- Email: [email protected]
Effects of retinol and retinol palmitate on glycolipid and glycoprotein galactosyltransferase activities of rat liver plasma membranes
Vol. 95, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1775-1780
August 29, 1980
EFFECTS OF RETINOL AND RETINOL PALMITATE ON GLYCOLIPID AND GLYCOPROTEIN GALACTOSYLTRANSFERASE ACTIVITIES OF RAT LIVER PLASMA MEMBRANES
Kim E. Creek and D. James Morre
Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, U.S.A. Received
July
14,1980 SUMMARY
Purified plasma membranes isolated from rat liver exhibited low levels of galaetosyltransferase activity toward endogenous glycolipid and glycoprotein acceptors. The addition of retinol (0.005 mM) stimulated the transfer of [14C]galactose from UDP-[14C]galactose to endogenous glycolipid and glycoprotein acceptors of plasma membranes while higher or lower concentrations of retinol were either inhibitory or had no effect. Retinol palmitate (0.005 mM) stimulated the transfer of [14C]galactose from UDP-[14C]galactose into glycolipid but not glycoprotein acceptors of isolated plasma membranes and was inhibitory at concentrations above 0.05 mM. The addition of ATP (0.5 mM) to incubations in the absence or presence of retinol (0.025 or 0.005 mM) inhibited incorporation of [14C]galactose into glycolipid and $1ycoprotein acceptors. Plasma membranes did not catalyze the synthesis of [±4C]galactosyl retinyl phosphate in incubations containing UDP-[14C]galactose and retinyl phosphate. The results of this study indicate that retinol stimulates incorporation of [14C]galactose into glycolipid and glycoprotein acceptors of isolated plasma membranes. The mechanism of stimulation is unknown but does not appear to be a simple detergent effect or the result of retinol acting as a galactosyl retinyl phosphate intermediate.
INTRODUCTION Ectoglycosyltransferases,
glycosyltransferases at the cell surface, of
NIL or BHK cells glycosylate glyeosphingolipids linked covalently to fine glass particles upon cell contact (I). This cell contact-mediated glycosylation was reported to be enhanced by retinol.
Additionally, isolated galactosyl retinyl
phosphate was a better galaetose donor than UDP-galactose in galactosylation of lactosylceramide on glass (2).
We found that plasma membranes isolated
from rat liver exhibited low levels of galactosyltransferase activity toward Abbreviations:
GL, glycolipid; GP, glycoprotein.
0006-291X/80/161775-06501.00/0 1775
Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.
Vol. 95, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
endogenous glycolipid (GL) and glycoprotein (GP) acceptors (3).
Therefore, we
began a study to determine whether this galactosyltransferase activity was also modulated by retinol.
MATERIALS AND METHODS Plasma membranes were isolated by the procedure of Yunghans and Morre (4) and endoplasmic reticulum by the method of Morre (5) from the livers of male Holtzman rats (Holtzman Co., Madison, WI) given food and water ad libitum. Proteins were determined by the method of Lowry et al. (6). Retinyl phosphate was kindly supplied bv Dr. L. M. De Luca, National Institutes of Health, Bethesda, MD. UDP-[l~C]galactose (347 mCi/mmol) and GDP-[14C]mannose (166 mCi/mmol) were obtained from Amersham-Searle (Arlington Heights, IL). The transfer of [14C]galactose from UDP-[14C]galactose to endogenous acceptors of rat liver plasma membranes was measured in reactions containing, in a final volume of 0.i ml; 40-60 ~g plasma membrane protein, 2.5 ~M UDP[14C]galactose, 15 mM MnCI 2, 5 mM MgCI 2, 3 mM NaF, 5 mM 2-mercaptoethanol, 25 mM Tris-HCl, pH 7.0, and varying concentrations of all trans retinol or retinol palmitate (250,000 USP units/g) (Sigma Chemical Co., St. Louis, MO). Reactions were at 37 ° and terminated after 5 min by the addition of 2 ml of chloroform-methanol 2:1 (v/v). Incorporation of [14C]galactose into a protein residue and a lipid phase was measured by the procedure of Waechter et al. (7) as modified by Merritt et al. (3).
RESULTS The addition of retinol at an optimum concentration of 0.005 ram stimulated the transfer of [14C]galactose from UDP-[14C]galactose to endogenous GL and GP acceptors of isolated plasma membranes (Fig. I).
Above or below this optimal
concentration the vitamin either inhibited enzymatic activity or had no significant effect.
Retinol palmitate, at a concentration of 0.005 mM,
stimulated the incorporation of [14C]galactose in GL but not GP acceptors (Fig. i).
Furthermore, retinol palmitate at concentrations higher than 0.05 mM
inhibited the incorporation of [14C]galactose into both GL and GP acceptors. The addition of ATP (0.5 mM), in the absence or in the presence of retinol (0.025 or 0.005 mM) inhibited the incorporation of [14C]galactose into both GL and GP acceptors (Table I). Incubations containing isolated plasma membranes, UDP-[14C]galactose, and retinyl phosphate failed to synthesize a product with the chromatographic properties of galactosyl retinyl phosphate (Table II).
1776
Controls with isolated
Voh 95, No. 4, 1 9 8 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7
Retinal
~ j
~
~
•
Protein Residue
x
Lipid
Phase
~4 No Vitamin
c
R ./ (- (
\
"~
.
1"
"~.
T
,
|
~2 I ~
I
0
I
I
I
I
I
Retinal Paimit ate 7
•
Protein Residue
x
Lipid
[
±
Phase
u. 6 14J
~5
N
m
.J 0 IE 0
I
No Vitamin
NO Vitamin 2
0
I -4
I
-3
LOG
Figure i:
I
-2
CONCENTRATION
-I
, ",,l--O
(mM
L--l-I
)
Effect of retinal or retinal palmitate on the incorporation of [14C]galaetose from UDP-[14C]galactose into endogenous acceptors of isolated plasma membranes. Plasma membranes (40-60 ~g protein) were incubated with UDP-[14C]galactose for 5 min in the presence of varying concentrations of retinal (top) or retinal palmitate (bottom). Exact assay conditions are described in methods. Reaction products were separated into a protein residue (e) and a lipid phase (X) as described. Results are the averages of three experiments ± standard deviation.
plasma membrane or endoplasmic reticulum demonstrated that mannosyl retinyl phosphate was actively synthesized under these conditions.
DISCUSSION Yogeeswaran e t a l .
(2) raised the possibility that a galactosyltransferase
at the cell surface of NIL cells may function in the galactosylation of lactosylceramide through a galactosyl retinyl phosphate intermediate.
1777
A
Vol. 95, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I EFFECTS OF RETINOL AND ATP ON THE INCORPORATION OF [14C]GALACTOSE INTO ENDOGENOUS ACCEPTORS OF RAT LIVER PLASMA MEMBRANES Concentration (mM) Retinol
pmoles galactose/min/mg protein
ATP
Lipid phase
Protein residue
0
0
3.0
3.4
0
0.5
0.6
0.5
0.025
0
3.1
4.8
0.025
0.5
0.5
0.4
0.005
0
4.1
7.0
0.005
0.5
0.9
0.8
Isolated plasma membranes (0.05 mg protein) were incubated for 5 min in the presence or absence of ATP and retinol at the concentrations indicated. Incorporation of [14C]galaetose from UDP-[14C]galactose into a lipid phase and a protein residue was determined as described in methods.
TABLE II TRANSFER OF [14C]MANNOSE FROM GDP-[14C]MANNOSE BUT NOT [14C]GALACTOSE FROM UDP-[14C]GALACTOSE TO RETINYL PHOSPHATE BY RAT LIVER MEMBRANES
retinyl phosphate glycoside, epm/h/mg protein Cellular fraction
UDP-[14C]galactose
GDP-[14C]mannose
30
610
175
22500
Plasma membrane Endoplasmic reticulum
Isolated plasma membranes (0.8 mg protein) or endoplasmic reticulum (1.5 mg protein) were incubated for 15 min at 37 ° in reactions containing 20 ~i of 0.25 M EDTA, 20 ~i of 0.3 M Tris-HCl buffer, pH 8.0, 20 ~i of 0.i M MnCI2, i0 ~I of an aqueous solution of 22 mg of ATP per ml, i0 ~i of retinyl phosphate (36 ~g) in dimethylsulfoxide, and 20 ~i of an aqueous solution of sugar nucleotide (0.5 uCi of UDP-[14C]galactose or 0.2 ~Ci of GDP-[14C]mannose). Reactions were terminated by the addition of 3 ml of chloroform-methanol 2:1 (v/v) and 0.6 ml of 0.9% NaCI. Purification of retinyl phosphate glycoside was by differential solvent extraction, DEAE cellulose column chromatography, and thin-layer chromatography as described by Silverman-Jones et al. (8).
1778
VOI. 95, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar mechanism may also function in mannosylation of glycoproteins at the surface of liver cells from embryonic chick (9).
To determine if retinol is
involved in galactosylation at the cell surface of rat hepatocytes we studied the effects of retinol on a galactosyltransferase associated with plasma membranes isolated from rat liver (3).
We demonstrated that retinol (0.005 mM)
stimulated the incorporation of [14C]galactose from UDP-[14C]galactose into endogenous GL and GP acceptors while retinol palmitate (0.005mM) only stimulated incorporation into GL acceptors.
Since the two vitamin A derivatives behaved
so differently we believe that the stimulation by retinol of [14C]galactose incorporation into GL and GP acceptors was more complicated than simple detergent activation of galactosyltransferase activity by the vitamin. Mouse mastocytoma cells (i0) and cultured mouse epidermal cells (ii) catalyzed the synthesis of galactosyl retinyl phosphate.
Retinyl phosphate (I0)
and not retinol was the substrate in the galactosyl retinyl phosphate synthetase reaction and the phosphate group was most likely donated to retinol via ATP (12). The possibility existed that retinol, in our system, was stimulating the synthesis Of galactose containing GL and GP acceptors in plasma membrane through the formation of a galactosyl retinyl phosphate intermediate.
If this were so
the addition of ATP to incubations containing plasma membrane and retinol should further stimulate galactose incorporation since more of the vitamin would be in the retinyl phosphate form.
However, the addition of ATP inhibited
the galactosyltransferase reaction both in the absence or the presence of added retinol.
Also, incubations containing isolated plasma membranes, UDP-galactose,
and retinyl phosphate failed to synthesize a compound with the chromatographic properties of galactosyl retinyl phosphate. mannosyl retinyl phosphate (13-16).
Rat liver membranes do synthesize
In control experiments we found that this
compound was actively synthesized under the same conditions that galactosyl retinyl phosphate formation could not be detected.
We therefore conclude that
the stimulation by retinol of the incorporation of [14C]galactose into endogenous GL and GP aeceptors in rat liver plasma membranes is not the result of retinol
1779
VOI. 95, No, 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
acting as a lipid intermediate in GL and GP biosynthesis at the cell surface and alternative mechanisms must be considered.
ACKNOWLEDGEMENTS We thank Keri Safranski for isolation of the plasma membrane fractions and Randy Canfield for expert technical assistance. Work supported in part by a grant from the National Institute of Health CA 18801.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii, 12. 13. 14. 15. 16.
Yogeeswaran, G., Laine, R. A., and Hakomori, S. (1974) Biochem. Biophys. Res. Comm. 59, 591-599. Yogeeswaran, G., Laine, R. A., and Hakomori, S. (1975) Fed. Proc. 34, 645. Merritt, W. D., Morre, D. J., Franke, W. W., and Keenan, T. W. (1977) Biochim. Biophys. Acta 497, 820-824. Yunghans, W, N., and Morre, D. J. (1973) Prep. Biochem. 3, 301-312. MorrO, D. J. (1973) In Molecular T echniquesapd Approaches in Developmental Biology (Chrispeels, M. J., ed.), pp. 1-27, Wiley, New York. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973) J. Biol. Chem. 248, 7570-7579. Silverman-Jones, C. S., Frot-Coutaz, J. P,, and De Luca, L. M. (1976) Anal. Biochem. 75, 664-667. Arnold, D., Hommel, E., and Risse, H. J. (1976) Mol. CeIL Biochem. ii, 137-14] Peterson, P. A., Rask, L., Helting, T., Ostberg, L., and Fernstedt, Y. (1976) J. Biol. Chem. 251, 4986-4995. Adamo, S., De Luca, L. M., Silverman-Jones, C. S., and Yuspa, S. H. (1979) J. Biol. Chem. 254, 3279-3287. De Luca, L., Maestri, N., Rosso, G., and Wolf, G. (1973) J. Biol. Chem. 248, 641-648. De Luca, L. M., Frot-Coutaz, J. P., Silverman-Jones, C. S., and Roller, P. R. (1977) J. Biol. Chem. 252, 2575-2579. Rosso, G. C., De Luca, L., Warren, C. D., and Wolf, G. (1975) J. Lipid Res. 16, 235-243. Bergman, A., Mankowski, T., Chojnacki, T., De Luca, L. M., Peterson, E., and Dallner, G. (1978) Biochem. J. 172, 123-127. Smith, M. J., Schreiber, J. B., and Wolf, G. (1979) Biochem. J. 180, 449-453.
1780
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1775-1780
August 29, 1980
EFFECTS OF RETINOL AND RETINOL PALMITATE ON GLYCOLIPID AND GLYCOPROTEIN GALACTOSYLTRANSFERASE ACTIVITIES OF RAT LIVER PLASMA MEMBRANES
Kim E. Creek and D. James Morre
Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, U.S.A. Received
July
14,1980 SUMMARY
Purified plasma membranes isolated from rat liver exhibited low levels of galaetosyltransferase activity toward endogenous glycolipid and glycoprotein acceptors. The addition of retinol (0.005 mM) stimulated the transfer of [14C]galactose from UDP-[14C]galactose to endogenous glycolipid and glycoprotein acceptors of plasma membranes while higher or lower concentrations of retinol were either inhibitory or had no effect. Retinol palmitate (0.005 mM) stimulated the transfer of [14C]galactose from UDP-[14C]galactose into glycolipid but not glycoprotein acceptors of isolated plasma membranes and was inhibitory at concentrations above 0.05 mM. The addition of ATP (0.5 mM) to incubations in the absence or presence of retinol (0.025 or 0.005 mM) inhibited incorporation of [14C]galactose into glycolipid and $1ycoprotein acceptors. Plasma membranes did not catalyze the synthesis of [±4C]galactosyl retinyl phosphate in incubations containing UDP-[14C]galactose and retinyl phosphate. The results of this study indicate that retinol stimulates incorporation of [14C]galactose into glycolipid and glycoprotein acceptors of isolated plasma membranes. The mechanism of stimulation is unknown but does not appear to be a simple detergent effect or the result of retinol acting as a galactosyl retinyl phosphate intermediate.
INTRODUCTION Ectoglycosyltransferases,
glycosyltransferases at the cell surface, of
NIL or BHK cells glycosylate glyeosphingolipids linked covalently to fine glass particles upon cell contact (I). This cell contact-mediated glycosylation was reported to be enhanced by retinol.
Additionally, isolated galactosyl retinyl
phosphate was a better galaetose donor than UDP-galactose in galactosylation of lactosylceramide on glass (2).
We found that plasma membranes isolated
from rat liver exhibited low levels of galactosyltransferase activity toward Abbreviations:
GL, glycolipid; GP, glycoprotein.
0006-291X/80/161775-06501.00/0 1775
Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.
Vol. 95, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
endogenous glycolipid (GL) and glycoprotein (GP) acceptors (3).
Therefore, we
began a study to determine whether this galactosyltransferase activity was also modulated by retinol.
MATERIALS AND METHODS Plasma membranes were isolated by the procedure of Yunghans and Morre (4) and endoplasmic reticulum by the method of Morre (5) from the livers of male Holtzman rats (Holtzman Co., Madison, WI) given food and water ad libitum. Proteins were determined by the method of Lowry et al. (6). Retinyl phosphate was kindly supplied bv Dr. L. M. De Luca, National Institutes of Health, Bethesda, MD. UDP-[l~C]galactose (347 mCi/mmol) and GDP-[14C]mannose (166 mCi/mmol) were obtained from Amersham-Searle (Arlington Heights, IL). The transfer of [14C]galactose from UDP-[14C]galactose to endogenous acceptors of rat liver plasma membranes was measured in reactions containing, in a final volume of 0.i ml; 40-60 ~g plasma membrane protein, 2.5 ~M UDP[14C]galactose, 15 mM MnCI 2, 5 mM MgCI 2, 3 mM NaF, 5 mM 2-mercaptoethanol, 25 mM Tris-HCl, pH 7.0, and varying concentrations of all trans retinol or retinol palmitate (250,000 USP units/g) (Sigma Chemical Co., St. Louis, MO). Reactions were at 37 ° and terminated after 5 min by the addition of 2 ml of chloroform-methanol 2:1 (v/v). Incorporation of [14C]galactose into a protein residue and a lipid phase was measured by the procedure of Waechter et al. (7) as modified by Merritt et al. (3).
RESULTS The addition of retinol at an optimum concentration of 0.005 ram stimulated the transfer of [14C]galactose from UDP-[14C]galactose to endogenous GL and GP acceptors of isolated plasma membranes (Fig. I).
Above or below this optimal
concentration the vitamin either inhibited enzymatic activity or had no significant effect.
Retinol palmitate, at a concentration of 0.005 mM,
stimulated the incorporation of [14C]galactose in GL but not GP acceptors (Fig. i).
Furthermore, retinol palmitate at concentrations higher than 0.05 mM
inhibited the incorporation of [14C]galactose into both GL and GP acceptors. The addition of ATP (0.5 mM), in the absence or in the presence of retinol (0.025 or 0.005 mM) inhibited the incorporation of [14C]galactose into both GL and GP acceptors (Table I). Incubations containing isolated plasma membranes, UDP-[14C]galactose, and retinyl phosphate failed to synthesize a product with the chromatographic properties of galactosyl retinyl phosphate (Table II).
1776
Controls with isolated
Voh 95, No. 4, 1 9 8 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7
Retinal
~ j
~
~
•
Protein Residue
x
Lipid
Phase
~4 No Vitamin
c
R ./ (- (
\
"~
.
1"
"~.
T
,
|
~2 I ~
I
0
I
I
I
I
I
Retinal Paimit ate 7
•
Protein Residue
x
Lipid
[
±
Phase
u. 6 14J
~5
N
m
.J 0 IE 0
I
No Vitamin
NO Vitamin 2
0
I -4
I
-3
LOG
Figure i:
I
-2
CONCENTRATION
-I
, ",,l--O
(mM
L--l-I
)
Effect of retinal or retinal palmitate on the incorporation of [14C]galaetose from UDP-[14C]galactose into endogenous acceptors of isolated plasma membranes. Plasma membranes (40-60 ~g protein) were incubated with UDP-[14C]galactose for 5 min in the presence of varying concentrations of retinal (top) or retinal palmitate (bottom). Exact assay conditions are described in methods. Reaction products were separated into a protein residue (e) and a lipid phase (X) as described. Results are the averages of three experiments ± standard deviation.
plasma membrane or endoplasmic reticulum demonstrated that mannosyl retinyl phosphate was actively synthesized under these conditions.
DISCUSSION Yogeeswaran e t a l .
(2) raised the possibility that a galactosyltransferase
at the cell surface of NIL cells may function in the galactosylation of lactosylceramide through a galactosyl retinyl phosphate intermediate.
1777
A
Vol. 95, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I EFFECTS OF RETINOL AND ATP ON THE INCORPORATION OF [14C]GALACTOSE INTO ENDOGENOUS ACCEPTORS OF RAT LIVER PLASMA MEMBRANES Concentration (mM) Retinol
pmoles galactose/min/mg protein
ATP
Lipid phase
Protein residue
0
0
3.0
3.4
0
0.5
0.6
0.5
0.025
0
3.1
4.8
0.025
0.5
0.5
0.4
0.005
0
4.1
7.0
0.005
0.5
0.9
0.8
Isolated plasma membranes (0.05 mg protein) were incubated for 5 min in the presence or absence of ATP and retinol at the concentrations indicated. Incorporation of [14C]galaetose from UDP-[14C]galactose into a lipid phase and a protein residue was determined as described in methods.
TABLE II TRANSFER OF [14C]MANNOSE FROM GDP-[14C]MANNOSE BUT NOT [14C]GALACTOSE FROM UDP-[14C]GALACTOSE TO RETINYL PHOSPHATE BY RAT LIVER MEMBRANES
retinyl phosphate glycoside, epm/h/mg protein Cellular fraction
UDP-[14C]galactose
GDP-[14C]mannose
30
610
175
22500
Plasma membrane Endoplasmic reticulum
Isolated plasma membranes (0.8 mg protein) or endoplasmic reticulum (1.5 mg protein) were incubated for 15 min at 37 ° in reactions containing 20 ~i of 0.25 M EDTA, 20 ~i of 0.3 M Tris-HCl buffer, pH 8.0, 20 ~i of 0.i M MnCI2, i0 ~I of an aqueous solution of 22 mg of ATP per ml, i0 ~i of retinyl phosphate (36 ~g) in dimethylsulfoxide, and 20 ~i of an aqueous solution of sugar nucleotide (0.5 uCi of UDP-[14C]galactose or 0.2 ~Ci of GDP-[14C]mannose). Reactions were terminated by the addition of 3 ml of chloroform-methanol 2:1 (v/v) and 0.6 ml of 0.9% NaCI. Purification of retinyl phosphate glycoside was by differential solvent extraction, DEAE cellulose column chromatography, and thin-layer chromatography as described by Silverman-Jones et al. (8).
1778
VOI. 95, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar mechanism may also function in mannosylation of glycoproteins at the surface of liver cells from embryonic chick (9).
To determine if retinol is
involved in galactosylation at the cell surface of rat hepatocytes we studied the effects of retinol on a galactosyltransferase associated with plasma membranes isolated from rat liver (3).
We demonstrated that retinol (0.005 mM)
stimulated the incorporation of [14C]galactose from UDP-[14C]galactose into endogenous GL and GP acceptors while retinol palmitate (0.005mM) only stimulated incorporation into GL acceptors.
Since the two vitamin A derivatives behaved
so differently we believe that the stimulation by retinol of [14C]galactose incorporation into GL and GP acceptors was more complicated than simple detergent activation of galactosyltransferase activity by the vitamin. Mouse mastocytoma cells (i0) and cultured mouse epidermal cells (ii) catalyzed the synthesis of galactosyl retinyl phosphate.
Retinyl phosphate (I0)
and not retinol was the substrate in the galactosyl retinyl phosphate synthetase reaction and the phosphate group was most likely donated to retinol via ATP (12). The possibility existed that retinol, in our system, was stimulating the synthesis Of galactose containing GL and GP acceptors in plasma membrane through the formation of a galactosyl retinyl phosphate intermediate.
If this were so
the addition of ATP to incubations containing plasma membrane and retinol should further stimulate galactose incorporation since more of the vitamin would be in the retinyl phosphate form.
However, the addition of ATP inhibited
the galactosyltransferase reaction both in the absence or the presence of added retinol.
Also, incubations containing isolated plasma membranes, UDP-galactose,
and retinyl phosphate failed to synthesize a compound with the chromatographic properties of galactosyl retinyl phosphate. mannosyl retinyl phosphate (13-16).
Rat liver membranes do synthesize
In control experiments we found that this
compound was actively synthesized under the same conditions that galactosyl retinyl phosphate formation could not be detected.
We therefore conclude that
the stimulation by retinol of the incorporation of [14C]galactose into endogenous GL and GP aeceptors in rat liver plasma membranes is not the result of retinol
1779
VOI. 95, No, 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
acting as a lipid intermediate in GL and GP biosynthesis at the cell surface and alternative mechanisms must be considered.
ACKNOWLEDGEMENTS We thank Keri Safranski for isolation of the plasma membrane fractions and Randy Canfield for expert technical assistance. Work supported in part by a grant from the National Institute of Health CA 18801.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii, 12. 13. 14. 15. 16.
Yogeeswaran, G., Laine, R. A., and Hakomori, S. (1974) Biochem. Biophys. Res. Comm. 59, 591-599. Yogeeswaran, G., Laine, R. A., and Hakomori, S. (1975) Fed. Proc. 34, 645. Merritt, W. D., Morre, D. J., Franke, W. W., and Keenan, T. W. (1977) Biochim. Biophys. Acta 497, 820-824. Yunghans, W, N., and Morre, D. J. (1973) Prep. Biochem. 3, 301-312. MorrO, D. J. (1973) In Molecular T echniquesapd Approaches in Developmental Biology (Chrispeels, M. J., ed.), pp. 1-27, Wiley, New York. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973) J. Biol. Chem. 248, 7570-7579. Silverman-Jones, C. S., Frot-Coutaz, J. P,, and De Luca, L. M. (1976) Anal. Biochem. 75, 664-667. Arnold, D., Hommel, E., and Risse, H. J. (1976) Mol. CeIL Biochem. ii, 137-14] Peterson, P. A., Rask, L., Helting, T., Ostberg, L., and Fernstedt, Y. (1976) J. Biol. Chem. 251, 4986-4995. Adamo, S., De Luca, L. M., Silverman-Jones, C. S., and Yuspa, S. H. (1979) J. Biol. Chem. 254, 3279-3287. De Luca, L., Maestri, N., Rosso, G., and Wolf, G. (1973) J. Biol. Chem. 248, 641-648. De Luca, L. M., Frot-Coutaz, J. P., Silverman-Jones, C. S., and Roller, P. R. (1977) J. Biol. Chem. 252, 2575-2579. Rosso, G. C., De Luca, L., Warren, C. D., and Wolf, G. (1975) J. Lipid Res. 16, 235-243. Bergman, A., Mankowski, T., Chojnacki, T., De Luca, L. M., Peterson, E., and Dallner, G. (1978) Biochem. J. 172, 123-127. Smith, M. J., Schreiber, J. B., and Wolf, G. (1979) Biochem. J. 180, 449-453.
1780