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Partial purification and properties of diglyceride kinase from Escherichia coli
201
Biochimica et Biophysics Acta, 441 (1976) 201~-212 @ Elsevier Scientific Publishing Company, Amsterdam
-Printed
in The Netherlands
BBA 56842
PARTIAL PURIFICATION AND PROPERTIES FROM ESCHERKHIA COLI
E. GAYLE
SCHNEIDER
Department (Received
* and EUGENE
OF DIGLYCERIDE
KXNASE
P. KENNEDY
of Biological Chemistry, Warvord Medical School, Boston, Mass. 022 15 (&~..%A.) December
23rd,
1975)
Summary
Diglyceride kinase (diacylglycerol kinase, E.C. 2.7-l.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycero~ or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.
Introduction
membranes of Esc~er~c~iu eoli actively catalyze the synthesis of phosphatidic acid by a direct phospho~lation of diacylglycerol with ATP [l--4]. However, the physiological function of diglyceride kinase is uncertain, since the studies of Chang and Kennedy [2] failed to reveal a metabolically active pool of diacylglycerol in this organism. Other biochemical and genetic studies, reviewed by Cronan and Vagelos [5] and Silbert [6], support the view that the acylation of glycerophosphate is the principal, if not the sole, pathway for the formation of phosphatidic acid in this organism. Diglyceride kinase may have a different substrate and perhaps a different physiological function in vivo. The purpose of this study was to purify and characterize the enzyme and to investigate the physiologic~ role of this activity in E. coli. Some aspects of this work
* Present address: Department of Physiological Medicine, Baltimore. Md. 21205 (U.S.A.).
Chemistry.
the Johns
Hopkins
University
School
of
202
were described in a preliminary report [ 41. In the present communication, details of the purification of diglyceride kinase are provided, together with entirely new information about the enzyme regarding its subcellular localization, stability, apparent size, and activity toward various lipid and nucleotide substrates. Materials
and Methods
[y-32P]ATP and [y-32P]GTP were products of the New England Nuclear Chemical Corp., Boston. DL-[3-3H]serine was obtained from the International Chemical and Nuclear Corp. (Irvine, Calif.); it was purified before use by preparative paper chromatography. Triton X-100 (octylphenoxypolyethoxyethanol) labeled with 14C in the ethoxy chain, was a gift of Rohm and Haas (Philadelphia). Beef heart cardiolipin (diphosphatidylglycerol) and sphingomyelin, products of the Sylvana Co. (Millburn, N.J.), were purified by chromatography on Whatman DEAE-52 cellulose and silicic acid, respectively. Phosphatidylethanolamine was isolated and purified from E. coli. Phosphatidylcholine was isolated from hen egg yolks and purified on alumina by the method of Singleton et al. [ 71. Diacylglycerol was prepared from egg phosphatidylcholine by treatment with a highly purified preparation of phospholipase C from Bacillus cereus (a gift of L.M.G. van Golde, Rijksuniversiteit te Utrecht; The Netherlands). Ceramide was prepared from sphingomyelin by treatment with phospholipase C from Clostridium perfringens (Worthington). Batyl and chimyl alcohols were obtained from Western Chemical Industries (Vancouver). Thiochimyl alcohol was a gift of D.D. Lawson (California Inst. Technol.; Pasadena). Selachyl alcohol was obtained from the California Corp. for Biochemical Research (Los Angeles). 2-Phenylethanol was purchased from Eastman; Dchloramphenico1 was obtained from Parke, Davis and Co. (Ann Arbor). Ficaprenol was a gift of R. Ginnis and J.L. Strominger (Harvard Univ.; Cambridge). Sphingosine was prepared from purified ceramide by the procedure of Karlsson [8]. N-Acetylsphingosine was prepared by Sribney and Kennedy [ 91. N-dodecanoyl2-amino-1-butanol was prepared by acylation of 2-amino-1-butanol (Aldrich Chemical Co., Milwaukee) with dodecanoyl chloride (Eastman) as described by Arora and Radin [lo]. The (lR, 2R) and (IS, 2s) isomers of N-dodecanoyl1-phenyl-2-amino-1,3-propanediol were synthesized by acylation of the amines (gifts of Parke, Davis and Co., Ann Arbor). The purity of the parent amines and the R- and s-acylated derivatives was assessed by measurement of their specific rotations in a Perkin-Elmer 141 Polarimeter at the laboratory of Dr. R.W. Jeanloz, Massachusetts General Hospital. At 23.5”C, (IS, 2s)-1-phenyl-2amino-1,3-propanediol exhibited an (Ye of + 3.31”; the (lR, 2R)-isomer, - 3.32”. The S and R acylated derivatives were found to be + 1.95” and 1.96”) respectively. Triton X-100 was obtained from Rohm and Haas; Cetab (trimethyloctadecylammonium chloride), from Armour and Co. (Chicago). Brij 35 (polyoxyethylene lauryl ether) and Brij 58 (polyoxyethylene cetyl ether) were products of Atlas Chemical Industries (Wilmington). Sodium dodecyl sulfate (SDS) was obtained from Schwartz-Mann; sodium deoxycholate, from Pierce (Rockford, Ill.). Cells of E. coli K-12 were purchased as a frozen paste from Grain Processing Co., Muscatine, Iowa.
203
Assay of diglyceride kinase Diglyceride kinase activity was measured under two similar sets of assay conditions, A and B, as reported previously [ 41. In the present work, System B contained 2 mM lipid substrate. The Tris-phosphate buffer of System A consists of 0.05 M phosphoric acid, neutralized to pH 6.6 with Fisher Tris free base; the concentration of Tris in this buffer is about 0.07 M. The reaction was stopped by the addition of chloroform/methanol (2 : 1, v/v) containing 0.016 M HCl. Activity is expressed as pmol of product per min. Specific activity is expressed as units per mg of enzyme protein. Protein determination Protein was determined by the method of Lowry et al. [ll], using bovine serum albumin as a standard. Triton X-100 was found to interfere in this determination by forming a flocculant yellow precipitate upon addition of the phenol reagent. This precipitate could be removed by centrifugation at 3000 X g for 10 min. The resulting supernatant yielded the same standard curve for bovine serum albumin when up to 5 mg of Triton X-100 were present in the protein sample before addition of reagents. As a further check on the validity of the method for membrane proteins, E. coli cells were grown on tritiated leucine. The specific activity of protein in broken cells and membranes was determined by measuring radioactivity of protein samples and protein concentration by the method of Lowry et al. [ll]. The protein concentration of a Triton X-100 extract of the tritiated membranes could then be determined from the specific activity of protein. For a given Triton extract of membrane proteins, the protein concentration determined by measuring tritium and that determined by the modified Lowry procedure agreed to within a few per cent. Polyacrylamide gel electrophoresis Acrylamide gel electrophoresis under nondenaturing conditions was carried out using the running buffer of pH 8.2 described by Davis [12] as modified by Dowhan et al. [13]. Of two identical gels containing 0.1% Triton X-100, one was stained with Coomassie blue to determine the position of protein bands; the other gel was sliced into 2-mm segments to determine the position of the kinase. Activity was eluted from the slices by an overnight incubation at 3°C in 0.1 ml of lo-fold concentrated Tris-phosphate buffer, pH 6.6, containing 2% Triton X-100. Results Purification of diglyceride kinase Step 1: preparation of cell-free extract. A frozen paste of E. coli K-12 cells (454 g) was suspended in 2 1 of 0.1 M potassium phosphate buffer, pH 6.4, containing 10 mM 2-mercaptoethanol and 1 mM EDTA. All procedures were carried out at 0-4°C unless otherwise stated; all suspensions were done in a Waring blendor. The cells were broken by four passages through a MantonGaulin press; unbroken cells were removed by centrifugation at 5000 X g for 20 min. Step 2: preparation of membrane fraction. The broken cell suspension,
204
which contained about 30 mg/ml of protein, was centrifuged at 44 000 X g for 3 h. The supernatant was discarded; the membrane pellet was suspended in 0.75 1 of buffer I (0.02 M acetic acid and 0.02 M sodium dihydrogen phosphate, neutralized with Fisher Tris-free base to pH 7.0; 10 mM 2mercaptoethanol and 0.1 mM EDTA). Step 3: extraction of membranes with Triton X-100. The membrane suspension was centrifuged at 44 000 X g for 1 h; the supernatant was discarded. The washed membranes were suspended in 0.75 1 of Buffer I also containing 5% (w/v) Triton X-100 for 1.5 h. Unextracted material was removed by centrifugation at 44 000 X g for 1 h. The resulting supernatant, the Triton X-100 extract, contained 2-3 mg protein per ml. Step 4: chromatography on phosphocellulos& The enzyme from Step 3 was adjusted to pH 5.0 with 0.5 M acetic acid; insoluble material was removed by a l-h centrifugation at 44 000 X g. The supernatant, which contained 8090% of the activity, was applied to a phosphocellulose (Whatman P, I) column (5.4 X 7.0 cm) at 25°C in 10 mM sodium citrate buffer, pH 5.0, containing 10 mM 2-mercaptoethanol and 5% Triton X-100. The detergent and mercaptoethanol concentrations were lowered by washing the column with 300 ml of the citrate buffer containing 1 mM 2-mercaptoethanol and 0.5% Triton (Buffer II). After an initial elution with 450 ml of Buffer I containing 0.3 M sodium dihydrogen phosphate (final pH 4.57), the kinase was eluted from the column with 750 ml of Buffer II containing 0.65 M sodium dihydrogen phosphate (final pH, 4.39). The enzyme preparation was concentrated lo-fold by Amicon filtration with a PM-10 membrane at room temperature. It was dialyzed in 10 mM sodium phosphate, pH 7, containing 1 mM 2-mercaptoethanol and 0.5% Triton X-100 (Buffer III). Insoluble material (which contained no kinase) was removed by centrifugation at 44 000 X g for 1 h. The dialyzed, concentrated supernatant contained about 1 mg protein per ml. Step 5: chromatography on hydroxyapatite. The purified enzyme from Step 4 was applied to a hydroxyapatite (Clarkson Hypatite C) column (2.5 X 7.0 cm) at 25°C in Buffer III containing 10 mM 2-mercaptoethanol and 5% Triton X-100. The column was washed with 70 ml Buffer III and eluted with 105 ml of the washing buffer containing 0.1 M sodium phosphate (pH 6.9). Diglyceride kinase activity was eluted with 140 ml Buffer III containing 0.5 M sodium phosphate (pH 6.6). Following dialysis against Buffer III, the enzyme preparation was concentrated 20-fold by Amicon filtration with a PM-10 membrane at room temperature. No material precipitated during this step. The concentrated hydroxyapatite-purified enzyme contained about 4 mg of protein per ml. Step 6: sucrose density gradient centrifugation. The enzyme from Step 5 was applied to sucrose density gradients (5-20% sucrose) in Buffer III containing 1% Triton X-100. The volume of each linear gradient (prepared in 12-ml polyallomer tubes) was 11.5 ml; 0.5 ml of the hydroxyapatite-purified enzyme was applied to each gradient. The gradients were centrifuged at 200 000 X g for 24 h at 20°C in an International B-60 preparative ultracentrifuge equipped with an SB 283 rotor. Fractions of 0.4 ml were collected, and those which contained the peak of the kinase activity were pooled and dialyzed against Buffer II? containing 1% Triton X-100. The sucrose-purified enzyme contained 0.2 mg protein per ml and was purified approx. 600-fold [ 41.
205
Purity of diglyceride kinase The purified kinase was analyzed by polyacrylamide gel electrophoresis in Triton X-100 and found to contain a large number of protein bands (Fig. 1). It was impossible to assign a single protein band to the observed band of kinase activity. Some unusual properties of this enzyme hampered attempts at further purification. Early attempts to purify the crude enzyme on ion exchange resins resulted in poor yields (30-50%) with gradient elution, as compared to high (> 80%) yields with batch elution. Low yields were also obtained upon gel filtration on Sephadex G-200. Control experiments revealed that these low yields could not be explained by instability of the kinase under the conditions of the experiment (temperature, pH, etc.) or by its adsorption on the resins over extended periods of time. Possible separation of the kinase from some essential cofactor or resolution of a multi-enzyme during its purification was considered. As yet, all attempts to recover kinase activity by combining fractions from the columns or adding boiled crude enzyme to the assay system have not been successful. The most purified preparation (Step 6) was examined for the presence of other membrane-bound enzymes, particularly those involved in phospholipid metabolism. Of the enzymes tested (phosphatidylserine decarboxylase, CDPdiglyceride pyrophosphatase, glycerophosphate phosphatidyltransferase, partic-
16
A
8
t TOP B
Distance Migrated
(cm)
1 BOTTOM
Fig. 1. Electrophoresis of diglyceride kinase on acrylamide gel. The Step 6 enzyme (70 !Jg protein and 310 munits of diglyceride kinase) was applied to each of two polyacrylamide gels. the distribution of protein and of diglyceride (assay A) and ceramide (assay B) kinase activities was determined as described in the text. (A) Distribution of kinase activity: DK, diglyceride kinase; CK, ceramidz kinase. Recovery of both activities was 11%. (B) Schematic representation of the distribution of the major protein bands (Coomassie blue stain) in the above preparation.
206
ulate adenosine triphosphatase, and isoprenoid alcohol kinase), none appeared to be associated with the diglyceride kinase. van den Bosch and Vagelos [14] have described a phosphatidate phosphatase (E.C. 3.1.3.4) in E. coli; such an activity could possibly interfere with assay of the kinase by degrading the phosphatidic acid product. However, under conditions of the diglyceride kinase assay with the crude enzyme of Step 3, no loss of 32P from [ 32P]phosphatidic acid was observed when the further synthesis of labeled phosphatidic acid was prevented by addition of large amounts of unlabeled ATP and the incubation was continued for additional periods of time. It would appear that phosphatidate phosphatase is either absent from the kinase preparation or not active under the assay conditions employed. Subcellular localization of diglyceride kinase The distribution of diglyceride kinase activity was examined in fractions of a discontinuous sucrose gradient of disrupted E. coli A-3245 (a K-12 strain), according to the procedure of Schnaitman [ 151, as modified by Hanson and Kennedy [ 161. These studies revealed that the enzyme is localized almost entirely in the inner, cytoplasmic membrane of E. coli. Diglyceride kinase was recovered with a 87% yield in the inner membrane fraction (spec. act. 47 munits/mg) and 3% yield in the outer membrane fraction (specific activity, 6.5 munits/mg). Similarly, phosphatidylserine decarboxylase, a marker enzyme for the inner membrane [17,18] was recovered with a 89% yield in the inner membrane (spec. act. 90.8 munits/mg) and 3% yield in the outer membrane (spec. act. 11.2 munits/mg). The distribution of azide-inhibitable adenosine triphosphatase, which is also localized in the inner membrane [16] closely followed the distribution of diglyceride kinase and phosphatidylserine decarboxylase. Stimulation by phospholipid Phosphorylation of diacylglycerol is stimulated by diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin as shown in Fig. 2. Although all phospholipids tested stimulated the enzyme, diphosphatidylglycerol was by far the best, since much lower concentrations of this lipid were required for half maximal stimulation of the kinase. Phosphorylation of ceramide [4] was also much more efficiently stimulated by diphosphatidylglycerol than by the other phospholipids tested in Fig. 2. Stability The purified enzyme (Step 6) is stable for several months when stored at 0°C in Buffer III containing 1% Triton X-100. The same enzyme preparation lost activity much more rapidly if stored frozen at -20°C. The purified enzyme retains at least 70% of its activity after 24 h at room temperature and 50% of its activity after 12 min at 57°C. As discussed above, many attempts to further purify this enzyme resulted in poor yields. Substrates and reagents in the assay system as well as glycerol, ethylene glycol, sucrose, and bovine serum albumin were tested for their ability to stabilize diglyceride kinase against heat inactivation. Only phospholipids protected the kinase; neither diacylglycerol nor ATP had any effect. Of the phospholipids tested (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylserine, sphingomyelin, phosphatidic acid,
207
PnosFnoLIF/o hwl Fig. 2. Stimulation of phosphorylation of diacylglycerol by phospholipid. The purified kinase (Step 6) was assayed according to assay B 141. except that the phosphoiipid concentration was varied. CL. cardiolipin (diphosphatidy~lycerol)~ PE. phosphatidylethanoiamine: PC, phosphatidylcholine~ SPM, sphingomy&n.
phosphatidylcholine, and phosphatidylethanolamine), all protected the enzyme, although diphosphatidylglycerol and phosphatidylglycerol appeared to be the best stabilizers. Effect of detergents For optimal activity, diglyceride kinase requires a combination of phospholipid such as diphosp~atidylglycerol and a suitable detergent, such as Triton X-100. A number of detergents added to the assay system were found to inhibit the enzyme. The ionic detergents Cetab (trimethyloctadecylammonium chloride) and SDS and the nonionic detergents Brij 35 and Brij 58 inhibited completely; the anionic detergent deoxycholate inhibited only slightly, if at all. Other nucleot~de substrates The ability of other nucleotides and phosphorus-containing compounds to serve as potential substrates for the enzyme was tested by their ability to inhibit phosphorylation of diacylglycerol with [32P]ATP. When 2 mM [32P]ATP was used, addition of 10 mM GTP, ITP, UTP, CTP, PEP, or PPi to the assay had no effect on the level of ~32P]phosphatidic acid synthesized. However, addition of 10 mM AMP, dATP, ADP, and ATP resulted in 30, 40,91, and 78% inhibition, respectively. GTP was tested directly as a possible substrate for diglyceride kinase by incubation of [T-~~P]GTP with enzyme and diacylglycerol (assay A) in the presence or absence of diphosphatidylglycerol. No production of [32P]phosphatidic acid could be detected. The apparent I(, for ATP under assay conditions A and B was found to be 0.18 mM. Phosphorylation of ceramide Preparations of diglyceride kinase from E. coli have been shown to catalyze the phosphorylation of ceramide to ceramide-l-phosphate 141. That both types of substrates are phosphorylated by the same enzyme is suggested by the obser-
208
vation that the two activities remain together throughout the 600-fold purification [4] and upon further fractionation by polyacrylamide gel electrophoresis (Fig. 1). The ratio of specific activities observed in Fig. 1 is identical to that observed throughout the six step purification. In addition, both enzymes exhibit identical labilities to freezing. Further evidence that phosphorylation of diacylglycerol and ceramide is catalyzed by the same enzyme is the previously reported observation of mutual inhibition when both types of substrate are added together [4]. Other lipid substrates
The phosphorylation of ceramide by diglyceride kinase prompted us to test a number of lipids as possible substrates for the enzyme (Table I). No phosphorylation of sphingosine was observed, as reported previously [4]. In addition, no phospho~iation of ficaprenol was detected with purified preparations
TABLE
I
ACTIVITY
OF THE KINASE
TOWARD
LIPID SUBSTRATES
The test substrates (2.0 mM) were incubated with purified enzyme from Step 6 and ATP according to assay B. In one set of experiments the usual chloroform/:! M KC1 partition was performed. In a parallel set of incubations, the reaction was stopped by the addition of 1 mg bovine serum albumin and 5 ml of 5% trichloroacetic acid (0°C) and centrifuged at 10 000 X P for 20 min. The precipitate was washed four times with 5% trichloroacetic acid and resuspended in 2 ml 1% Triton X-100. The suspension was neutralized with Tris-free base to pH 7; a l.O-ml sample was counted. The trichloroacetic acid procedure was used to precipitate those products which might be appreciably soluble in Hz0 (namely the phosphoEylated derivatives of I-phenylethanol and l-octanol) and atso to cheek the chloroform/2 M KC1 partition method for recovery of phosphory~ted products. The two procedures gave identical results. Test substrate ___ 1 Ceramide
,_ _...-_.__I_________
[(2S,
2 ~-acetylsphin~osine 3 Sphingosine
~__
_ _ ._
-._
~~_ __ _ -._
3R)-N-acyl-2-amino-4-octadecene-l.3-diolJ [(ZS,
3R)-~-ac~tyl-Z-amino-4-octadecene-l.3-diolJ
[ (2S, 3R)-2-amino-4-octadecene-1.3-dioll
_._
Activity relative to ceramide 1.0 0.24 0
4 S-Ceramide
analog
[(lS,
2S)-N-dodecanoyl-l-phenyl-2-amino-l,3-propanedioll
2.3
5 R-Ceramide
analog
[(lR,
2R)-N-dodecanoyl-l-phenyl-2-amino-l,3-propanediol
0.27
6 ~-dodec~oyl-2-amino-l-butanol
0.73
7 2-Phenylethanol
0
8 S-1.2-Diacylglycerol
3.1
9 Batyl alcohol
0.10
10 Chimyl
alcohol
11 Thiochimyl 12 Selachyl 13 1-octanol
[S-l-octadecylglycerol~ [S-l-hexadecylgiycerol]
alcohol
alcohol
0.18
[S-l-hexadecyl-1-thioglvcemll
[S-1-(9-octadecenyl)-glycerol]
__..~.
I-______ ..__.--_-.---.-___
0.17 0.23 0
209
of the kinase (data not shown). In ceramide analogs, replacement of a portion of the ceramide long chain base (last 15 carbons) with a phenyl group (No. 4, Table I) or even with a methyl group (No. 6) does not affect the reactivity of the substrate, as long as the 2-amino group is acylated by a long-chain fatty acid. The high degree of phosphorylation of N-dodecanoyl-2-amino-l-butanol (No. 6), which has no hydroxyl at carbon 3, further supports the observation that ceramides are phosphorylated at C-l, the location of the primary hydroxyl timUP [41. The purified enzyme was found to catalyze the phosphorylation of both S- and R-isomers of a ceramide analog (Nos. 4 and 5, Table I). The s-isomer was ten times more active than the R. Substrate saturation curves for both isomers followed typical Michaelis-Menten kinetics with K, = 0.3 mM, V = 2 units/mg protein for the s analog and K, = 0.6 mM, V = 0.2 units/mg for the R-analog. Addition of R-isomer to the assay system resulted in inhibition of phosphorylation of the S-isomer. Moreover, mutual inhibition was observed between diacylglycerol and each isomer. However, D-chloramphenicol, which is structurally similar to the R-ceramide analog, had no effect on the phosphorylation of either diacylglycerol or ceramide. The parent amines of these ceramide analogs were inactive, as would be expected from the inactivity of free sphingosine. The phosphorylation of these compounds, like that of ceramide, was highly dependent on the addition of diphosphatidylglycerol to the assay system. A further illustration of the broad substrate specificity of diglyceride kinase is its apparent ability to catalyze the phosphorylation of even the detergent Triton X-100. Incubation of the purified enzyme with [‘4C]Triton X-100, [“‘PI ATP, and phospholipid resulted in the synthesis of an alkali-stable [ 14C, 32P]lipid, which was partially purified and tentatively identified as [ 14C]Triton X-l 00 [32P] phosphate. Diacyiglycerol severely inhibited phosphorylation of the detergent, suggesting that it is the diglyceride kinase which is responsible for the observed activity. Under normal assay conditions, the phosphorylation of detergent is not detected, since diacylglycerol is about 300 times more active than Triton X-100. Apparent
size of diglyceride
kinase
When the step 5 enzyme was chromatographed on Sephadex G-200 (in 50% yield), its apparent molecular weight relative to globular protein standards was found to be about 240 000. This is in contrast to a value of 120 000 determined for the same enzyme preparation by sedimentation in sucrose gradients. This anomaly has also been observed for two other solubilized membrane enzymes of E. coli, the phosphatidylserine decarboxylase [13] and the CDPdiglyceride pyrophosphatase (Raetz, C.R.H. and Kennedy, E.P., in preparation). The discrepancy may result from the binding of large amounts of Triton X-100 to the solubilized enzyme, thus giving it a lower buoyant density than the globular protein standards, which bind very little detergent [ 133. A similar behavior was earlier noted by Meunier et al. [ 191 for the cholinergic receptor of Electrophorus. Search
for additional
endogenous
Since the physiological
function
lipid substrates
of the diglyceride
kinase of E. coli is uncer-
210
tain and since this enzyme efficiently catalyzes the phosphorylation of a wide variety of lipid substrates in vitro, we searched in E. coli for possible endogenous substrates in addition to diacylgly~erol. Neither ~eramide-like substances nor sphingolipids were detected in E. co& and no evidence was found for the existence of macromolecular substrates precipitable by trichloroacetic acid. Discussion The purification of E. coii diglyceride kinase described here represents the most extensive purification to date of this membrane-bound enzyme. During the course of this work, we became aware of a report by Horvath and Pieringer [ 201 describing a partial purification of E. coli diglyceride kinase to a watersoluble, detergent-free state. The partially purified enzyme described by these workers possessed a specific activity (2 munits/mg) less than that of their Triton X-loo-extracted enzyme (17 munits/mg); the harsh treatments (ammonium sulfate precipitation followed by alkaline hydrolysis at pH 11.5) during the purification presumably led to inactivation of the enzyme. More significant was the observation that the purified kinase emerged very early from Biogel 200 and DEAE-cellulose columns and migrated very slowly on polyacrylamide gels. These obse~ations suggest that the diglyceride kinase obtained by this procedure may be aggregated with other membrane proteins. The purified kinase described in the present report does not appear to be aggregated; it is observed to move well into polyacrylamide gels and to display a discrete peak of kinase activity, while the bulk protein is displayed throughout the gel (Fig. 1). The detergent Triton X-100 was necessary throughout all stages of the purification; the enzyme could not be eluted from ion exchange resins without it. On Sephadex G-ZOO or sucrose gradients in the absence of detergent, the kinase appeared to aggregate with other membrane proteins, although enzyme activity was often recovered in good yield. The apparent K, of 0.02 mM for diacylglycerol [4] and 0.18 mM for ATP agree well with the values obtained by Pieringer and Kunnes [I] for the membrane-bound enzyme. However, the apparent diglyceride kinase activity in vesicles of E. coli described by Weissbach et al. [3] is reported to have a K, for ATP of 1.3 - lO+ mM, a much lower value than that observed in assay systems of the enzyme containing exogenous diacylglycerol and detergent. Moreover, the enzyme described by these workers is reported to be dependent on Mn”, to use GTP as well as ATP as nucleotide substrate, and to form [32P]ATP from [ 3zP]phosphatidic acid and ADP. Purified diglyceride kinase assayed under our conditions did not utilize GTP as a substrate; Pieringer and Kunnes [l J have reported Mg2+ to be more effective than Mn*+ as a metal ion co-factor. Finally, apparent reversiblity of the kinase reaction is not observed with our purified enzyme preparation. Upon addition of large amounts of unlabeled ATP, no loss of 32P from [ 32P]phosphatidic acid was detected; if the reaction were freely reversible, the specific activity of phosphatidic acid would decrease, as a reflection of the decrease in specific activity of the ATP pool, with which it is in equilibrium. The studies of Weissbach et al. [3] were carried out on unfractionated vesicles, and these workers offered no strong evidence that a single enzyme was re-
211
sponsible for the reaction studied. A recent report by Thomas [Zl] suggests that the previous observations in membrane vesicles [3,22,23] may be accounted for by the concerted action of two enzyines, succinyl-CoA synthetase and diglyceride kinase. The phosphorylation of ceramide by “diglyceride kinase” preparations from E. coli [4] represents the first report of such an enzyme activity in any organism. Neither sphingosine nor ficaprenol are substrates for diglyceride kinase. Thus, the activity described here which catalyzes the phosphorylation of ceramides is distinctly different from the sphingosine kinase reported in animal tissues [24-261 and in Tetruhymena [27] and from the isoprenoid alcohol kinase reported in Staphylococcus [28]. Keenan [27] reported that sphingosine kinase of Tetrahymena requires a substrate with a free amino group; the enzyme is inactive toward N-acylated or N-acetylsphingosine. In contrast, the diglyceride kinase requires an acylated substrate, as shown by its activity toward even N-acetylsphingosine but not toward sphingosine. Despite the ability of diglyceride kinase to utilize a wide variety of lipid substrates in vitro, no endogenous substrates in addition to diacylglycerol have as yet been detected in E. coli. Although Chang and Kennedy [2] were unable to find a pool of metabolically active diacylglycerol in E. coli, it is possible that such a pool exists smaller than could be detected by the most sensitive of their methods. Their results, however, together with the genetic studies already cited, make it very unlikely that the phosphorylation of diacylglycerol is a major route for the synthesis of phosphatidic acid in this organism. It is possible, nevertheless, that diacylglycerols arising during the metabolism of glycerophosphatides formed via the glycerophosphate pathway may be phosphorylated by this enzyme. Acknowledgements We are indebted to W. Dowhan, C.R.H. Raetz, and R. Ginnis for assay of phosphatidylserine decarboxylase, CDPdiglyceride pyrophosphatase and glycerophosphate phosphatidyltransferase, and isoprenoid alcohol kinase, respectively. We thank R. Hanson for fractionation of disrupted E. co/i on discontinuous sucrose gradients and for assay of azide-inhibitable adenosine triphosphatase. This research was supported by grants from the National Institute of General Medical Sciences, National Institutes of Health, GM 19822-15; GM 18731-03; and GM 13952-09. E.G.S. was a Predoctoral Fellow of the National Institute of General Medical Sciences GM 00451. The work described here forms part of a dissertation that was submitted to the Faculty of Arts and Sciences of Harvard University in partial fulfillment of the requirements for the Ph.D. degree. References 1
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Biochimica et Biophysics Acta, 441 (1976) 201~-212 @ Elsevier Scientific Publishing Company, Amsterdam
-Printed
in The Netherlands
BBA 56842
PARTIAL PURIFICATION AND PROPERTIES FROM ESCHERKHIA COLI
E. GAYLE
SCHNEIDER
Department (Received
* and EUGENE
OF DIGLYCERIDE
KXNASE
P. KENNEDY
of Biological Chemistry, Warvord Medical School, Boston, Mass. 022 15 (&~..%A.) December
23rd,
1975)
Summary
Diglyceride kinase (diacylglycerol kinase, E.C. 2.7-l.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycero~ or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.
Introduction
membranes of Esc~er~c~iu eoli actively catalyze the synthesis of phosphatidic acid by a direct phospho~lation of diacylglycerol with ATP [l--4]. However, the physiological function of diglyceride kinase is uncertain, since the studies of Chang and Kennedy [2] failed to reveal a metabolically active pool of diacylglycerol in this organism. Other biochemical and genetic studies, reviewed by Cronan and Vagelos [5] and Silbert [6], support the view that the acylation of glycerophosphate is the principal, if not the sole, pathway for the formation of phosphatidic acid in this organism. Diglyceride kinase may have a different substrate and perhaps a different physiological function in vivo. The purpose of this study was to purify and characterize the enzyme and to investigate the physiologic~ role of this activity in E. coli. Some aspects of this work
* Present address: Department of Physiological Medicine, Baltimore. Md. 21205 (U.S.A.).
Chemistry.
the Johns
Hopkins
University
School
of
202
were described in a preliminary report [ 41. In the present communication, details of the purification of diglyceride kinase are provided, together with entirely new information about the enzyme regarding its subcellular localization, stability, apparent size, and activity toward various lipid and nucleotide substrates. Materials
and Methods
[y-32P]ATP and [y-32P]GTP were products of the New England Nuclear Chemical Corp., Boston. DL-[3-3H]serine was obtained from the International Chemical and Nuclear Corp. (Irvine, Calif.); it was purified before use by preparative paper chromatography. Triton X-100 (octylphenoxypolyethoxyethanol) labeled with 14C in the ethoxy chain, was a gift of Rohm and Haas (Philadelphia). Beef heart cardiolipin (diphosphatidylglycerol) and sphingomyelin, products of the Sylvana Co. (Millburn, N.J.), were purified by chromatography on Whatman DEAE-52 cellulose and silicic acid, respectively. Phosphatidylethanolamine was isolated and purified from E. coli. Phosphatidylcholine was isolated from hen egg yolks and purified on alumina by the method of Singleton et al. [ 71. Diacylglycerol was prepared from egg phosphatidylcholine by treatment with a highly purified preparation of phospholipase C from Bacillus cereus (a gift of L.M.G. van Golde, Rijksuniversiteit te Utrecht; The Netherlands). Ceramide was prepared from sphingomyelin by treatment with phospholipase C from Clostridium perfringens (Worthington). Batyl and chimyl alcohols were obtained from Western Chemical Industries (Vancouver). Thiochimyl alcohol was a gift of D.D. Lawson (California Inst. Technol.; Pasadena). Selachyl alcohol was obtained from the California Corp. for Biochemical Research (Los Angeles). 2-Phenylethanol was purchased from Eastman; Dchloramphenico1 was obtained from Parke, Davis and Co. (Ann Arbor). Ficaprenol was a gift of R. Ginnis and J.L. Strominger (Harvard Univ.; Cambridge). Sphingosine was prepared from purified ceramide by the procedure of Karlsson [8]. N-Acetylsphingosine was prepared by Sribney and Kennedy [ 91. N-dodecanoyl2-amino-1-butanol was prepared by acylation of 2-amino-1-butanol (Aldrich Chemical Co., Milwaukee) with dodecanoyl chloride (Eastman) as described by Arora and Radin [lo]. The (lR, 2R) and (IS, 2s) isomers of N-dodecanoyl1-phenyl-2-amino-1,3-propanediol were synthesized by acylation of the amines (gifts of Parke, Davis and Co., Ann Arbor). The purity of the parent amines and the R- and s-acylated derivatives was assessed by measurement of their specific rotations in a Perkin-Elmer 141 Polarimeter at the laboratory of Dr. R.W. Jeanloz, Massachusetts General Hospital. At 23.5”C, (IS, 2s)-1-phenyl-2amino-1,3-propanediol exhibited an (Ye of + 3.31”; the (lR, 2R)-isomer, - 3.32”. The S and R acylated derivatives were found to be + 1.95” and 1.96”) respectively. Triton X-100 was obtained from Rohm and Haas; Cetab (trimethyloctadecylammonium chloride), from Armour and Co. (Chicago). Brij 35 (polyoxyethylene lauryl ether) and Brij 58 (polyoxyethylene cetyl ether) were products of Atlas Chemical Industries (Wilmington). Sodium dodecyl sulfate (SDS) was obtained from Schwartz-Mann; sodium deoxycholate, from Pierce (Rockford, Ill.). Cells of E. coli K-12 were purchased as a frozen paste from Grain Processing Co., Muscatine, Iowa.
203
Assay of diglyceride kinase Diglyceride kinase activity was measured under two similar sets of assay conditions, A and B, as reported previously [ 41. In the present work, System B contained 2 mM lipid substrate. The Tris-phosphate buffer of System A consists of 0.05 M phosphoric acid, neutralized to pH 6.6 with Fisher Tris free base; the concentration of Tris in this buffer is about 0.07 M. The reaction was stopped by the addition of chloroform/methanol (2 : 1, v/v) containing 0.016 M HCl. Activity is expressed as pmol of product per min. Specific activity is expressed as units per mg of enzyme protein. Protein determination Protein was determined by the method of Lowry et al. [ll], using bovine serum albumin as a standard. Triton X-100 was found to interfere in this determination by forming a flocculant yellow precipitate upon addition of the phenol reagent. This precipitate could be removed by centrifugation at 3000 X g for 10 min. The resulting supernatant yielded the same standard curve for bovine serum albumin when up to 5 mg of Triton X-100 were present in the protein sample before addition of reagents. As a further check on the validity of the method for membrane proteins, E. coli cells were grown on tritiated leucine. The specific activity of protein in broken cells and membranes was determined by measuring radioactivity of protein samples and protein concentration by the method of Lowry et al. [ll]. The protein concentration of a Triton X-100 extract of the tritiated membranes could then be determined from the specific activity of protein. For a given Triton extract of membrane proteins, the protein concentration determined by measuring tritium and that determined by the modified Lowry procedure agreed to within a few per cent. Polyacrylamide gel electrophoresis Acrylamide gel electrophoresis under nondenaturing conditions was carried out using the running buffer of pH 8.2 described by Davis [12] as modified by Dowhan et al. [13]. Of two identical gels containing 0.1% Triton X-100, one was stained with Coomassie blue to determine the position of protein bands; the other gel was sliced into 2-mm segments to determine the position of the kinase. Activity was eluted from the slices by an overnight incubation at 3°C in 0.1 ml of lo-fold concentrated Tris-phosphate buffer, pH 6.6, containing 2% Triton X-100. Results Purification of diglyceride kinase Step 1: preparation of cell-free extract. A frozen paste of E. coli K-12 cells (454 g) was suspended in 2 1 of 0.1 M potassium phosphate buffer, pH 6.4, containing 10 mM 2-mercaptoethanol and 1 mM EDTA. All procedures were carried out at 0-4°C unless otherwise stated; all suspensions were done in a Waring blendor. The cells were broken by four passages through a MantonGaulin press; unbroken cells were removed by centrifugation at 5000 X g for 20 min. Step 2: preparation of membrane fraction. The broken cell suspension,
204
which contained about 30 mg/ml of protein, was centrifuged at 44 000 X g for 3 h. The supernatant was discarded; the membrane pellet was suspended in 0.75 1 of buffer I (0.02 M acetic acid and 0.02 M sodium dihydrogen phosphate, neutralized with Fisher Tris-free base to pH 7.0; 10 mM 2mercaptoethanol and 0.1 mM EDTA). Step 3: extraction of membranes with Triton X-100. The membrane suspension was centrifuged at 44 000 X g for 1 h; the supernatant was discarded. The washed membranes were suspended in 0.75 1 of Buffer I also containing 5% (w/v) Triton X-100 for 1.5 h. Unextracted material was removed by centrifugation at 44 000 X g for 1 h. The resulting supernatant, the Triton X-100 extract, contained 2-3 mg protein per ml. Step 4: chromatography on phosphocellulos& The enzyme from Step 3 was adjusted to pH 5.0 with 0.5 M acetic acid; insoluble material was removed by a l-h centrifugation at 44 000 X g. The supernatant, which contained 8090% of the activity, was applied to a phosphocellulose (Whatman P, I) column (5.4 X 7.0 cm) at 25°C in 10 mM sodium citrate buffer, pH 5.0, containing 10 mM 2-mercaptoethanol and 5% Triton X-100. The detergent and mercaptoethanol concentrations were lowered by washing the column with 300 ml of the citrate buffer containing 1 mM 2-mercaptoethanol and 0.5% Triton (Buffer II). After an initial elution with 450 ml of Buffer I containing 0.3 M sodium dihydrogen phosphate (final pH 4.57), the kinase was eluted from the column with 750 ml of Buffer II containing 0.65 M sodium dihydrogen phosphate (final pH, 4.39). The enzyme preparation was concentrated lo-fold by Amicon filtration with a PM-10 membrane at room temperature. It was dialyzed in 10 mM sodium phosphate, pH 7, containing 1 mM 2-mercaptoethanol and 0.5% Triton X-100 (Buffer III). Insoluble material (which contained no kinase) was removed by centrifugation at 44 000 X g for 1 h. The dialyzed, concentrated supernatant contained about 1 mg protein per ml. Step 5: chromatography on hydroxyapatite. The purified enzyme from Step 4 was applied to a hydroxyapatite (Clarkson Hypatite C) column (2.5 X 7.0 cm) at 25°C in Buffer III containing 10 mM 2-mercaptoethanol and 5% Triton X-100. The column was washed with 70 ml Buffer III and eluted with 105 ml of the washing buffer containing 0.1 M sodium phosphate (pH 6.9). Diglyceride kinase activity was eluted with 140 ml Buffer III containing 0.5 M sodium phosphate (pH 6.6). Following dialysis against Buffer III, the enzyme preparation was concentrated 20-fold by Amicon filtration with a PM-10 membrane at room temperature. No material precipitated during this step. The concentrated hydroxyapatite-purified enzyme contained about 4 mg of protein per ml. Step 6: sucrose density gradient centrifugation. The enzyme from Step 5 was applied to sucrose density gradients (5-20% sucrose) in Buffer III containing 1% Triton X-100. The volume of each linear gradient (prepared in 12-ml polyallomer tubes) was 11.5 ml; 0.5 ml of the hydroxyapatite-purified enzyme was applied to each gradient. The gradients were centrifuged at 200 000 X g for 24 h at 20°C in an International B-60 preparative ultracentrifuge equipped with an SB 283 rotor. Fractions of 0.4 ml were collected, and those which contained the peak of the kinase activity were pooled and dialyzed against Buffer II? containing 1% Triton X-100. The sucrose-purified enzyme contained 0.2 mg protein per ml and was purified approx. 600-fold [ 41.
205
Purity of diglyceride kinase The purified kinase was analyzed by polyacrylamide gel electrophoresis in Triton X-100 and found to contain a large number of protein bands (Fig. 1). It was impossible to assign a single protein band to the observed band of kinase activity. Some unusual properties of this enzyme hampered attempts at further purification. Early attempts to purify the crude enzyme on ion exchange resins resulted in poor yields (30-50%) with gradient elution, as compared to high (> 80%) yields with batch elution. Low yields were also obtained upon gel filtration on Sephadex G-200. Control experiments revealed that these low yields could not be explained by instability of the kinase under the conditions of the experiment (temperature, pH, etc.) or by its adsorption on the resins over extended periods of time. Possible separation of the kinase from some essential cofactor or resolution of a multi-enzyme during its purification was considered. As yet, all attempts to recover kinase activity by combining fractions from the columns or adding boiled crude enzyme to the assay system have not been successful. The most purified preparation (Step 6) was examined for the presence of other membrane-bound enzymes, particularly those involved in phospholipid metabolism. Of the enzymes tested (phosphatidylserine decarboxylase, CDPdiglyceride pyrophosphatase, glycerophosphate phosphatidyltransferase, partic-
16
A
8
t TOP B
Distance Migrated
(cm)
1 BOTTOM
Fig. 1. Electrophoresis of diglyceride kinase on acrylamide gel. The Step 6 enzyme (70 !Jg protein and 310 munits of diglyceride kinase) was applied to each of two polyacrylamide gels. the distribution of protein and of diglyceride (assay A) and ceramide (assay B) kinase activities was determined as described in the text. (A) Distribution of kinase activity: DK, diglyceride kinase; CK, ceramidz kinase. Recovery of both activities was 11%. (B) Schematic representation of the distribution of the major protein bands (Coomassie blue stain) in the above preparation.
206
ulate adenosine triphosphatase, and isoprenoid alcohol kinase), none appeared to be associated with the diglyceride kinase. van den Bosch and Vagelos [14] have described a phosphatidate phosphatase (E.C. 3.1.3.4) in E. coli; such an activity could possibly interfere with assay of the kinase by degrading the phosphatidic acid product. However, under conditions of the diglyceride kinase assay with the crude enzyme of Step 3, no loss of 32P from [ 32P]phosphatidic acid was observed when the further synthesis of labeled phosphatidic acid was prevented by addition of large amounts of unlabeled ATP and the incubation was continued for additional periods of time. It would appear that phosphatidate phosphatase is either absent from the kinase preparation or not active under the assay conditions employed. Subcellular localization of diglyceride kinase The distribution of diglyceride kinase activity was examined in fractions of a discontinuous sucrose gradient of disrupted E. coli A-3245 (a K-12 strain), according to the procedure of Schnaitman [ 151, as modified by Hanson and Kennedy [ 161. These studies revealed that the enzyme is localized almost entirely in the inner, cytoplasmic membrane of E. coli. Diglyceride kinase was recovered with a 87% yield in the inner membrane fraction (spec. act. 47 munits/mg) and 3% yield in the outer membrane fraction (specific activity, 6.5 munits/mg). Similarly, phosphatidylserine decarboxylase, a marker enzyme for the inner membrane [17,18] was recovered with a 89% yield in the inner membrane (spec. act. 90.8 munits/mg) and 3% yield in the outer membrane (spec. act. 11.2 munits/mg). The distribution of azide-inhibitable adenosine triphosphatase, which is also localized in the inner membrane [16] closely followed the distribution of diglyceride kinase and phosphatidylserine decarboxylase. Stimulation by phospholipid Phosphorylation of diacylglycerol is stimulated by diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin as shown in Fig. 2. Although all phospholipids tested stimulated the enzyme, diphosphatidylglycerol was by far the best, since much lower concentrations of this lipid were required for half maximal stimulation of the kinase. Phosphorylation of ceramide [4] was also much more efficiently stimulated by diphosphatidylglycerol than by the other phospholipids tested in Fig. 2. Stability The purified enzyme (Step 6) is stable for several months when stored at 0°C in Buffer III containing 1% Triton X-100. The same enzyme preparation lost activity much more rapidly if stored frozen at -20°C. The purified enzyme retains at least 70% of its activity after 24 h at room temperature and 50% of its activity after 12 min at 57°C. As discussed above, many attempts to further purify this enzyme resulted in poor yields. Substrates and reagents in the assay system as well as glycerol, ethylene glycol, sucrose, and bovine serum albumin were tested for their ability to stabilize diglyceride kinase against heat inactivation. Only phospholipids protected the kinase; neither diacylglycerol nor ATP had any effect. Of the phospholipids tested (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylserine, sphingomyelin, phosphatidic acid,
207
PnosFnoLIF/o hwl Fig. 2. Stimulation of phosphorylation of diacylglycerol by phospholipid. The purified kinase (Step 6) was assayed according to assay B 141. except that the phosphoiipid concentration was varied. CL. cardiolipin (diphosphatidy~lycerol)~ PE. phosphatidylethanoiamine: PC, phosphatidylcholine~ SPM, sphingomy&n.
phosphatidylcholine, and phosphatidylethanolamine), all protected the enzyme, although diphosphatidylglycerol and phosphatidylglycerol appeared to be the best stabilizers. Effect of detergents For optimal activity, diglyceride kinase requires a combination of phospholipid such as diphosp~atidylglycerol and a suitable detergent, such as Triton X-100. A number of detergents added to the assay system were found to inhibit the enzyme. The ionic detergents Cetab (trimethyloctadecylammonium chloride) and SDS and the nonionic detergents Brij 35 and Brij 58 inhibited completely; the anionic detergent deoxycholate inhibited only slightly, if at all. Other nucleot~de substrates The ability of other nucleotides and phosphorus-containing compounds to serve as potential substrates for the enzyme was tested by their ability to inhibit phosphorylation of diacylglycerol with [32P]ATP. When 2 mM [32P]ATP was used, addition of 10 mM GTP, ITP, UTP, CTP, PEP, or PPi to the assay had no effect on the level of ~32P]phosphatidic acid synthesized. However, addition of 10 mM AMP, dATP, ADP, and ATP resulted in 30, 40,91, and 78% inhibition, respectively. GTP was tested directly as a possible substrate for diglyceride kinase by incubation of [T-~~P]GTP with enzyme and diacylglycerol (assay A) in the presence or absence of diphosphatidylglycerol. No production of [32P]phosphatidic acid could be detected. The apparent I(, for ATP under assay conditions A and B was found to be 0.18 mM. Phosphorylation of ceramide Preparations of diglyceride kinase from E. coli have been shown to catalyze the phosphorylation of ceramide to ceramide-l-phosphate 141. That both types of substrates are phosphorylated by the same enzyme is suggested by the obser-
208
vation that the two activities remain together throughout the 600-fold purification [4] and upon further fractionation by polyacrylamide gel electrophoresis (Fig. 1). The ratio of specific activities observed in Fig. 1 is identical to that observed throughout the six step purification. In addition, both enzymes exhibit identical labilities to freezing. Further evidence that phosphorylation of diacylglycerol and ceramide is catalyzed by the same enzyme is the previously reported observation of mutual inhibition when both types of substrate are added together [4]. Other lipid substrates
The phosphorylation of ceramide by diglyceride kinase prompted us to test a number of lipids as possible substrates for the enzyme (Table I). No phosphorylation of sphingosine was observed, as reported previously [4]. In addition, no phospho~iation of ficaprenol was detected with purified preparations
TABLE
I
ACTIVITY
OF THE KINASE
TOWARD
LIPID SUBSTRATES
The test substrates (2.0 mM) were incubated with purified enzyme from Step 6 and ATP according to assay B. In one set of experiments the usual chloroform/:! M KC1 partition was performed. In a parallel set of incubations, the reaction was stopped by the addition of 1 mg bovine serum albumin and 5 ml of 5% trichloroacetic acid (0°C) and centrifuged at 10 000 X P for 20 min. The precipitate was washed four times with 5% trichloroacetic acid and resuspended in 2 ml 1% Triton X-100. The suspension was neutralized with Tris-free base to pH 7; a l.O-ml sample was counted. The trichloroacetic acid procedure was used to precipitate those products which might be appreciably soluble in Hz0 (namely the phosphoEylated derivatives of I-phenylethanol and l-octanol) and atso to cheek the chloroform/2 M KC1 partition method for recovery of phosphory~ted products. The two procedures gave identical results. Test substrate ___ 1 Ceramide
,_ _...-_.__I_________
[(2S,
2 ~-acetylsphin~osine 3 Sphingosine
~__
_ _ ._
-._
~~_ __ _ -._
3R)-N-acyl-2-amino-4-octadecene-l.3-diolJ [(ZS,
3R)-~-ac~tyl-Z-amino-4-octadecene-l.3-diolJ
[ (2S, 3R)-2-amino-4-octadecene-1.3-dioll
_._
Activity relative to ceramide 1.0 0.24 0
4 S-Ceramide
analog
[(lS,
2S)-N-dodecanoyl-l-phenyl-2-amino-l,3-propanedioll
2.3
5 R-Ceramide
analog
[(lR,
2R)-N-dodecanoyl-l-phenyl-2-amino-l,3-propanediol
0.27
6 ~-dodec~oyl-2-amino-l-butanol
0.73
7 2-Phenylethanol
0
8 S-1.2-Diacylglycerol
3.1
9 Batyl alcohol
0.10
10 Chimyl
alcohol
11 Thiochimyl 12 Selachyl 13 1-octanol
[S-l-octadecylglycerol~ [S-l-hexadecylgiycerol]
alcohol
alcohol
0.18
[S-l-hexadecyl-1-thioglvcemll
[S-1-(9-octadecenyl)-glycerol]
__..~.
I-______ ..__.--_-.---.-___
0.17 0.23 0
209
of the kinase (data not shown). In ceramide analogs, replacement of a portion of the ceramide long chain base (last 15 carbons) with a phenyl group (No. 4, Table I) or even with a methyl group (No. 6) does not affect the reactivity of the substrate, as long as the 2-amino group is acylated by a long-chain fatty acid. The high degree of phosphorylation of N-dodecanoyl-2-amino-l-butanol (No. 6), which has no hydroxyl at carbon 3, further supports the observation that ceramides are phosphorylated at C-l, the location of the primary hydroxyl timUP [41. The purified enzyme was found to catalyze the phosphorylation of both S- and R-isomers of a ceramide analog (Nos. 4 and 5, Table I). The s-isomer was ten times more active than the R. Substrate saturation curves for both isomers followed typical Michaelis-Menten kinetics with K, = 0.3 mM, V = 2 units/mg protein for the s analog and K, = 0.6 mM, V = 0.2 units/mg for the R-analog. Addition of R-isomer to the assay system resulted in inhibition of phosphorylation of the S-isomer. Moreover, mutual inhibition was observed between diacylglycerol and each isomer. However, D-chloramphenicol, which is structurally similar to the R-ceramide analog, had no effect on the phosphorylation of either diacylglycerol or ceramide. The parent amines of these ceramide analogs were inactive, as would be expected from the inactivity of free sphingosine. The phosphorylation of these compounds, like that of ceramide, was highly dependent on the addition of diphosphatidylglycerol to the assay system. A further illustration of the broad substrate specificity of diglyceride kinase is its apparent ability to catalyze the phosphorylation of even the detergent Triton X-100. Incubation of the purified enzyme with [‘4C]Triton X-100, [“‘PI ATP, and phospholipid resulted in the synthesis of an alkali-stable [ 14C, 32P]lipid, which was partially purified and tentatively identified as [ 14C]Triton X-l 00 [32P] phosphate. Diacyiglycerol severely inhibited phosphorylation of the detergent, suggesting that it is the diglyceride kinase which is responsible for the observed activity. Under normal assay conditions, the phosphorylation of detergent is not detected, since diacylglycerol is about 300 times more active than Triton X-100. Apparent
size of diglyceride
kinase
When the step 5 enzyme was chromatographed on Sephadex G-200 (in 50% yield), its apparent molecular weight relative to globular protein standards was found to be about 240 000. This is in contrast to a value of 120 000 determined for the same enzyme preparation by sedimentation in sucrose gradients. This anomaly has also been observed for two other solubilized membrane enzymes of E. coli, the phosphatidylserine decarboxylase [13] and the CDPdiglyceride pyrophosphatase (Raetz, C.R.H. and Kennedy, E.P., in preparation). The discrepancy may result from the binding of large amounts of Triton X-100 to the solubilized enzyme, thus giving it a lower buoyant density than the globular protein standards, which bind very little detergent [ 133. A similar behavior was earlier noted by Meunier et al. [ 191 for the cholinergic receptor of Electrophorus. Search
for additional
endogenous
Since the physiological
function
lipid substrates
of the diglyceride
kinase of E. coli is uncer-
210
tain and since this enzyme efficiently catalyzes the phosphorylation of a wide variety of lipid substrates in vitro, we searched in E. coli for possible endogenous substrates in addition to diacylgly~erol. Neither ~eramide-like substances nor sphingolipids were detected in E. co& and no evidence was found for the existence of macromolecular substrates precipitable by trichloroacetic acid. Discussion The purification of E. coii diglyceride kinase described here represents the most extensive purification to date of this membrane-bound enzyme. During the course of this work, we became aware of a report by Horvath and Pieringer [ 201 describing a partial purification of E. coli diglyceride kinase to a watersoluble, detergent-free state. The partially purified enzyme described by these workers possessed a specific activity (2 munits/mg) less than that of their Triton X-loo-extracted enzyme (17 munits/mg); the harsh treatments (ammonium sulfate precipitation followed by alkaline hydrolysis at pH 11.5) during the purification presumably led to inactivation of the enzyme. More significant was the observation that the purified kinase emerged very early from Biogel 200 and DEAE-cellulose columns and migrated very slowly on polyacrylamide gels. These obse~ations suggest that the diglyceride kinase obtained by this procedure may be aggregated with other membrane proteins. The purified kinase described in the present report does not appear to be aggregated; it is observed to move well into polyacrylamide gels and to display a discrete peak of kinase activity, while the bulk protein is displayed throughout the gel (Fig. 1). The detergent Triton X-100 was necessary throughout all stages of the purification; the enzyme could not be eluted from ion exchange resins without it. On Sephadex G-ZOO or sucrose gradients in the absence of detergent, the kinase appeared to aggregate with other membrane proteins, although enzyme activity was often recovered in good yield. The apparent K, of 0.02 mM for diacylglycerol [4] and 0.18 mM for ATP agree well with the values obtained by Pieringer and Kunnes [I] for the membrane-bound enzyme. However, the apparent diglyceride kinase activity in vesicles of E. coli described by Weissbach et al. [3] is reported to have a K, for ATP of 1.3 - lO+ mM, a much lower value than that observed in assay systems of the enzyme containing exogenous diacylglycerol and detergent. Moreover, the enzyme described by these workers is reported to be dependent on Mn”, to use GTP as well as ATP as nucleotide substrate, and to form [32P]ATP from [ 3zP]phosphatidic acid and ADP. Purified diglyceride kinase assayed under our conditions did not utilize GTP as a substrate; Pieringer and Kunnes [l J have reported Mg2+ to be more effective than Mn*+ as a metal ion co-factor. Finally, apparent reversiblity of the kinase reaction is not observed with our purified enzyme preparation. Upon addition of large amounts of unlabeled ATP, no loss of 32P from [ 32P]phosphatidic acid was detected; if the reaction were freely reversible, the specific activity of phosphatidic acid would decrease, as a reflection of the decrease in specific activity of the ATP pool, with which it is in equilibrium. The studies of Weissbach et al. [3] were carried out on unfractionated vesicles, and these workers offered no strong evidence that a single enzyme was re-
211
sponsible for the reaction studied. A recent report by Thomas [Zl] suggests that the previous observations in membrane vesicles [3,22,23] may be accounted for by the concerted action of two enzyines, succinyl-CoA synthetase and diglyceride kinase. The phosphorylation of ceramide by “diglyceride kinase” preparations from E. coli [4] represents the first report of such an enzyme activity in any organism. Neither sphingosine nor ficaprenol are substrates for diglyceride kinase. Thus, the activity described here which catalyzes the phosphorylation of ceramides is distinctly different from the sphingosine kinase reported in animal tissues [24-261 and in Tetruhymena [27] and from the isoprenoid alcohol kinase reported in Staphylococcus [28]. Keenan [27] reported that sphingosine kinase of Tetrahymena requires a substrate with a free amino group; the enzyme is inactive toward N-acylated or N-acetylsphingosine. In contrast, the diglyceride kinase requires an acylated substrate, as shown by its activity toward even N-acetylsphingosine but not toward sphingosine. Despite the ability of diglyceride kinase to utilize a wide variety of lipid substrates in vitro, no endogenous substrates in addition to diacylglycerol have as yet been detected in E. coli. Although Chang and Kennedy [2] were unable to find a pool of metabolically active diacylglycerol in E. coli, it is possible that such a pool exists smaller than could be detected by the most sensitive of their methods. Their results, however, together with the genetic studies already cited, make it very unlikely that the phosphorylation of diacylglycerol is a major route for the synthesis of phosphatidic acid in this organism. It is possible, nevertheless, that diacylglycerols arising during the metabolism of glycerophosphatides formed via the glycerophosphate pathway may be phosphorylated by this enzyme. Acknowledgements We are indebted to W. Dowhan, C.R.H. Raetz, and R. Ginnis for assay of phosphatidylserine decarboxylase, CDPdiglyceride pyrophosphatase and glycerophosphate phosphatidyltransferase, and isoprenoid alcohol kinase, respectively. We thank R. Hanson for fractionation of disrupted E. co/i on discontinuous sucrose gradients and for assay of azide-inhibitable adenosine triphosphatase. This research was supported by grants from the National Institute of General Medical Sciences, National Institutes of Health, GM 19822-15; GM 18731-03; and GM 13952-09. E.G.S. was a Predoctoral Fellow of the National Institute of General Medical Sciences GM 00451. The work described here forms part of a dissertation that was submitted to the Faculty of Arts and Sciences of Harvard University in partial fulfillment of the requirements for the Ph.D. degree. References 1
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