 0.23 mM, 0; 0.43 mM, 0; 0.90 mM, A; 1.80 mM, 0 (30 pg purified enzyme protein assay). Reciprocal plots of the data are presented.
28
MACHER
AND
MUDD
i / /
z z 0’ aI E g Y/ E
I : : :’ /--A AA--. \:9.20 t \- * : : 730 -------a---- .--___CL-.. dimethylethanolamine > choline. ADP also inhibited ethanolamine kinase. This inhibition was most likely produced by MgADP. Since the partially purified ethanolamine kinase retained choline kinase activity, another method was used to separate these enzymes. Ethanolamine kinase and choline kinase were separated by polyacrylamide electrophoresis (Fig. 5). Ethanolamine kinase moved to a position in the gel between two areas of choline kinase activity.
ETHANOLAMINE
KINASE
FROM
From studies with animal cells (23) and tissue (251, it is clear that the protein responsible for ethanolamine phosphorylaTABLE
II
OF METAL-ION ETHANOLAMINE
-~ Additions
Concentration
CHELATORS KINASE”
ON
Nanomoles per minute
Percentage of control
(IIIM)
Pione
-
0.12
100
EDTA
0.4 0.9 1.8
0.13 0.11
108 92 42
0.4 0.9
0.20
166
0.18
1.8
0.09
150 75
0.4
0.10 0.10 0.10
83
0.5 0.9
0.10
83
0.07
1.8 9.1
0.06 0.02
58 50 17
EGTA
o-Phenanthroline
0.9 1.8
c92+
0.05
--
83 83
FIG. 5. Polyacrylamide gel electrophoresis separation of ethanolamine kinase and choline kinase (8% resolving gel, 630 pg of the postmicrosomal supernatant preparation). Preparation of gel slices and assay conditions are given under Experimental Procedure (ethanolamine kinase, 0; choline kinase,
(i Each reaction mixture contained 1.80 mM ATP, and 520 1.80 mM Mg**, 0.11 mM [‘“Clethanolamine, pg of protein of the 100,OOOg supernatant.
l ).
TABLE EFFECT
OF N-METHYL
Additions
DERIVATIVES
0.03 0.13 0.21
0.10 1.00 5.00
Dimethylethanolamine
0.10 1.00
Choline
102 74
reaction mixture of the 100,OOOg
contained supernatant.
72 44
30
0.080 0.032 0.026
64 26
89
0.108 0.076
87 61 31
62 32
20
1.80 mM ATP.
100
0.186
..--____ ’ Each of protein
0.120 0.135
31 65 26 21
101 100 97 59 49
3.64 18.20
KINASE”
Percentage control
0.189
0.10 1.00 1.80
ON ETHANOLAMINE
Ethanolamine (nmolesimin)
45
5.00
5.00
ADP
AND ADP
Disintegrations per minute (x10-:‘) ._______--.
0.05
Monomethylethanolamine
III
OF ETHANOLAMINE
Concentration (mM)
None Ethanolamine
29
LEAF
tion is not the same protein which phosphorylates choline. Sung and Johnstone (23) differentiated ethanolamine kinase and choline kinase, in Ehrlich Ascites cell extracts, on the basis of a number of kinetic and physical properties. For example, ethanolamine kinase was less stable during storage than choline kinase; ethanolamine kinase is more sensitive to thio reagents and calcium than choline kinase; and choline analogs, which stimulate choline kinase, inhibit ethanolamine kinase. Weinhold and Rethy (25) demonstrated two forms of ethanolamine kinase. Etha-
DISCUSSION
EFFECT
SPINACH
1.80 mM Mg”,
0.038 0.122
0.121 0.117
0.072 0.059 0.024
-.-____ 0.03 mM 1°C lethanolamine,
of
21
99 98 95 58 48 20
and 520 kg
-
30
MACHER
nolamine kinase I was physically separated from choline kinase by DEAE-cellulose column chromatography. Ethanolamine kinase II could be differentiated from choline kinase by its stability during storage and its kinetic properties. Comparable studies with plant tissues have not been reported previously. However, some comparisons of our results can be made to the available literature on choline kinase. Ramasarma and Wetter (20) have purified choline kinase from rapeseed. The purified enzyme did not phosphorylate ethanolamine, but no data were presented on ethanolamine phosphorylation by crude enzyme preparations. Choline kinase has also been purified from spinach (24), but no data were presented for ethanolamine phosphorylation. Choline kinase from spinach and rapeseed had pH curves similar to that found in this study for ethanolamine kinase. However, the choline kinases were stable to freezing and required equal amounts of magnesium and ATP for maximum activity. This is not true for ethanolamine kinase from spinach. The partially purified preparation of ethanolamine kinase is unstable to freezing and requires an excess of magnesium for maximum activity, suggesting that the enzyme requires both MgATP and free magnesium. The fact that ethanolamine kinase is inhibited by monomethylethanolamine and dimethylethanolamine but not choline also suggests that ethanolamine kinase and choline kinase are different proteins. The separation of the two activities by disc gel polyacrylamide electrophoresis confirms the theory that the two activities are due to separate proteins. It is interesting to point out that Weinhold and Rethy (25) demonstrated two ethanolamine kinase activities in rat liver, while we have found two choline kinase activities in spinach. With the data presented in this report, it would be premature to propose a reaction mechanism for ethanolamine kinase . However, it is clear that ethanolamine and MgATP produce a sequential initial velocity pattern. It is also important to consider the fact that free magnesium is essential for maximum velocity, suggesting that magnesium plays two roles.
AND
MUDD ACKNOWLEDGMENTS
The authors are indebted to Dr. Paul Cook for his assistance with some of the data analyses and interpretation.
REFERENCES 1. ANDREWS, P. (1964) Biochemistry 91, 222-233. 2. BAHL, J., GUILLOT-SALOMON, T., AND DOUCE, R. (1970) Physiol. Veg. 8, 55-74. 3. BORKENHAGEN, L. F., AND KENNEDY, E. P. (1957) J. Biol. Chem. 227, 951-962. 4. BORKENHAGEN, L. F., KENNEDY, E. P., AND FIELDING, A. (1961)J. Biol. Chem. 236, 28-30. W. W. (1970) in The Enzymes (Boyer, 5. CLELAND, P. D., ed.), Vol. 2, pp. l-65, Academic Press, New York. 6. CLELAND, W. W. (1967)Adu. Enzymol. 29, l-32. 7. DENNIS, E. A., AND KENNEDY, E. P. (1972) J. Lipid Res. 13, 263-267. 8. FURLONG, C. E., CIRAKOGLU, C., WILLIS, R. C., AND SANTY, P. A. (1973) Anal. Biochem. 5, 297-311. 9. KANFER, J., AND KENNEDY, E. P. (1964)J. Biol. Chem. 239, 1720-1726. E. P. (1956) J. Biol. Chem. 222, 185 10. KENNEDY, 192. 11. KENNEDY, E. P., AND WEISS, S. B. (1956)5. Biol. Chem. 222, 193-214. 12. LOWRY, 0. M., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. MACHER, B. A., AND MUDD, J. B. (1974) Plant Physiol. 53, 171-175. 14. MARSHALL, M. O., AND KATES, M. (1974) Canad. J. Biochem. 52, 469-482. 15. MARSHALL, M. O., AND KATES, M. (1973) FEBS Lett. 31, 199-202. 16. MOORE, T. S., LORD, J. M., KAGAWA, T., AND BEEVERS, H. (1973) Plant Physiol. 52,50-53. 17. PLOWMAN, K. M. (1972) Enzyme Kinetics, pp. 40-55, McGraw-Hill, New York. 18. PREISS, J., SHEN, L., GREENBERG, E., AND GENTNER, N. (1966) Biochemistry 5, 1833-1845. 19. RAETZ, C. R. M., AND KENNEDY, E. P. (1972) J. Biol. Chem. 247, 2008-2014. 20. RAMASAMRA, T., AND WETTER, L. (1957) Canod. J. Biochem. Physiol. 35, 853-863. W. C. (1969) in Methods in Enzy21. SCHNEIDER, mology, Vol. 14, pp. 684-690, Academic Press, New York. 22. SUMIDA, S., AND MUDD, J. B. (1970) Plant Physiol. 45, 719-722. 23. SUNG, C., AND JOHNSTONE, R. M. (1967) Biothem. J. 105, 497-503. 24. TANAKA, K., TOLBERT, N. E., AND GOHLKE, A. F. (1966) Plant Physiol. 41, 307-312. 25. WEINHOLD, P. A., AND RETHY, V. B. (1974) Biochemistry 13, 5135-5141.
