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Enzymic studies of glycol and glycerol lipids containing O-alkyl bonds in liver and tumor tissues
ARCHIVES
OF
Enzymic
BIOCHEMISTRY
AND
Studies
of Glycol Bonds
FRED Medical
Division,
SNYDER,2 Oak
Ridge
Medicinal
161, lu)2-407 (1974)
BIOPHYSICS
and
in Liver BOYD
Glycerol and
MALONE,
Lipids
Tumor AND
Containing
0-Alkyl
Tissues’ CLAUDE
PIANTADOS13
Universities, Oak Ridge, Tennessee 97830, and the Department of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27614
Associated
of
Chemistry, School
Received September
17, 1973
The utilization of [1-14C]hexadecyl-[2-3H]ethyleneglycol and [1-‘%?]hexadecyl[2-3HJglycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and 0-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg2+; although the small quantities formed precluded absolute identification, its thin-layer chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The 0-alkyl moiety of hexadecylethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.
tissues (2), and alk-1-enyl ethane phosphorylcholine has been found in rat liver (3). Nothing is known about the metabolism of the ether-linked diol lipids. However, since the diol lipids, including the ethyleneglycol type, occur as analogs of their glycero1 This work was supported in part by the United lipid counterparts, it is possible that such States Atomic Energy Commission; American molecules are utilized as substrates by the Cancer Society, Grant BC-70E; National Cancer same enzymes that metabolize the glyceroInstitute, National Institutes of Health, Grant lipid analogs. We have tested this reasoning CA11949.04; and the National Institute of Arby using enzyme sources from two mamthritis, Metabolism and Digestive Diseases, Namalian tissues that are involved in the tional Institutes of Health, Grant AM-15172-10. metabolism of ether-linked glycerolipids. 2 Author to whom inquires should be made: Dr. In these experiments, acyltransferase, phosFred Snyder, Medical Division, Oak Ridge Asphotransferase, phosphorylcholine and phossociated Universities, P. 0. Box 117, Oak Ridge, phorylethanolamine transferase, and 0-alkyl Tennessee 37830. cleavage activities were investigated using 8 Department of Medicinal Chemistry, School [l-14C]hexadecyl-[2-3H]ethyleneglycol and [lof Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27514. 14C]hexadecyl-[2-3H]glycerol as substrates. 402
A number of types of diol lipids are known to occur in nature (l), including those that contain 0-alkyl (2) and 0-alk-1-enyl (3) moieties. Alkylacyland alk-l-enylacylethyleneglycols have been found in starfish
Copyright AU rights
@ 1974 by Aeademio Pm, of reproduction in any form
Inc. reserved.
GLYCOL EXPERIMENTAL
AND
GLYCEROL
PROCEDURES
Cell-free preparations. Liver microsomes were prepared in the usual manner (4) from Charles River female rats; the excised livers (4 g/10 ml buffer) were homogenized in 0.25 M sucrose/O.125 M Tris buffer (pH 7.5) containing 0.001 M EDTA, using a Potter-Elvehjem apparatus. A portion of the homogenate was sedimented at 15,000g in a centrifuge, and the supernatant fraction was then centrifuged at 100,OOOg for 60 min. The pellet, representing the microsomes, was then washed in the buffered sucrose solution and resedimented at 100,OOOg; this washing procedure was repeated three times. Samples were maintained at approximately 4°C during all operations, and after the final washing step they were frozen at -20°C until used. Microsomes from preputial gland tumors (ESR-586), grown subcutaneously in C57BL/6 mice, were prepared and stored in a manner identical to that outlined for the liver. The microsomal preparations were used in the experiments involving the 0-alkyl cleavage enzyme and the phosphorylcholine and phosphorylethanolamine transferases. For the acyltransferase and phosphotransferase assays, a 5009 supernatant preparation (referred to as homogenate in this article) was prepared using a 0.25 M sucrose solution (4 g/10 ml). Incubation systems. The assay systems for the various enzymes investigated have previously been described: acyltransferases (5), kinase (6), phosphorylcholine and phosphorylethanolamine transferases (7, 8), and the tetrahydropteridine (PteHa)-linked 0-alkyl cleavage enzyme (4). Where appropriate, the complete systems are described in the legends of the tables. [l-14C]Hexadecyl-[2-3Hlethyleneglycol or rat-[1-l%]hexadecyl-[2-3H]glycerol were used as substrates in all assays. All incubations were terminated by extracting the total lipids (9). l-11 -%]Hexadecyl[2 - 3Hlethyleneglycol was prepared from rat-[1-‘4C]hexadecyl-[2-3Hlglycerol (10) (3H/‘% = 0.42) by treating the latter with 907, acetic acid saturated with NaI04 for 30 min at room temperature (11). The labeled hexadecylglycolic aldehyde (3H/14C = 0.42) was then reduced with Vitride [NaAlHz(OCH&H20CH,)2] as described earlier (12); the product had the same 3H/14C ratio as the starting material. Chromatographic and analytical procedures. Total lipid extracts from the incubated samples and standards were chromatographed in the following solvent systems (v/v): (A) hexane-diethyl ether (9O:lO); (B) hexane-diethyl ether (95:5); (C) hexane-diethyl ether-acetic acid (60:40:1); (D) chloroform-methanol-acetic acid (98:2:1); (E) chloroform-methanol-acetic acid (50:25:8);
LIPID
403
METABOLISM
(F) chloroform-methanol-acetic acid-water (50:25:8:4); and (G) chloroform-methanol-ammonium hydroxide (65:35:5). Solvent systems A-D for separating the relatively nonpolar lipids were used with Silica Gel G as the absorbent, whereas solvent systems E-G for separating the polar lipids were used with Silica Gel HR. For each sample, the distribution of radioactivity along the entire chromatographic lane was determined by zonal profile analysis (13). In some samples, the products (purified by thin-layer chromatography in one of the above systems) and the substrate were analyzed by gas-liquid chromatography of the following derivatives: methyl esters (14) of fatty acids, acetates (14) of fatty alcohols and alkylethyleneglycols, and dimethylacetals (12) of fatty aldehydes. Gas-liquid chromatography of intact alkylacylethyleneglycols (purified by thinlayer chromatography) was carried out isothermally at 280°C under conditions previously described for alkyldiacylglycerols (15). In one experiment the purified alkylatiylethyleneglycols, isolated from the lipid extracts of the liver and tumor incubation systems, were treated with pancreatic lipase (Calbiochem) as descirbed before (16). Total protein (17) in each cell-free preparation was also determined. RESULTS
AND
DISCUSSION
Studies related to the transfer of acyl, phosphate, and phosphorylbase groups to alkylethyleneglycol. Data in Table I and Figs. 1 and 2 show that [lJ4C]hexadecyl-[23H]ethyleneglycol is acylated by homogenates of rat livers and preputial gland tumors. With the liver homogenates, it was not possible to demonstrate a requirement for ATP and CoA, since apparently sufficient quantities of these cofactors are present. However, with the tumor homogenate, the need for activation of fatty acids is seen because significant acylation occurred only when ATP and CoA were present. With the complete system, boiled preparations from both tissues produced insignificant quantities of the acylated products. As observed in previous work (5, 18) with alkyl glycerolipids, there is a sufficient quantity of endogenous free fatty acids present in the cell-free preparations to serve as precursors of the acyl moieties. The acylated product (alkylacylethyleneglycol) was easily separated from the substrate (alkylethyleneglycol), and the triacylglycerols and alkyldiacylglycerols on Silica Gel G layers
404
SNYDER,
MALONE
AND
TABLE ACYLATION
OF
Homogenate
Complete ATP,
Complete,
CoA except
homogenate
was
boiled*
Complete Minus
ATP,
Complete,
CoA except
homogenate
was boiled*
Rat
liver
Rat
liver
Rat
liver
source
Preputial
gland
tumor
Preputial
gland
tumor
Preputial
gland
tumor
a Acylation system contained Tris buffer (100 mM, NaF (42 mM), cysteine (8.33 mM), CoA (100 PM), 0.185 pCi 14C, 0.075 &i 3H) suspended in 10 11 95% nate (15 mg protein) or rat liver homogenate (21 mg 20 min at 37°C. * Homogenate was heated in a boiling water bath H&014CH,(CH2),,CH 1 t
I
1-0-[1-'4C]HEXADECYL-[2-3H]ETHYLENEGLYCOL
System”
Minus
PIANTADOSI
Alkylacylethyleneglycol formed (nmoles) Based on “C dpm
Based on 3H dpm
10.2 9.8 11.5 11.3 0.12 0.18 19.9 20.9 3.2 1.7 0.20 0.22
11.6 11.7 11.4 10.3 0 0 19.9 21.0 3.1 1.4 0.26 0.0
pH 7.1), KC1 (26 mM), ATP (10 mM), MgClz (4 mM) J l-O-[l-14Clhexadecyl-~2-3H1ethyleneglycol (33.3 pM; ethyl alcohol, and preputial gland tumor homogeprotein) in a total volume of 3.0 ml; incubated for for
15 min.
3 15 min (1OOY) 2 N ethanolic KOH
H,C014CH,(CH,),,CH, '
3H2COCR
I 3H,COH
(1) 45 min acetic
/ H2C014CH,(CH,),4CH
(1OOT) anhydride
3
I! 3H,COCCH3 (III)
1. Identification of 1-[1-‘*C]hexadecy1-2-acy1-[2-3Hjethy1eneg1yco1 formed by acyltransferases in tumor homogenates. Radioassay of I, II, and III, after thin-layer chromatography, gave a 3H/W ratio of 0.42 for each compound. Radioassay of Compound III, after collection by gas-liquid chromatography, demonstrated that 96% of the 1% was present as the 16 :0 derivative; the 3H,“4C ratio of III under these conditions was 0.46. Similar results were obtained with homogenates of liver. FIG.
developed in hexane-diethyl ether (95 : 5, v/v). The [l-14C]hexadecyl-acyl-[2-3H]ethyleneglycol synthesized by the homogenates was identified after it had been purified by thinlayer chromatography in system B, according to the scheme depicted in Fig. 1. Gas-
liquid chromatography revealed that the final acetate derivative of labeled hexadecylethyleneglycol from the tumor and liver samples contained 96% and 85% of the radioactivity, respectively. Additional proof for the identity of the labeled alkyIacylethyleneglycols formed by
GLYCOL
SNI>
GLYCEROL
LIPID
Rat
E
METABOLISM
405
liver
801
2 . I
1200-
: Preputial
b I
ZONE
gland gland
tumor
NO.
of a total lipid extract obtained after incubation of with homogenates of rat liver or preputial gland tumors; the complete system contained added ATP, CoA, and Mg*+ (see footnote to Table I for details). Numbers associated with each peak designate: unidentified polar lipid (peak l), the unreacted substrate, hexadecylethyleneglycol (peak 2), and hexadecylacylethyleneglycol (peak 3). The solid line represents 1% activity, and the broken line represents 3H activity. Chromatography was done in solvent system B. The difference in Rp of the component designated peak 3 is due to day-to-day differences in laboratory humidity; when the two samples were cochromatographed, an identical migration pattern was obtained. FIG. 2. A typical tonal [l-‘4C]hexadecyl-[2-3H]ethyleneglycol
both the tumor obtained
from
profile
scan
and liver preparations was gas-liquid
chromat,ographie
data which demonstrated that their retention times were equivalent to authenic alkylacylethyleneglycol standards. Under
these conditions the 3H/14Cratio of the acylated products collected was identical to that in the substrate used as the acyl acceptor. With the liver samples, 45% of the radioactivity was collected wilth the hexadecyl-
406
SNYDER,
MALONE
palmitoyl-ethyleneglycol standard, whereas 51% was collected after this peak. With the tumor sample, 21% of the radioactivity was collected with the hexadecylpalmitoyl-ethyleneglycol standard, whereas 74% was collected after this peak. The differences in radioactivity distributions in these samples are undoubtedly related to the endogenous fatty acids available to the acyl transferases in these tissues. When the purified fraction of alkylacylethyleneglycols was treated with pancreatic lipase for 1 hr, deacylation was almost quantitative (91% and 92 %, respectively, for liver and tumor samples). These data provide additional proof of the structure and also demonstrate that acylated glycol lipids can serve as substrates for lipases. Thin-layer chromatographic behavior of the labeled 3H and 14Cproducts formed in the incubations is illustrated in Fig. 2; in rat liver homogenates, only the alkylacylethyleneglycol (peak 3) was formed, whereas in the homogenate from the preputial gland tumor, an additional more polar component (peak 1) was also detected. However, beTABLE ENZYMIC
CLEAVAGE
AND PIANTADOSI
cause of the limited quantities formed, it was not possible to rigorously identify the polar component. It had chromatographic properties like hexadecylethyleneglycol phosphate, since its behavior in acidic (system E) and basic (system G) developing solvents used for separating phospholipids indicated that a free phosphate group was present, i.e., in the basic system the polar labeled component (3H/14C = 0.48) migrated only slightly from the origin at essentially the same Rf as phosphatidic acid, whereas in the acid system it migrated at an Rf of 0.66, similar to that of phosphatidylethanolamine. Previous experzments with alkylglycerols as substrates have demonstrated that a phosphotransferase is present in the preputial gland tumor that can phosphorylate the primary hydroxyl moiety (6) and our data suggest that a similar reaction occurs with diol lipids. When CDP-choline or CDP-ethanolamine was included as a cofactor in the incubations, no label was found in the choline or ethanolamine phospholipids. As expected, phosphorylbase transfer to the alkylethyleneII OF 0-ALICYL
Systema
LIPIDS
Microsomal
source
[1-W]Hexadecanal (nmoles)
l-O-[l-14C]Hexadecyl-[2-3H]ethyleneglycol Complete Minus PteHl Complete, except Complete Minus PteHc Complete, except
microsomes
microsomes
were
were
boiledb
boiledb
Rat liver Rat liver Rat liver Preputial Preputial Preputial
gland gland gland
tumor tumor tumor
22.5; 19.8 0.02; 0.46 0.10; 0 0.03; 0.35 0.06; 0.07 0.11; 0.11
tumor tumor tumor
16.0; 0.47; 0.86; 0; 0.43; 0.75;
1-0-[1-14C]Hexadecyl-[2-3H]glycerol Complete Minus PteHd Complete, except Complete Minus PteH4 Complete, except
microsomes
microsomes
were
were
boiledb
boiled6
Rat liver Rat liver Rat liver Preputial Preputial Preputial
gland gland gland
15.0 0.42 0.87 0.70 0.48 0.88
n Complete system contained borate-KC1 buffer (40 mM each, pH 9.0), (NHd)rSO, (24 mM), GSH (10 mM), PteH, (0.75 mM), 1-0-[1-W]hexadecyl-[2-3H]ethyleneglycol (33.3 NM; 0.185&i W, 0.75 pCi 3H) or 1-0-[1-Wlhexadecyl-[2-*HJglycerol (33.3 PM; 0.185 pCi W, 0.075 pCi aH) suspended in 10 ~195% ethyl alcohol, and rat liver microsomes washed four times (0.22 mg protein) or preputial gland tumor microsomes washed three times (0.50 mg protein), in a total volume of 1.0 ml; incubated for 10 min at 37°C; under these conditions zero order kinetics were maintained for both substrates. b Microsomes were heated in a boiling water bath for 15 min.
GLYCOL
BND
GLYCEROL
glycol or alkylglycerol did not occur. However, when diacyl- or alkyla’cyl-glycerols are used as the substrates, the microsomes from both the liver (7) and preputial gland tumor (8) catalyze the transferase reaction. Tetrahydropteridin,e-dependent
cleavage en-
zymefor 0-alkyl lipids. The [l-S]hexadecyl123H]ethyleneglycol was cleaved to a somewhat greater extent than the ]l-14C]hexadecyl-[2-3Hlglycerol by rat liver microsomes in the presence of tetrahydropteridine (Table II). W-Labeled hexadecanal was isolated from the incubation mixtures where 0-alkyl cleavage occurred; the dimethylacetal derivative of the aldehyde was identified by gas-liquid chromatography. In contrast, the microsomes of the preputial gland tumor, like other tumors (19), did not oxidize hhe ether linkage of either the glycol or glycerol substrate. Negligible cleavage was also observed for all samples that did not contain tetrahydropteridine and for those samplescontaining microsomes that had been heated in a boiling water bath for 15 min. Under these conditions, almost all the radioactivit)y was recovered as unreacted substrate. Conclusion. Our data indicate that alkylethyleneglycols are metabolized in much the same way as alkylgIycerols, and perhaps by the same enzymes. However, the enzyme(s) involved must be isolated and purified to prove whether a single catalytic protein is responsible. REFERENCES L. D. (1969) in Progress in the Chemistry of Fats and Other Lipids (R. T. Holman, ed.), Vol. 10, pp. 230-286, Pergamon Press, Oxford.
1. BERGELSON,
LlPID
407
METABOLISM
2. VAVER, V. A., PISAREVA, N. A., ROZYNOV, B. V., USHAKOV, A. N., AND BERGELSON, L. D. (1971) Chem. Phys. Lipids 7, 75-02. 3. BERGELSON, L. D., VAVER, V. A., PROK.IZOVA, N. V., USHAKOV, A. N., ROZYNOV, B. V., STEFANOV, K., ILUKHINA, L. I., AND SIMONOV~, T. N. (1972) Biochim. Biophys. Acta 260, 571-582. 4. SOODSMA, J. F., PIANTBDOSI, C., BND SNYDER, F. (1972) J. Biol. Chem. 247,3923-3929, 5. SNYDER, F., PIANTADOSI, C., AND MALONE, B. (1970) Biochim. Biophys. Acta 202,244-249. 6. CHAE, K., PIANTADOSI, C., AND SNYDER, F. (1973) Biochem. Biophys. Res. Commun. 61, 119-124. 7. KENNEDY, E. P., AND WEISS, S. B. (1956) J. Biol. Chem. 222, 193-214. 8. SNYDER, F., BLANK, M. L., AND MALONE, B. (1970) J. Riot Chem. 246,4016-4018. 9. BLIGH, E. G., AND DYER, W. J. (1959) Can. J.
Biochem. Physiol. 37,911-017. 10. OSWALD,
C. E.
E. AND
246. 11. SNYDER,
F.,
BLANK,
R.
WYKLE,
O., PIANTADOSI, SNYDER, F. (1966)
L.
180&1805. 12. SNYDER, F., BLANK, (1971)
M.
(1070) M.
C., ANDERSON, Lipids 1, 241-
L., MALONE,
J. Biol. L., AND
B.,
.~ND
Chem. 246,
WYKLE,
K. L.
Biol. Chem. 246, 36393645. F., AND SMITH, D. (1966) Sep.
J.
13. SNYDER, Sci. I, 7W722. 14. SNYDER, F., AND BLANK, M. L. (1969) Arch. Biochem. Biophys. 130, 101-110. 15. BLANK, M. L., KASAMA, K., AND SNYDER, F, (1972) J. Lipid Res. 13,39&395. 16. SNYDER, F., AND PIANTADOSI, C. (1968) Biochim. Biophys. Acta 162, 794-797. 17. LOWRY, 0. H., R.OSEBROUGH, N. J., FARR, A. L., .~ND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. WYKLE, R. L., AND SNYDER, F. (1970) J. Biol.
Chem. 246, 3047-3058. 19.
SOODSM~,
F. (1970)
C., A4~~ Cancer Res. 30, 30+311.
J. E‘., PIANTADOSI,
SNYDER,
OF
Enzymic
BIOCHEMISTRY
AND
Studies
of Glycol Bonds
FRED Medical
Division,
SNYDER,2 Oak
Ridge
Medicinal
161, lu)2-407 (1974)
BIOPHYSICS
and
in Liver BOYD
Glycerol and
MALONE,
Lipids
Tumor AND
Containing
0-Alkyl
Tissues’ CLAUDE
PIANTADOS13
Universities, Oak Ridge, Tennessee 97830, and the Department of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27614
Associated
of
Chemistry, School
Received September
17, 1973
The utilization of [1-14C]hexadecyl-[2-3H]ethyleneglycol and [1-‘%?]hexadecyl[2-3HJglycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and 0-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg2+; although the small quantities formed precluded absolute identification, its thin-layer chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The 0-alkyl moiety of hexadecylethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.
tissues (2), and alk-1-enyl ethane phosphorylcholine has been found in rat liver (3). Nothing is known about the metabolism of the ether-linked diol lipids. However, since the diol lipids, including the ethyleneglycol type, occur as analogs of their glycero1 This work was supported in part by the United lipid counterparts, it is possible that such States Atomic Energy Commission; American molecules are utilized as substrates by the Cancer Society, Grant BC-70E; National Cancer same enzymes that metabolize the glyceroInstitute, National Institutes of Health, Grant lipid analogs. We have tested this reasoning CA11949.04; and the National Institute of Arby using enzyme sources from two mamthritis, Metabolism and Digestive Diseases, Namalian tissues that are involved in the tional Institutes of Health, Grant AM-15172-10. metabolism of ether-linked glycerolipids. 2 Author to whom inquires should be made: Dr. In these experiments, acyltransferase, phosFred Snyder, Medical Division, Oak Ridge Asphotransferase, phosphorylcholine and phossociated Universities, P. 0. Box 117, Oak Ridge, phorylethanolamine transferase, and 0-alkyl Tennessee 37830. cleavage activities were investigated using 8 Department of Medicinal Chemistry, School [l-14C]hexadecyl-[2-3H]ethyleneglycol and [lof Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27514. 14C]hexadecyl-[2-3H]glycerol as substrates. 402
A number of types of diol lipids are known to occur in nature (l), including those that contain 0-alkyl (2) and 0-alk-1-enyl (3) moieties. Alkylacyland alk-l-enylacylethyleneglycols have been found in starfish
Copyright AU rights
@ 1974 by Aeademio Pm, of reproduction in any form
Inc. reserved.
GLYCOL EXPERIMENTAL
AND
GLYCEROL
PROCEDURES
Cell-free preparations. Liver microsomes were prepared in the usual manner (4) from Charles River female rats; the excised livers (4 g/10 ml buffer) were homogenized in 0.25 M sucrose/O.125 M Tris buffer (pH 7.5) containing 0.001 M EDTA, using a Potter-Elvehjem apparatus. A portion of the homogenate was sedimented at 15,000g in a centrifuge, and the supernatant fraction was then centrifuged at 100,OOOg for 60 min. The pellet, representing the microsomes, was then washed in the buffered sucrose solution and resedimented at 100,OOOg; this washing procedure was repeated three times. Samples were maintained at approximately 4°C during all operations, and after the final washing step they were frozen at -20°C until used. Microsomes from preputial gland tumors (ESR-586), grown subcutaneously in C57BL/6 mice, were prepared and stored in a manner identical to that outlined for the liver. The microsomal preparations were used in the experiments involving the 0-alkyl cleavage enzyme and the phosphorylcholine and phosphorylethanolamine transferases. For the acyltransferase and phosphotransferase assays, a 5009 supernatant preparation (referred to as homogenate in this article) was prepared using a 0.25 M sucrose solution (4 g/10 ml). Incubation systems. The assay systems for the various enzymes investigated have previously been described: acyltransferases (5), kinase (6), phosphorylcholine and phosphorylethanolamine transferases (7, 8), and the tetrahydropteridine (PteHa)-linked 0-alkyl cleavage enzyme (4). Where appropriate, the complete systems are described in the legends of the tables. [l-14C]Hexadecyl-[2-3Hlethyleneglycol or rat-[1-l%]hexadecyl-[2-3H]glycerol were used as substrates in all assays. All incubations were terminated by extracting the total lipids (9). l-11 -%]Hexadecyl[2 - 3Hlethyleneglycol was prepared from rat-[1-‘4C]hexadecyl-[2-3Hlglycerol (10) (3H/‘% = 0.42) by treating the latter with 907, acetic acid saturated with NaI04 for 30 min at room temperature (11). The labeled hexadecylglycolic aldehyde (3H/14C = 0.42) was then reduced with Vitride [NaAlHz(OCH&H20CH,)2] as described earlier (12); the product had the same 3H/14C ratio as the starting material. Chromatographic and analytical procedures. Total lipid extracts from the incubated samples and standards were chromatographed in the following solvent systems (v/v): (A) hexane-diethyl ether (9O:lO); (B) hexane-diethyl ether (95:5); (C) hexane-diethyl ether-acetic acid (60:40:1); (D) chloroform-methanol-acetic acid (98:2:1); (E) chloroform-methanol-acetic acid (50:25:8);
LIPID
403
METABOLISM
(F) chloroform-methanol-acetic acid-water (50:25:8:4); and (G) chloroform-methanol-ammonium hydroxide (65:35:5). Solvent systems A-D for separating the relatively nonpolar lipids were used with Silica Gel G as the absorbent, whereas solvent systems E-G for separating the polar lipids were used with Silica Gel HR. For each sample, the distribution of radioactivity along the entire chromatographic lane was determined by zonal profile analysis (13). In some samples, the products (purified by thin-layer chromatography in one of the above systems) and the substrate were analyzed by gas-liquid chromatography of the following derivatives: methyl esters (14) of fatty acids, acetates (14) of fatty alcohols and alkylethyleneglycols, and dimethylacetals (12) of fatty aldehydes. Gas-liquid chromatography of intact alkylacylethyleneglycols (purified by thinlayer chromatography) was carried out isothermally at 280°C under conditions previously described for alkyldiacylglycerols (15). In one experiment the purified alkylatiylethyleneglycols, isolated from the lipid extracts of the liver and tumor incubation systems, were treated with pancreatic lipase (Calbiochem) as descirbed before (16). Total protein (17) in each cell-free preparation was also determined. RESULTS
AND
DISCUSSION
Studies related to the transfer of acyl, phosphate, and phosphorylbase groups to alkylethyleneglycol. Data in Table I and Figs. 1 and 2 show that [lJ4C]hexadecyl-[23H]ethyleneglycol is acylated by homogenates of rat livers and preputial gland tumors. With the liver homogenates, it was not possible to demonstrate a requirement for ATP and CoA, since apparently sufficient quantities of these cofactors are present. However, with the tumor homogenate, the need for activation of fatty acids is seen because significant acylation occurred only when ATP and CoA were present. With the complete system, boiled preparations from both tissues produced insignificant quantities of the acylated products. As observed in previous work (5, 18) with alkyl glycerolipids, there is a sufficient quantity of endogenous free fatty acids present in the cell-free preparations to serve as precursors of the acyl moieties. The acylated product (alkylacylethyleneglycol) was easily separated from the substrate (alkylethyleneglycol), and the triacylglycerols and alkyldiacylglycerols on Silica Gel G layers
404
SNYDER,
MALONE
AND
TABLE ACYLATION
OF
Homogenate
Complete ATP,
Complete,
CoA except
homogenate
was
boiled*
Complete Minus
ATP,
Complete,
CoA except
homogenate
was boiled*
Rat
liver
Rat
liver
Rat
liver
source
Preputial
gland
tumor
Preputial
gland
tumor
Preputial
gland
tumor
a Acylation system contained Tris buffer (100 mM, NaF (42 mM), cysteine (8.33 mM), CoA (100 PM), 0.185 pCi 14C, 0.075 &i 3H) suspended in 10 11 95% nate (15 mg protein) or rat liver homogenate (21 mg 20 min at 37°C. * Homogenate was heated in a boiling water bath H&014CH,(CH2),,CH 1 t
I
1-0-[1-'4C]HEXADECYL-[2-3H]ETHYLENEGLYCOL
System”
Minus
PIANTADOSI
Alkylacylethyleneglycol formed (nmoles) Based on “C dpm
Based on 3H dpm
10.2 9.8 11.5 11.3 0.12 0.18 19.9 20.9 3.2 1.7 0.20 0.22
11.6 11.7 11.4 10.3 0 0 19.9 21.0 3.1 1.4 0.26 0.0
pH 7.1), KC1 (26 mM), ATP (10 mM), MgClz (4 mM) J l-O-[l-14Clhexadecyl-~2-3H1ethyleneglycol (33.3 pM; ethyl alcohol, and preputial gland tumor homogeprotein) in a total volume of 3.0 ml; incubated for for
15 min.
3 15 min (1OOY) 2 N ethanolic KOH
H,C014CH,(CH,),,CH, '
3H2COCR
I 3H,COH
(1) 45 min acetic
/ H2C014CH,(CH,),4CH
(1OOT) anhydride
3
I! 3H,COCCH3 (III)
1. Identification of 1-[1-‘*C]hexadecy1-2-acy1-[2-3Hjethy1eneg1yco1 formed by acyltransferases in tumor homogenates. Radioassay of I, II, and III, after thin-layer chromatography, gave a 3H/W ratio of 0.42 for each compound. Radioassay of Compound III, after collection by gas-liquid chromatography, demonstrated that 96% of the 1% was present as the 16 :0 derivative; the 3H,“4C ratio of III under these conditions was 0.46. Similar results were obtained with homogenates of liver. FIG.
developed in hexane-diethyl ether (95 : 5, v/v). The [l-14C]hexadecyl-acyl-[2-3H]ethyleneglycol synthesized by the homogenates was identified after it had been purified by thinlayer chromatography in system B, according to the scheme depicted in Fig. 1. Gas-
liquid chromatography revealed that the final acetate derivative of labeled hexadecylethyleneglycol from the tumor and liver samples contained 96% and 85% of the radioactivity, respectively. Additional proof for the identity of the labeled alkyIacylethyleneglycols formed by
GLYCOL
SNI>
GLYCEROL
LIPID
Rat
E
METABOLISM
405
liver
801
2 . I
1200-
: Preputial
b I
ZONE
gland gland
tumor
NO.
of a total lipid extract obtained after incubation of with homogenates of rat liver or preputial gland tumors; the complete system contained added ATP, CoA, and Mg*+ (see footnote to Table I for details). Numbers associated with each peak designate: unidentified polar lipid (peak l), the unreacted substrate, hexadecylethyleneglycol (peak 2), and hexadecylacylethyleneglycol (peak 3). The solid line represents 1% activity, and the broken line represents 3H activity. Chromatography was done in solvent system B. The difference in Rp of the component designated peak 3 is due to day-to-day differences in laboratory humidity; when the two samples were cochromatographed, an identical migration pattern was obtained. FIG. 2. A typical tonal [l-‘4C]hexadecyl-[2-3H]ethyleneglycol
both the tumor obtained
from
profile
scan
and liver preparations was gas-liquid
chromat,ographie
data which demonstrated that their retention times were equivalent to authenic alkylacylethyleneglycol standards. Under
these conditions the 3H/14Cratio of the acylated products collected was identical to that in the substrate used as the acyl acceptor. With the liver samples, 45% of the radioactivity was collected wilth the hexadecyl-
406
SNYDER,
MALONE
palmitoyl-ethyleneglycol standard, whereas 51% was collected after this peak. With the tumor sample, 21% of the radioactivity was collected with the hexadecylpalmitoyl-ethyleneglycol standard, whereas 74% was collected after this peak. The differences in radioactivity distributions in these samples are undoubtedly related to the endogenous fatty acids available to the acyl transferases in these tissues. When the purified fraction of alkylacylethyleneglycols was treated with pancreatic lipase for 1 hr, deacylation was almost quantitative (91% and 92 %, respectively, for liver and tumor samples). These data provide additional proof of the structure and also demonstrate that acylated glycol lipids can serve as substrates for lipases. Thin-layer chromatographic behavior of the labeled 3H and 14Cproducts formed in the incubations is illustrated in Fig. 2; in rat liver homogenates, only the alkylacylethyleneglycol (peak 3) was formed, whereas in the homogenate from the preputial gland tumor, an additional more polar component (peak 1) was also detected. However, beTABLE ENZYMIC
CLEAVAGE
AND PIANTADOSI
cause of the limited quantities formed, it was not possible to rigorously identify the polar component. It had chromatographic properties like hexadecylethyleneglycol phosphate, since its behavior in acidic (system E) and basic (system G) developing solvents used for separating phospholipids indicated that a free phosphate group was present, i.e., in the basic system the polar labeled component (3H/14C = 0.48) migrated only slightly from the origin at essentially the same Rf as phosphatidic acid, whereas in the acid system it migrated at an Rf of 0.66, similar to that of phosphatidylethanolamine. Previous experzments with alkylglycerols as substrates have demonstrated that a phosphotransferase is present in the preputial gland tumor that can phosphorylate the primary hydroxyl moiety (6) and our data suggest that a similar reaction occurs with diol lipids. When CDP-choline or CDP-ethanolamine was included as a cofactor in the incubations, no label was found in the choline or ethanolamine phospholipids. As expected, phosphorylbase transfer to the alkylethyleneII OF 0-ALICYL
Systema
LIPIDS
Microsomal
source
[1-W]Hexadecanal (nmoles)
l-O-[l-14C]Hexadecyl-[2-3H]ethyleneglycol Complete Minus PteHl Complete, except Complete Minus PteHc Complete, except
microsomes
microsomes
were
were
boiledb
boiledb
Rat liver Rat liver Rat liver Preputial Preputial Preputial
gland gland gland
tumor tumor tumor
22.5; 19.8 0.02; 0.46 0.10; 0 0.03; 0.35 0.06; 0.07 0.11; 0.11
tumor tumor tumor
16.0; 0.47; 0.86; 0; 0.43; 0.75;
1-0-[1-14C]Hexadecyl-[2-3H]glycerol Complete Minus PteHd Complete, except Complete Minus PteH4 Complete, except
microsomes
microsomes
were
were
boiledb
boiled6
Rat liver Rat liver Rat liver Preputial Preputial Preputial
gland gland gland
15.0 0.42 0.87 0.70 0.48 0.88
n Complete system contained borate-KC1 buffer (40 mM each, pH 9.0), (NHd)rSO, (24 mM), GSH (10 mM), PteH, (0.75 mM), 1-0-[1-W]hexadecyl-[2-3H]ethyleneglycol (33.3 NM; 0.185&i W, 0.75 pCi 3H) or 1-0-[1-Wlhexadecyl-[2-*HJglycerol (33.3 PM; 0.185 pCi W, 0.075 pCi aH) suspended in 10 ~195% ethyl alcohol, and rat liver microsomes washed four times (0.22 mg protein) or preputial gland tumor microsomes washed three times (0.50 mg protein), in a total volume of 1.0 ml; incubated for 10 min at 37°C; under these conditions zero order kinetics were maintained for both substrates. b Microsomes were heated in a boiling water bath for 15 min.
GLYCOL
BND
GLYCEROL
glycol or alkylglycerol did not occur. However, when diacyl- or alkyla’cyl-glycerols are used as the substrates, the microsomes from both the liver (7) and preputial gland tumor (8) catalyze the transferase reaction. Tetrahydropteridin,e-dependent
cleavage en-
zymefor 0-alkyl lipids. The [l-S]hexadecyl123H]ethyleneglycol was cleaved to a somewhat greater extent than the ]l-14C]hexadecyl-[2-3Hlglycerol by rat liver microsomes in the presence of tetrahydropteridine (Table II). W-Labeled hexadecanal was isolated from the incubation mixtures where 0-alkyl cleavage occurred; the dimethylacetal derivative of the aldehyde was identified by gas-liquid chromatography. In contrast, the microsomes of the preputial gland tumor, like other tumors (19), did not oxidize hhe ether linkage of either the glycol or glycerol substrate. Negligible cleavage was also observed for all samples that did not contain tetrahydropteridine and for those samplescontaining microsomes that had been heated in a boiling water bath for 15 min. Under these conditions, almost all the radioactivit)y was recovered as unreacted substrate. Conclusion. Our data indicate that alkylethyleneglycols are metabolized in much the same way as alkylgIycerols, and perhaps by the same enzymes. However, the enzyme(s) involved must be isolated and purified to prove whether a single catalytic protein is responsible. REFERENCES L. D. (1969) in Progress in the Chemistry of Fats and Other Lipids (R. T. Holman, ed.), Vol. 10, pp. 230-286, Pergamon Press, Oxford.
1. BERGELSON,
LlPID
407
METABOLISM
2. VAVER, V. A., PISAREVA, N. A., ROZYNOV, B. V., USHAKOV, A. N., AND BERGELSON, L. D. (1971) Chem. Phys. Lipids 7, 75-02. 3. BERGELSON, L. D., VAVER, V. A., PROK.IZOVA, N. V., USHAKOV, A. N., ROZYNOV, B. V., STEFANOV, K., ILUKHINA, L. I., AND SIMONOV~, T. N. (1972) Biochim. Biophys. Acta 260, 571-582. 4. SOODSMA, J. F., PIANTBDOSI, C., BND SNYDER, F. (1972) J. Biol. Chem. 247,3923-3929, 5. SNYDER, F., PIANTADOSI, C., AND MALONE, B. (1970) Biochim. Biophys. Acta 202,244-249. 6. CHAE, K., PIANTADOSI, C., AND SNYDER, F. (1973) Biochem. Biophys. Res. Commun. 61, 119-124. 7. KENNEDY, E. P., AND WEISS, S. B. (1956) J. Biol. Chem. 222, 193-214. 8. SNYDER, F., BLANK, M. L., AND MALONE, B. (1970) J. Riot Chem. 246,4016-4018. 9. BLIGH, E. G., AND DYER, W. J. (1959) Can. J.
Biochem. Physiol. 37,911-017. 10. OSWALD,
C. E.
E. AND
246. 11. SNYDER,
F.,
BLANK,
R.
WYKLE,
O., PIANTADOSI, SNYDER, F. (1966)
L.
180&1805. 12. SNYDER, F., BLANK, (1971)
M.
(1070) M.
C., ANDERSON, Lipids 1, 241-
L., MALONE,
J. Biol. L., AND
B.,
.~ND
Chem. 246,
WYKLE,
K. L.
Biol. Chem. 246, 36393645. F., AND SMITH, D. (1966) Sep.
J.
13. SNYDER, Sci. I, 7W722. 14. SNYDER, F., AND BLANK, M. L. (1969) Arch. Biochem. Biophys. 130, 101-110. 15. BLANK, M. L., KASAMA, K., AND SNYDER, F, (1972) J. Lipid Res. 13,39&395. 16. SNYDER, F., AND PIANTADOSI, C. (1968) Biochim. Biophys. Acta 162, 794-797. 17. LOWRY, 0. H., R.OSEBROUGH, N. J., FARR, A. L., .~ND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. WYKLE, R. L., AND SNYDER, F. (1970) J. Biol.
Chem. 246, 3047-3058. 19.
SOODSM~,
F. (1970)
C., A4~~ Cancer Res. 30, 30+311.
J. E‘., PIANTADOSI,
SNYDER,