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Origin of the glyceryl component of α-glyceryl ethers
ARCHIVES
OF
BIOCIIEhfISTHY
AXD
96,
BIOPHYSICS
Letters Origin
of the Glyceryl a-Glyceryl
Component
(1962)
to the Editors
of
Ethers1
Earlier work carried out in this laboratory on the lipids of bovine erythrocytes has demonstrated that the ethanolamine phospholipid fraction from these cells is composed largely of a’-alkoxy&acyla-glycerophosphorylethanolamine, I, (1). Probable derivatives of I have been isolated from nerve tissue (2-4), egg yolk (5), calf brain (B), and hu man brain, liver, kidney, and spleen (6). However, no evidence is available on the origin of these compounds. During a study on the chemical nat,ure of bovine hone marrow lipids, a concomitant investigation of the biosynthetic route to glyceryl ethers was carried out. The result,s of preliminary experiments are reported here. The lipid composition of bovine sternal (hematopoietic) marrow and also of “yellow marrow” taken from the long bones of the leg was determined by chromatography on silicic acid (7).2 Interestingly, in sternal marrow t,he ratio of neut)ral lipid t,o phospholipid was found to be 12-14, whereas in t.he “yellow marrow” only neutral lipids, predominantly t,riglycerides, were present. Analysis of the neutral lipids of the long bones con firmed the reported presence of glyceryl ethers (8), and it was further established by silicic acid chromatography that these compounds are associated wit,h t,he triglyceride components. The neutral fraction of i.he sternal lipids was similarly const,ituted, while the phospholipid fraction could be shown to have the composition outlined in Table 1. (ilyceryl ether phospholipids were present in t)he phosphatidyl et)hanolamine and the phosphatidyl choline (lecithin) fractions, but could not be separated chromatographically from their diacyl counterparts. A semi-quantitative plasmalogen assay by the procedure of Kort)on (9) revealed a plasmalogen to phosphorus rat,io of 0.05-0.08 for the tota, phospholipid fraction. In order to study the rate of lipid formation by marrow tissue, experiments were performed in 1 This investigation was supported by grants from the Life Insurance Medical Research Fund, and the American Cancer Society. * The principal difference between circulating erythrocyte and marrow phospholipids lies in the very low (0 to 37;) lecithin content of the former (7).
which glucose-(i-C14 (3.56 mc./mM; Nuclear~Chicage Corporation) was incubated with fresh marrow- tissue.3 In experiment I marrow was isolated by the following procedure: The large bones were removed from the legs of a three-week-old milk-fed calf4 under nitrous oxide and anectine chloride anesthesia. FortyT-nine grams of hematopoietic marrow were removed and centrifuged in saline at 1000 X 9 for 20 min. at 4°C. Two fractions were isolated: one as a light pink floating pellet (Fraction A) and the other as a small amount of red sediment, (Fraction B). Fractions A and B were each suspended in saline at pH 7.4 and glucose-t04 was added. After 15 min. of gentle shaking at 37°C. unlaheled glucose was added, and the incubation was cont,inued for 165 min. Lipids were extracted and the radioact,ivity of various thromatographic fractions assayed with a Packard Tri-Carb scintillation counter. Fraction A incorporated 1.47’; of the label from 5 PC. glucose-t-Cl4 into lipids. Of this total uptake 94(;:, appeared in neutral lipids. Fraction B catalyzed the conversion of 1.75,;. of t.he 10 MC. glucose(i-Cl4 srrbstrate into lipids, with 69’x of t,he total incorporation being into neutral lipids. When hydrolyzed, less than Ir,;, of t,he neutral lipid radioactivity was found in the fatt,y acid fraction, indic:tt,ing that glucose provided only the glycerol unit for the triglycerides. ;2 control experiment in which labeled glucose ~vas added to a lipid extract just prior to chromatography demonstrated that 9fi’;l of the recovered radioactivity was cluted wit.h t,he phosphat idyl swine-phosphatidyl inositol fraction and that, unlike the incubated s:m~ples, this radioactivity could be almost quantitatively removed by washing of the lipid ext,ract with a dilute KC1 solution. The phospholipid portion (1.9 mg. 1’) from Fraction 1%was diluted I:3 with unlaheled marrow phospholipids and chromatographed. The distribution of radioactivity is shown in Table I. .4n analysis of the phosphatidyl ethanolamine fraction for its fat,ty acid ester content made it clear that approximately GO’:;, of the phospholipid was a glyceryl et,her derivative. The glyceryl 3 We are indebt)ed to Mrs. Carolyn Mitchell for her expert assistance in the initial phases of these experiments. 4 In the very young animal, these hones contain marrow active in blood formation.
671
6’72
T,ETTERS
TO THb:
EI)ITORS
1. H.UXAHAN, (‘hem.
T,ipid applied to column contained 7.2 mg. phospholipid phosphorus and 131,000 cts/min. CY*. Rccovery in chromatographic fractions: 04’;; of phospholipid phosphorus :~pplied and !Xi”;, of radioactivity applied.
PC
.IOHN,
“Phosphatidic acid” I’hosphatidyl ethanolamine Phosph;tt,idyl serine, phostidy1 inositol Phosphatidyl choline (lecithin) Sphingomyelin” ‘6 Contained
a slight
I3.,
28.1 1.7
8.6 11 .(i 2.0
42.
i5.0
13.4
I).,
./.
5 Kindly
donated
WATTS,
NORTON.
310 &NNEIJY,
/~FSfXwf;h
CORBET, N.,
Sot.
ANI)
63, 2607
W. T., Hiochim.
contaminant
hy the Seatt,le Parking
C:oln-
J.
Hid.
(l%io).
C:EIGER,
W.
W.. .I.
AIJXANDER, (1911).
et Hiophys.
dlclu
38,
(1960).
I’;. I’., .I. Niol. (‘hem.
2.7 of lecithin
1, 121
11. E.,
C;IT II.
It.,
(l!Kil).
I,ipid
S.,
KORNBLI-Y,
.1,m. (‘hem.
9. 10.
ethers were isolated from this material 1)~ acetolyais and subjected to periodate oxidation (I). The a-carbon of the glyceryl moiety was recovered as the dimedon derivative of formaldehyde, which possessed :I specific activity of 21.6 cts./min./mg. (corrected for dimedon quenching) as compared to 25.6 cts./min./mg. expected if all the radioactivit,> of the glyceryl ether were found in this carbon atom. The specific activity of the other portion (long chain aldehyde) of the molecule after periodate oxidation was 1 ,I cts./min./mg. The lecithin fraction from this experiment also contained radioactive glyceryl ethers, hut in a somewhat lowe caoncentration than found in the ethanolamine 1)hospholipid fraction. The presence of a biosynthetic system for glyccry1 ct,hcr phospholipids in marrow KU confirmed by :I second experiment utilizing fresh sternal marro\v from steers3 and glucose-G-V. Again the glyceryl et.hers of the phosphnlidyl ethanolamine and the lecit,hin fractions tvere radioactive. The experimental data presented here indicate that in bovine marrow the glyceryl moiet,y of glyccrpl ethers can he formed front glucose. The dist rihlltion of label bvould strongly suggest that the glucose metalmlitr directly involved in I.-ol-glycof erophosphate, as occurs in the hiosgnthesis phosphat idic acid (IO), or a metnl~olic:~lly anulogalls rompound. p:t11y.
ANI)
59
2. I)awsos. It. 11. C., Hiochew. J. 56, 621 (1951). 3. E;rJcAR, c. w. F., .lCfU Anal. 27, 210 (1!15li). 4. WEISS, B., J. Hiol. (‘hem. 253, 523 (1956). H. E., SMITH, I). I<., .%.\I” JOKES:, I). 5. CARTER, N., ./. Viol. Phrm. 232, 681 (1958). Ii. S\-ENNERHOLM, I,., ASD THORIN, H., Riochivr. et Riophys. Alrfn 41, 371 (IMO). I). .J., WATTS, 1~. Ll., ANI) PAI~I~A7. HASAHAS, 8. HOLMES. II.
I
J.,
1).
236,
‘4.
I)ONALI)
201, 399 (1953). JH. d. HANAHAS
‘THOMPSON
Department OJ” Biochemistry I ‘niwrsit y of IITashington
Seattle, M.ashington Received December 21, 1961
Effect
of Dietary
Cytochrome
Protein
on the DPNH
c Reductase
Activity
of Rat Liver It has been reported from t)his laboratory (1) that DPNH-cytochrome c reductsse activit,ies in mitochondria and microsomes of livers from rats on ‘I’orula yeast diets are greatly reduced, as COIC pared t,o those from animals on an optimal wheat casein ration (McCollmn’s lactating diet). The difference was not (*aused by the lack of vitamin I<: or Factor 3.selenium in the ?‘orrt/a yeast ratio11 which is in use here for the induction of dietar! necrotic liver degeneration (2). Sincue the reducaetl activity could have been related to a hitherto ~11. rerognized trace factor requirement, we have flvt.her investigated the dietary conditions affrcting l)PSH-cytochrorne c reductase levels. It has lwen found that the addition of 107,; casein or an amino acid mixtlwe to the l’orula diet reconstitrlted the mitochondrial l)PiVH-cyt,ochrome c reduct,ase levels to normal (Tahle I). Sources of accessor! fact,ors, sucbh as brewers dried yeast, liver rcsi(Iue (Wilson Laboratories), whole milk powder, or sea salts were without influence. ?‘orula yeast diets are quantitatively and qualitatively poor in protein, as is evident, from the growth rates of animals on such rations. The pro-
OF
BIOCIIEhfISTHY
AXD
96,
BIOPHYSICS
Letters Origin
of the Glyceryl a-Glyceryl
Component
(1962)
to the Editors
of
Ethers1
Earlier work carried out in this laboratory on the lipids of bovine erythrocytes has demonstrated that the ethanolamine phospholipid fraction from these cells is composed largely of a’-alkoxy&acyla-glycerophosphorylethanolamine, I, (1). Probable derivatives of I have been isolated from nerve tissue (2-4), egg yolk (5), calf brain (B), and hu man brain, liver, kidney, and spleen (6). However, no evidence is available on the origin of these compounds. During a study on the chemical nat,ure of bovine hone marrow lipids, a concomitant investigation of the biosynthetic route to glyceryl ethers was carried out. The result,s of preliminary experiments are reported here. The lipid composition of bovine sternal (hematopoietic) marrow and also of “yellow marrow” taken from the long bones of the leg was determined by chromatography on silicic acid (7).2 Interestingly, in sternal marrow t,he ratio of neut)ral lipid t,o phospholipid was found to be 12-14, whereas in t.he “yellow marrow” only neutral lipids, predominantly t,riglycerides, were present. Analysis of the neutral lipids of the long bones con firmed the reported presence of glyceryl ethers (8), and it was further established by silicic acid chromatography that these compounds are associated wit,h t,he triglyceride components. The neutral fraction of i.he sternal lipids was similarly const,ituted, while the phospholipid fraction could be shown to have the composition outlined in Table 1. (ilyceryl ether phospholipids were present in t)he phosphatidyl et)hanolamine and the phosphatidyl choline (lecithin) fractions, but could not be separated chromatographically from their diacyl counterparts. A semi-quantitative plasmalogen assay by the procedure of Kort)on (9) revealed a plasmalogen to phosphorus rat,io of 0.05-0.08 for the tota, phospholipid fraction. In order to study the rate of lipid formation by marrow tissue, experiments were performed in 1 This investigation was supported by grants from the Life Insurance Medical Research Fund, and the American Cancer Society. * The principal difference between circulating erythrocyte and marrow phospholipids lies in the very low (0 to 37;) lecithin content of the former (7).
which glucose-(i-C14 (3.56 mc./mM; Nuclear~Chicage Corporation) was incubated with fresh marrow- tissue.3 In experiment I marrow was isolated by the following procedure: The large bones were removed from the legs of a three-week-old milk-fed calf4 under nitrous oxide and anectine chloride anesthesia. FortyT-nine grams of hematopoietic marrow were removed and centrifuged in saline at 1000 X 9 for 20 min. at 4°C. Two fractions were isolated: one as a light pink floating pellet (Fraction A) and the other as a small amount of red sediment, (Fraction B). Fractions A and B were each suspended in saline at pH 7.4 and glucose-t04 was added. After 15 min. of gentle shaking at 37°C. unlaheled glucose was added, and the incubation was cont,inued for 165 min. Lipids were extracted and the radioact,ivity of various thromatographic fractions assayed with a Packard Tri-Carb scintillation counter. Fraction A incorporated 1.47’; of the label from 5 PC. glucose-t-Cl4 into lipids. Of this total uptake 94(;:, appeared in neutral lipids. Fraction B catalyzed the conversion of 1.75,;. of t.he 10 MC. glucose(i-Cl4 srrbstrate into lipids, with 69’x of t,he total incorporation being into neutral lipids. When hydrolyzed, less than Ir,;, of t,he neutral lipid radioactivity was found in the fatt,y acid fraction, indic:tt,ing that glucose provided only the glycerol unit for the triglycerides. ;2 control experiment in which labeled glucose ~vas added to a lipid extract just prior to chromatography demonstrated that 9fi’;l of the recovered radioactivity was cluted wit.h t,he phosphat idyl swine-phosphatidyl inositol fraction and that, unlike the incubated s:m~ples, this radioactivity could be almost quantitatively removed by washing of the lipid ext,ract with a dilute KC1 solution. The phospholipid portion (1.9 mg. 1’) from Fraction 1%was diluted I:3 with unlaheled marrow phospholipids and chromatographed. The distribution of radioactivity is shown in Table I. .4n analysis of the phosphatidyl ethanolamine fraction for its fat,ty acid ester content made it clear that approximately GO’:;, of the phospholipid was a glyceryl et,her derivative. The glyceryl 3 We are indebt)ed to Mrs. Carolyn Mitchell for her expert assistance in the initial phases of these experiments. 4 In the very young animal, these hones contain marrow active in blood formation.
671
6’72
T,ETTERS
TO THb:
EI)ITORS
1. H.UXAHAN, (‘hem.
T,ipid applied to column contained 7.2 mg. phospholipid phosphorus and 131,000 cts/min. CY*. Rccovery in chromatographic fractions: 04’;; of phospholipid phosphorus :~pplied and !Xi”;, of radioactivity applied.
PC
.IOHN,
“Phosphatidic acid” I’hosphatidyl ethanolamine Phosph;tt,idyl serine, phostidy1 inositol Phosphatidyl choline (lecithin) Sphingomyelin” ‘6 Contained
a slight
I3.,
28.1 1.7
8.6 11 .(i 2.0
42.
i5.0
13.4
I).,
./.
5 Kindly
donated
WATTS,
NORTON.
310 &NNEIJY,
/~FSfXwf;h
CORBET, N.,
Sot.
ANI)
63, 2607
W. T., Hiochim.
contaminant
hy the Seatt,le Parking
C:oln-
J.
Hid.
(l%io).
C:EIGER,
W.
W.. .I.
AIJXANDER, (1911).
et Hiophys.
dlclu
38,
(1960).
I’;. I’., .I. Niol. (‘hem.
2.7 of lecithin
1, 121
11. E.,
C;IT II.
It.,
(l!Kil).
I,ipid
S.,
KORNBLI-Y,
.1,m. (‘hem.
9. 10.
ethers were isolated from this material 1)~ acetolyais and subjected to periodate oxidation (I). The a-carbon of the glyceryl moiety was recovered as the dimedon derivative of formaldehyde, which possessed :I specific activity of 21.6 cts./min./mg. (corrected for dimedon quenching) as compared to 25.6 cts./min./mg. expected if all the radioactivit,> of the glyceryl ether were found in this carbon atom. The specific activity of the other portion (long chain aldehyde) of the molecule after periodate oxidation was 1 ,I cts./min./mg. The lecithin fraction from this experiment also contained radioactive glyceryl ethers, hut in a somewhat lowe caoncentration than found in the ethanolamine 1)hospholipid fraction. The presence of a biosynthetic system for glyccry1 ct,hcr phospholipids in marrow KU confirmed by :I second experiment utilizing fresh sternal marro\v from steers3 and glucose-G-V. Again the glyceryl et.hers of the phosphnlidyl ethanolamine and the lecit,hin fractions tvere radioactive. The experimental data presented here indicate that in bovine marrow the glyceryl moiet,y of glyccrpl ethers can he formed front glucose. The dist rihlltion of label bvould strongly suggest that the glucose metalmlitr directly involved in I.-ol-glycof erophosphate, as occurs in the hiosgnthesis phosphat idic acid (IO), or a metnl~olic:~lly anulogalls rompound. p:t11y.
ANI)
59
2. I)awsos. It. 11. C., Hiochew. J. 56, 621 (1951). 3. E;rJcAR, c. w. F., .lCfU Anal. 27, 210 (1!15li). 4. WEISS, B., J. Hiol. (‘hem. 253, 523 (1956). H. E., SMITH, I). I<., .%.\I” JOKES:, I). 5. CARTER, N., ./. Viol. Phrm. 232, 681 (1958). Ii. S\-ENNERHOLM, I,., ASD THORIN, H., Riochivr. et Riophys. Alrfn 41, 371 (IMO). I). .J., WATTS, 1~. Ll., ANI) PAI~I~A7. HASAHAS, 8. HOLMES. II.
I
J.,
1).
236,
‘4.
I)ONALI)
201, 399 (1953). JH. d. HANAHAS
‘THOMPSON
Department OJ” Biochemistry I ‘niwrsit y of IITashington
Seattle, M.ashington Received December 21, 1961
Effect
of Dietary
Cytochrome
Protein
on the DPNH
c Reductase
Activity
of Rat Liver It has been reported from t)his laboratory (1) that DPNH-cytochrome c reductsse activit,ies in mitochondria and microsomes of livers from rats on ‘I’orula yeast diets are greatly reduced, as COIC pared t,o those from animals on an optimal wheat casein ration (McCollmn’s lactating diet). The difference was not (*aused by the lack of vitamin I<: or Factor 3.selenium in the ?‘orrt/a yeast ratio11 which is in use here for the induction of dietar! necrotic liver degeneration (2). Sincue the reducaetl activity could have been related to a hitherto ~11. rerognized trace factor requirement, we have flvt.her investigated the dietary conditions affrcting l)PSH-cytochrorne c reductase levels. It has lwen found that the addition of 107,; casein or an amino acid mixtlwe to the l’orula diet reconstitrlted the mitochondrial l)PiVH-cyt,ochrome c reduct,ase levels to normal (Tahle I). Sources of accessor! fact,ors, sucbh as brewers dried yeast, liver rcsi(Iue (Wilson Laboratories), whole milk powder, or sea salts were without influence. ?‘orula yeast diets are quantitatively and qualitatively poor in protein, as is evident, from the growth rates of animals on such rations. The pro-