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Intracellular distribution of the free and esterified forms of dolichol in pig liver
.zRCHI”ES
OF
BIOCHEMISTRY
Intracellular
.ZND
BIOPHYSICS
Distribution
503-508 (1968)
128,
of the Free and
Dolichol
of Biochemistry,
Forms
of
in Pig Liver’
I’. H. W. BUTTERWORTH? The Depariment
Esterified
AND
The Universiiy
F.
\Ic’.
HEXIMISG
of Liverpool,
Liverpool,
England
Received May 3, 1968; accepted July 17, 1968 Pig liver (1 kg) yielded 69 mg of dolichols and of this G3c/bwas in a combined form, presumably as a fatty acid ester. A second portion of liver (500 g) from a different pig yielded 74.3 mg of dolichol of which 52% was in a combined form. Analysis of the cell fractions of the second sample of liver indicated that most (76.7y0) of the free dolichols was located in the mitochondria, while the major part (52.752) of the combined form was associated with a “cell debris plus nuclei” fraction. The subcellular distribution of ubiquinone, free sterol, and sterol ester was essentially the same as reported in the literature for rat liver. The proportion of individual doliehols in the mixture of dolichols was essentially the same whether t.he source was the mixture of esterified or free dolichols or whether the tissue was liver, kidney, or spleen.
The structure of dolichol-20, isolated from the unsaponifiable lipid of human kidney and pig liver has been shown (1) to be: C& c3
in the number of int,ernal cis-isoprene residues. The \vork of Burgos and Morton (4) showed that the dolichol preparat,ion is
CK
CH3
-C=CH-CHQ-[CH,-C=C~-CH~h-CH~-C~-~H?-C~z~~
It. was reported that of the eighteen internal isoprene units, fifteen or sixteen were in the cis configuration. This \vas the first report of the isolation of a predominantly cis-polyisoprenoid compound from animal tissues. It is now knoTsn (J. E’. Pennock and 3’. W. Hemming, unpublished work; see also (2) and (3)) that this polyprenol is accompanied by a number of isoprenologs ranging from C&oto ClOsand that t,he Cggpolyprenol is the predominant. isoprenolog of the mixture. Each isoprenolog differs from the other only i The work was aided by Grant AM05282-02 from t,he United Sates Public Health Service. * P. H. W. B. was in receipt of an Agricultural Research Council Research Fellowship. Present. address: 1)epartment. of Chemistry, Harvard University, Cambridge, Massachrrsetts 02138.
fairly evenIy distributed between the subcellular fractions of pig liver. In view of an observation that a significant portion of the dolichols in pig liver might be present as an ester (J. Gaskell andP. W. Hemming, unpublished work, 1961) it became important to reexamine the intracellular distribution of the polyprenols. In this paper the authors put the essentially qualitat.ive observat,ion of Gaskell and Hemming (unpublished work, 1961) onto a quantitative footing and also report on the distribution pattern of free and combined dolichols between the cell fragments of pig liver. EXPERIMENTAL Solvenls and nialerials. Diethy ether (referred to in this paper as ether or simply E) was dried with sodium wire and freshly distilled over reduced 503
504
BUTTERWORTH
iron. Petroleum ether, hoiling pc’int 1O-C,O” (referred to lat,er as “petrol” or simply P) was also dried and redistilled before usr. Alumina was purchased from P. Spence and Sons Ltd., Widnes, Lanes., and was then washed with acid (4). Sufficient water was added to the dried alumina immediately before use, to reduce its activity t,o Brockmann Grade III or II as required. Silica gel G (E. Merck, A. G. Darmstadt, Germany) was used for thin-layer chromatography. For reversed-phase partition chromatography the adsorbent was first coated with paraffin by immersing the prepared layer in a sollition of 5% paraflin in light petroleum and allowing the solvent to evaporate. Preparafion of tissue jar cell jracfionation. Fresh pig liver tissue (500 g) was obtained from a local slaughter house and immediately cooled to approximately 3”. The tissue was cut into small pieces and then homogenized with 0.25 M sucrose (3 vol.) in an all-steel Ultra-Turrax homogenizer (model TP18/2, Junke and Kunkel, K. G., Staufen im Breslau, Germany). Small samples of tissue were homogenized at a time and each for a period of only 3 sec. The combined homogenates were made up t,o 3 liters with 0.25 M sucrose. All operations were conducted at O-5’. Differedal cenfri~ugafion. The homogenate of pig liver was submitted to a scheme of differential centrifugation based on the method of Schneider and Hogeboom (5) carried out at O-5”. A fraction termed “cell debris and nuclei” was sedimented by centrifuging at l,OOOg for 10 min. Mitochondria were than recovered from the supernatant by spinning at 10,OOOgfor 10 min. The mitochondrial pellet was resuspended in 0.25 M sucrose and the washed mitochondria were resedimented. The supernatants from both mitochondrial pellets were hulked. I.ipid erfracfion. The lipid of whole liver tissue were obtained by boiling finely chopped tissue Cl kg) with acetone (1 liter) for 10 min. (The tissue was from a different pig from that used for cell farctionation.) After standing for a further 10 min the acetone sol&on was decanted. This process was repeated twice with aret,one and three times with ether. Further extraction of the same 1 kg of tissue removed negligible amounts of lipid. The extracts were hulked and the acetone was removed by repeated washing with water in a separat,ing funnel. The resulting ethereal solution was dried with anhydrous sodium sulfate and the ether removed, finally in z’uc?lo. The lipid from the mitochondrial fraction was obtained by first boiling for 10 min a suspension of the fraction in ethanol (500 ml). The denatured protein was sedimented by centrifugation and was
AND
HEMMING
ground to a fine powder with anhydrous NapSO?. This dry powder was then extracted with boiling acetone and et,her in a manner essentially the same as that used for the whole liver tissue. The ethanolic supernatant resulting frcm the centrif\tgation was diluted with water (2,500 ml) and extracted with ether (4 X 500 ml). The ether extract was added to that obtained from the NavS04powder, before washing with water. The lipid was recovered from this solution as described above for whole pig liver. The lipid from the “cell debris and nuclei” fraction was obtained in a similar way to that fraction. from the mitochondrial The final bulked srlpernatants from the differential centrifugation was boiled to denatllre the protein and the lipid was extracted in a similar way to that used for the mitochondrial ethanolic suspension. Frncfionation, o.f fhe lipid. The lipid was aarmed with “petrol” and the solution was chromatographed (approximately 4 g lipid each time) on 200 g alumina (Brockmann Grade II) in a glass column (i.d. 4.2 cm) fitted with a glass center rod (o.d. 1.5 cm). Elution was stepwise and the followingeluents (21iters each) were used (in this order) : pet,rol, 1, 2! 3, 4, 5, 6, and 100/h ether in “petrol,” and finally ether. In the rase of the lipid from whole liver the “petrol” fraction was collected in liter portions and when the lipids frc m the cell fractions were chromatographed two separate 2 lit,er portions of 27; ether m “petrol” R-ere run and collected and minor changes were ma.de lat.er in the chromatography. Each chromatographic fraction was rendered free of solvent and a portion was then t,aken up in ethanol and examined in a uv spectrophotometer for rlbiquinotle (see Specfrophofometq below). Each chromatographic fraction was then hoiled for 5 min under reflux with sodium methoxide (20 g Na in 250 ml methanol) using 25 ml reagent per 100 mg lipid. Pyrogallol (0.59>) was also present. The cooled solution was diluted with water and the unsaponifiable lipid extracted with ether. The unsaponifiable lipid from each chromatographic fract,ion was then further chromatographed on Brockmann Grade III alumina (4). The resulting chromatographic fractions were examined for dolichols by ir spectroscopy (see Specfrophotometry). All chromatographic fractions from an.y one unsaponifiable lipid sample that showed the presence of dolichols were hldked, and the mixture was isolated by preparative chromatography as a line on thin layers of silica gel G, using authentic markers. Occasionally, it was necessary to purify the preparation before thin-layer chromatography by chromatography of the acebte on columns of alumina (4).
FI
AN11 COMBINEI>
1WLICHOL
The purified dolichol preparation was weighed and its purit,y checked by comparing its ir spectrum with that of pure dolichol preparatiolrs aljd 1)~ thiwlayer chromatography. The pattern of isoprencllogs ill the free and combined forms of dolichol mixtllre was checked qlmlitat ively by reversed-phase part ition thiw layer rhromat ogrnphy of the samples ot)t ained as indicated above. Paraffin \?‘its used as statiollar) phase and dry acetotle as mobile phase (2). Spec(,opho(o,,,e/r,,/. Ubictllinolle was estimated by following spwt roph~)tomrtricnlly the redrlction by :LIL ethanolic solu~ iolr of the rompound by horohydride (see e.g., ref. 6). The presence of dolichol in a fract iolr was estxhlishetl by observing in their spectrum of a film of the fraction absorptiotl I-xulds at 2.9~ (O--II stretchillg), 6.0~ (unconjllgated tlollble tmlrds), 9.5, ((:-OH) and at 11.9 p (trisubstituted olefin). I’~,o/ein esiimn/ion. The proleill colrtellt, of small samples of sl~l~wllrdar fractiolrs \vas estimated accorditlg 11) a modification (7) of the qlmi~iitalivt: t)illret method. RESUI,TS
AN11
1)lSCUSSION
Whole pig liver (1 kg) yielded 18.7 g of lipid. The results of the subsequent analysis of t,his for dolichols and ubiquinone are presented in Table I. It can bc seen that t’he dolichol preparation was cluted from the column in t\\-o main bands. The first band was principally in fractions 3 and 4 and the second band was principally in fract,ion 6. The most, logical explanation of the biphasic elution is that the earlier band contains dolichols in a bound form (presumably an ester) and that the later band cont,ains dolichol in the free form. Preliminary work on the nature of t,he bound form of these polyprenols suggest
IN
CELL
.io:i
FI:ACTIONS TABLE
~~ISrTHIl~UTION CHR~M,\T~G~~APHIC
X0.
AKiU
FRXT~ON~
WHOLE IJraction
I
OF ~k~LIC!EIOLS PIG
Eluent (70 E in P)
LIVER
CHIQVISONE FROM
IAITTI
IN 0~
(1 kg)
Dolichols (mr)
Ubiquinone (ma .-
1 2 3 4 5 (i 7 8 9 10
0 0 1 2 3 4 5 ti 10 100
0.0 1.3 23.2 lfi.8 3.5 17.4 3 .3 1.4 1.9 0.0
0.00 0.00 0.00 0.44 0.40 4.81 1.18 0.54 0.00 0.00
experiment reported above was 69 mg and of this 63 5% \\-as in the csterified form. Dolichols have also been isolated from several other pig tissues (11). Two of these tissues, kidney and spleen, have been examined furt,her for the presence of dolichol esters. The concentration of total dolichols in each tissue was approximat,ely 25 5% of that in liver. In kidney almost all of the prenol preparation was present as free alcohol whereas in spleen approximately one-fourth was present as ester. lteversed-phase partition thin-layer chromatograpby. shoned that t*hc proportions of t,he individual prenols in the mixt,ure of prenols derived from t’he pool of esters mere the same as from the pool of unesterified prenols. It was also clear that the chromatographic patterns for t’he spleen and kidney preparations were essentially the same as for the liver preparation. In earh case the Cs, alcohol provided L&40% of the mixture with the C&, and Cl,,0 alcohols each providing 20-25 ‘X3and t,he Cs5 and C105alcohols each acwunting for 5-12%‘. Only :l trace of the Cso isoprenolog was presenL Details of t,he analysis of the lipid of each cell fraction from liver are given in Tables II, III, and IV. Also included are the results of gravimctric detJerminations of the “sterol” content of each chromatographic fraction after saponification. These “sterol” figures should bc used as a guide of sterol distribution rather than as an absolute assay of sterol cwntent, of each fract)ion. It is reason-
506
BUTTERWORTH
TABLE II OF UBIQUINOKE, DOLICHOLS ANI) DISTRIIII.TION 'Y~TEROL" IN CHROMATOGRAPHIC FRKTIONS ok' LIPID FROM "CELL DEBRIS + NUCLEI" Fnx~r~os
AND
HEMMING
Ubipuye
Dolichols
0 1 2 2
0 0 0 0
0 <0.5 19.2 1.2
31 4 5 G 8 10 12 Ether
0
<0.5
0
rized. The distribution of ubiyuinone can bc taken as a guide to the efficiency of the disruption of the tissue and of the fractionation of the cell fractions so liberated. It is widely accepted that the major part of the ubiyuinone of the cells of rat liver and kidney occurs in the mitochondria (12, 13). IJbiyuinone can also be isolated from the other cell fractions but t,he results of Sastri et al. (IX), who also &died t,he distribution of succinoxidase, show that some of this extramitochondrial ubiquinone is associated with
0.70 0.95 0.14 0 0
1.4 3.3 <0.5 0 0
0 0 0 0 354.0
DISTRIBUTION OF UBIQUINOKE, DOLICHOLS AND "STEROL" IN CHROMATOGKAPHIC FRACTIONS or LIPID FROM SuPERNaTaNT FRACTION
Eluent (9% E in P)
In
TABLE
bfi)
m “sEY 0 13.0 19.5 0
III
DIS'I~RIBu'I~ION OF UBIQUINOXE, DOLICHOLS AND "STEROL" IN CHROMATOGRAPHIC FRICTIONS OF LWID FROM MITOCHONDRIAL FRACTION Eluent (% E in PI
Ubipu$on In
0 i\
0 0
0 7.5
2.0 8.0
21
0
2.8
0
3 4 :I
0 0.04 2.53
<0.5 <0.5 8.5
0 0 0
71
2.74
18.8
m D”?%‘s
‘ ‘S kml” (md
5.8
81 12j Ether
0
0
TABLE
Eluent (% E in P)
Ubiquinone ha
0 1 2 2 3 4 5 G 8 10 12 100
0 0 0 0 0.15
IV
m Do:icFY
“Sterol” (WT)
<0.5 3.6 <0.5 0 0 0
0 0 0 0 45.4 222.0
0.42 1.40 0.84 0.07 0 0
TABLE
V
~LECOVERY OF FREE AND ESTERIFIED DOLICHOLS AND OF UBIQUINONE FROM SUBCELLULAR FRACTIONS FROM 500 G I'IG LIVER
58.8
able to assume that the sterol recovered in the early chromatographic fractions (“petrol,” 1 and 2 % ether in ‘Lpetrol” fractions) was present in the original lipid as an ester. It should again be noted that in each cell fraction the elution pattern of the dolichol mixture was biphasic, similar to that of sterol but different from the monophasic elution pattern of ubiyuinone. The intracellular distribution of the lipid constituents is made more clear in Table V, where the detailed results of Tables II, III, and IV are brought together and summa-
Ubiquinone (mg) Free dolichols (mg) Esterified dolichols bd Ubiquinone (% t’otal Free dolichols (2 total) Esterified dolichols (70 total) “sterol” (‘s Free total) Esterified “sterol”
1.79 4.7 20.4
5.46 27.3 10.3
2.88 3.6 8.0
10.13 35.6 38.7
17.7 13.2
53.9 76.7
28.4 10.1
100 100
52.7
26.6
20.7
100
44
34
22
100
27
8
65
100
FI
iz?jl)
COMBINET:,
L)OLICI~OI,
succinoxidase activity and may, therefore, be of mitochondrial origin. The distribution patt,ern for ubiyuinone in rat liver (13) and pig liver (4) is recorded in Table VI, together w&h that obtained by the authors. The three sets of figures are in reasonably good agreement and indicate that the preparation of the cell fractions reported in this paper was satisfactory. One of the most striking results in Tables V and VII is the marked difference between the distribution of the free and esterified forms of dolichols. About, half of the dolichols was in the free form and of this three-fourths was present in t’he mitochondria, t,he rest being equally divided between the ot)her two fractions. On the ot’her hand only onefourth of t’he esterified alcohols was present, in the mitochondrial fraction, the major portion (one-half) being located in the “cell debris + nuclei” fraction. It, is not without significance that t,his distribution pattern is ver!’ similar to t,hat for the closely related hexnhydropolyprenols of Aspe~gillus furnigatus (14). The Aspergillus prenols are predominant~ly cz’s, ront,ain 90 t,o 120 carbon atoms, and differ from the dolic*hols principally in that the W- and + isoprene residues are sat,urated (15). The two sets of results are compared in Table VIII. The figures for the free dolichol indicate that, this compound is as well qualified as ubiquinone to be considered as (sonfined to the mitochondrial caompartment of the ~11. It may bc that such a situation is consistent, wit,11 a structural role. It) is also relevant that, rwently a cofactor role for polyisoprcnoid alcohols in bacterial biosynthesis of pcptidoglycan units and in the transfer of thcsc units across the bwterial cell memTABLE COMI>ARIS~N
OF INTR.WELLUIAR
UBIQUINONE
WITH THE
(ref 13) Pig (ref 4) Pig (this paper)
l~ISTRII3IJTIoK
TH.\.L’
OF
REPORTEI)
IN
Xtochondria
Sllpernatant
LITER.\T~,RE
Celld~xk
Rat,
VI
37.5q;, 27, GS;, 17.7’j;,
+
41 0”’/(’ 54,9(‘f,P 53.9’)‘;)
21.57:, 17.5”~/” 28.‘g]
IN CELL
307
Fl:ilCT10SS TABLE
COI\‘CENTRATIOS
OF
DOLICHOLS
VII
FREE
AND
OF
.\SD
I+XERIIYELI
UBIQUINONE
IN
SIJIWELIAJLAR FK.ICTIONS
____Uhiquinone (mg/g protein) Free dolichols (mg/ g protein) Esterified dolichols (mg/g protein)
0.095
0.360
0.070
0.24
1.82
0.09
1.06
0.G9
0.20
TABLE
VIII
ESTERIFIED POLYPRENOL IS CELL FRACTIONS OF PIG LIVEI~ .\SD A. ~umiyat7cs AS a PEKCESTAGE OF THE TOTAL IN EACH TISSUE
DISTRIMJ,PION
OF FREE
ANI)
.4. .f2lmigalas (ref. 14)
Pig liver
~.
Cell fraction Free
Nuclei and cell debris Mitochorldria Sttpernatant
Esterified
Free
Esterified
13.2
52.7
9.2
34.3
76.7
2G.G
77.0
24.1
10.1
20.7
13.8
41.5
brane was proposed (16, 17). The results reported in this paper raise the possibility of a relat)ed role for dolichols in particulate fractions of pig liver. It is interesting t,hat the distribution of the est,erified form of dolichol in no way resembles that found for stem1 esters. The results for sterol est,er are fundamentally t#he same as those obtained by Schotz et al. (10) when working with rat liver. In both casts by far t’he major port’ion of the ester is to be found in the supernatal& fraction. :I similar picture holds for vitamin X ester (IS) and it, has been suggested (17) that t,his is the st)orage form of t,he alcohol. Inasmuch as the distribution pattern of esterified dolichols is very different from that of sterol and vitamirl A esters it may be that t,his is not simpl! a storage form of dolicahols.
1. Pig liver (1 kg) yielded 69 mg of dolichols of which 63 ‘Z was in a wmbined form, presumably as :I fatty acid ester.
508
BUTTERWORTH
2. A sample of liver (500 g) from a different pig yielded 74.3 mg of dolichols of which 52 % was in a combined form. 3. Most (76.7%) of the free dolichols was located in the mitochondria, Lvhile the major part (52.7%)) of the combined form was associated with a “cell debris plus nuclei” fraction. 4. The subcellular distribution of ubiyuinone, free st)erol, and sterol ester \vas essentially the same as reported in the literature for rat liver. 5. The proportion of individual dolichols in the mixture of dolicholx was essentially the same whether the source was the mixture of esterified or free dolichols or whether the tissue was liver, kidney, or spleen. ACKNOWLEDGMENT8 The authors are grateful for useful discussions with Professor R. A. Morton and I)r. J. F. Pcnnock and also for the valllable technical assistance of Miss Brenda Williams in part of the work. REFERENCES 1. BURGOS,J., IIIS:MMING,F. W., PENNOCK,J. F., AND MORTON, R. A. Biochem, J., 88, -I70 (1963). 2. DCNPHY, P. J., KERR, J. I)., PENNOCK, J. F., WHITTLE, I<., >IND FEENEY, J., Biochim. Biophys. Ada, 136, 136 (1967). 3. FEENEY, J., .~ND HEMUING, F. W., Anal. Biochem., 20, 1 (1967).
AND
HEMMING
4. BURGOS,J., AND ~UORTON,R. A.,
Biochern.
J.,
82, 454 (1962). 5. SCHNEIDER, W. C., \ND HO~~EIJOOM,G. II., J. Biol. Chcm., 183, 123 (1950). 6.
7. CHANCE, II., AXU I~EDFEIRS,
IX. II.,
Hiochem.
J., 80, 632 (1961). A.” London and New 8. MOORE, T., “Vit,amin 1’ork, I+:lsevier (1957). 9. IIAXUAHAS, I). J., “Lipide Chemistry,” p. 201. Wiley, New \I-ork (1960). 10. SCHOTZ,~~.C., I
202,
227
(1953).
19. GLOVER, J., (;OODWN, T. W., END MORTON, 11. A. Biochem. J., 41, 97 (1947).
OF
BIOCHEMISTRY
Intracellular
.ZND
BIOPHYSICS
Distribution
503-508 (1968)
128,
of the Free and
Dolichol
of Biochemistry,
Forms
of
in Pig Liver’
I’. H. W. BUTTERWORTH? The Depariment
Esterified
AND
The Universiiy
F.
\Ic’.
HEXIMISG
of Liverpool,
Liverpool,
England
Received May 3, 1968; accepted July 17, 1968 Pig liver (1 kg) yielded 69 mg of dolichols and of this G3c/bwas in a combined form, presumably as a fatty acid ester. A second portion of liver (500 g) from a different pig yielded 74.3 mg of dolichol of which 52% was in a combined form. Analysis of the cell fractions of the second sample of liver indicated that most (76.7y0) of the free dolichols was located in the mitochondria, while the major part (52.752) of the combined form was associated with a “cell debris plus nuclei” fraction. The subcellular distribution of ubiquinone, free sterol, and sterol ester was essentially the same as reported in the literature for rat liver. The proportion of individual doliehols in the mixture of dolichols was essentially the same whether t.he source was the mixture of esterified or free dolichols or whether the tissue was liver, kidney, or spleen.
The structure of dolichol-20, isolated from the unsaponifiable lipid of human kidney and pig liver has been shown (1) to be: C& c3
in the number of int,ernal cis-isoprene residues. The \vork of Burgos and Morton (4) showed that the dolichol preparat,ion is
CK
CH3
-C=CH-CHQ-[CH,-C=C~-CH~h-CH~-C~-~H?-C~z~~
It. was reported that of the eighteen internal isoprene units, fifteen or sixteen were in the cis configuration. This \vas the first report of the isolation of a predominantly cis-polyisoprenoid compound from animal tissues. It is now knoTsn (J. E’. Pennock and 3’. W. Hemming, unpublished work; see also (2) and (3)) that this polyprenol is accompanied by a number of isoprenologs ranging from C&oto ClOsand that t,he Cggpolyprenol is the predominant. isoprenolog of the mixture. Each isoprenolog differs from the other only i The work was aided by Grant AM05282-02 from t,he United Sates Public Health Service. * P. H. W. B. was in receipt of an Agricultural Research Council Research Fellowship. Present. address: 1)epartment. of Chemistry, Harvard University, Cambridge, Massachrrsetts 02138.
fairly evenIy distributed between the subcellular fractions of pig liver. In view of an observation that a significant portion of the dolichols in pig liver might be present as an ester (J. Gaskell andP. W. Hemming, unpublished work, 1961) it became important to reexamine the intracellular distribution of the polyprenols. In this paper the authors put the essentially qualitat.ive observat,ion of Gaskell and Hemming (unpublished work, 1961) onto a quantitative footing and also report on the distribution pattern of free and combined dolichols between the cell fragments of pig liver. EXPERIMENTAL Solvenls and nialerials. Diethy ether (referred to in this paper as ether or simply E) was dried with sodium wire and freshly distilled over reduced 503
504
BUTTERWORTH
iron. Petroleum ether, hoiling pc’int 1O-C,O” (referred to lat,er as “petrol” or simply P) was also dried and redistilled before usr. Alumina was purchased from P. Spence and Sons Ltd., Widnes, Lanes., and was then washed with acid (4). Sufficient water was added to the dried alumina immediately before use, to reduce its activity t,o Brockmann Grade III or II as required. Silica gel G (E. Merck, A. G. Darmstadt, Germany) was used for thin-layer chromatography. For reversed-phase partition chromatography the adsorbent was first coated with paraffin by immersing the prepared layer in a sollition of 5% paraflin in light petroleum and allowing the solvent to evaporate. Preparafion of tissue jar cell jracfionation. Fresh pig liver tissue (500 g) was obtained from a local slaughter house and immediately cooled to approximately 3”. The tissue was cut into small pieces and then homogenized with 0.25 M sucrose (3 vol.) in an all-steel Ultra-Turrax homogenizer (model TP18/2, Junke and Kunkel, K. G., Staufen im Breslau, Germany). Small samples of tissue were homogenized at a time and each for a period of only 3 sec. The combined homogenates were made up t,o 3 liters with 0.25 M sucrose. All operations were conducted at O-5’. Differedal cenfri~ugafion. The homogenate of pig liver was submitted to a scheme of differential centrifugation based on the method of Schneider and Hogeboom (5) carried out at O-5”. A fraction termed “cell debris and nuclei” was sedimented by centrifuging at l,OOOg for 10 min. Mitochondria were than recovered from the supernatant by spinning at 10,OOOgfor 10 min. The mitochondrial pellet was resuspended in 0.25 M sucrose and the washed mitochondria were resedimented. The supernatants from both mitochondrial pellets were hulked. I.ipid erfracfion. The lipid of whole liver tissue were obtained by boiling finely chopped tissue Cl kg) with acetone (1 liter) for 10 min. (The tissue was from a different pig from that used for cell farctionation.) After standing for a further 10 min the acetone sol&on was decanted. This process was repeated twice with aret,one and three times with ether. Further extraction of the same 1 kg of tissue removed negligible amounts of lipid. The extracts were hulked and the acetone was removed by repeated washing with water in a separat,ing funnel. The resulting ethereal solution was dried with anhydrous sodium sulfate and the ether removed, finally in z’uc?lo. The lipid from the mitochondrial fraction was obtained by first boiling for 10 min a suspension of the fraction in ethanol (500 ml). The denatured protein was sedimented by centrifugation and was
AND
HEMMING
ground to a fine powder with anhydrous NapSO?. This dry powder was then extracted with boiling acetone and et,her in a manner essentially the same as that used for the whole liver tissue. The ethanolic supernatant resulting frcm the centrif\tgation was diluted with water (2,500 ml) and extracted with ether (4 X 500 ml). The ether extract was added to that obtained from the NavS04powder, before washing with water. The lipid was recovered from this solution as described above for whole pig liver. The lipid from the “cell debris and nuclei” fraction was obtained in a similar way to that fraction. from the mitochondrial The final bulked srlpernatants from the differential centrifugation was boiled to denatllre the protein and the lipid was extracted in a similar way to that used for the mitochondrial ethanolic suspension. Frncfionation, o.f fhe lipid. The lipid was aarmed with “petrol” and the solution was chromatographed (approximately 4 g lipid each time) on 200 g alumina (Brockmann Grade II) in a glass column (i.d. 4.2 cm) fitted with a glass center rod (o.d. 1.5 cm). Elution was stepwise and the followingeluents (21iters each) were used (in this order) : pet,rol, 1, 2! 3, 4, 5, 6, and 100/h ether in “petrol,” and finally ether. In the rase of the lipid from whole liver the “petrol” fraction was collected in liter portions and when the lipids frc m the cell fractions were chromatographed two separate 2 lit,er portions of 27; ether m “petrol” R-ere run and collected and minor changes were ma.de lat.er in the chromatography. Each chromatographic fraction was rendered free of solvent and a portion was then t,aken up in ethanol and examined in a uv spectrophotometer for rlbiquinotle (see Specfrophofometq below). Each chromatographic fraction was then hoiled for 5 min under reflux with sodium methoxide (20 g Na in 250 ml methanol) using 25 ml reagent per 100 mg lipid. Pyrogallol (0.59>) was also present. The cooled solution was diluted with water and the unsaponifiable lipid extracted with ether. The unsaponifiable lipid from each chromatographic fract,ion was then further chromatographed on Brockmann Grade III alumina (4). The resulting chromatographic fractions were examined for dolichols by ir spectroscopy (see Specfrophotometry). All chromatographic fractions from an.y one unsaponifiable lipid sample that showed the presence of dolichols were hldked, and the mixture was isolated by preparative chromatography as a line on thin layers of silica gel G, using authentic markers. Occasionally, it was necessary to purify the preparation before thin-layer chromatography by chromatography of the acebte on columns of alumina (4).
FI
AN11 COMBINEI>
1WLICHOL
The purified dolichol preparation was weighed and its purit,y checked by comparing its ir spectrum with that of pure dolichol preparatiolrs aljd 1)~ thiwlayer chromatography. The pattern of isoprencllogs ill the free and combined forms of dolichol mixtllre was checked qlmlitat ively by reversed-phase part ition thiw layer rhromat ogrnphy of the samples ot)t ained as indicated above. Paraffin \?‘its used as statiollar) phase and dry acetotle as mobile phase (2). Spec(,opho(o,,,e/r,,/. Ubictllinolle was estimated by following spwt roph~)tomrtricnlly the redrlction by :LIL ethanolic solu~ iolr of the rompound by horohydride (see e.g., ref. 6). The presence of dolichol in a fract iolr was estxhlishetl by observing in their spectrum of a film of the fraction absorptiotl I-xulds at 2.9~ (O--II stretchillg), 6.0~ (unconjllgated tlollble tmlrds), 9.5, ((:-OH) and at 11.9 p (trisubstituted olefin). I’~,o/ein esiimn/ion. The proleill colrtellt, of small samples of sl~l~wllrdar fractiolrs \vas estimated accorditlg 11) a modification (7) of the qlmi~iitalivt: t)illret method. RESUI,TS
AN11
1)lSCUSSION
Whole pig liver (1 kg) yielded 18.7 g of lipid. The results of the subsequent analysis of t,his for dolichols and ubiquinone are presented in Table I. It can bc seen that t’he dolichol preparation was cluted from the column in t\\-o main bands. The first band was principally in fractions 3 and 4 and the second band was principally in fract,ion 6. The most, logical explanation of the biphasic elution is that the earlier band contains dolichols in a bound form (presumably an ester) and that the later band cont,ains dolichol in the free form. Preliminary work on the nature of t,he bound form of these polyprenols suggest
IN
CELL
.io:i
FI:ACTIONS TABLE
~~ISrTHIl~UTION CHR~M,\T~G~~APHIC
X0.
AKiU
FRXT~ON~
WHOLE IJraction
I
OF ~k~LIC!EIOLS PIG
Eluent (70 E in P)
LIVER
CHIQVISONE FROM
IAITTI
IN 0~
(1 kg)
Dolichols (mr)
Ubiquinone (ma .-
1 2 3 4 5 (i 7 8 9 10
0 0 1 2 3 4 5 ti 10 100
0.0 1.3 23.2 lfi.8 3.5 17.4 3 .3 1.4 1.9 0.0
0.00 0.00 0.00 0.44 0.40 4.81 1.18 0.54 0.00 0.00
experiment reported above was 69 mg and of this 63 5% \\-as in the csterified form. Dolichols have also been isolated from several other pig tissues (11). Two of these tissues, kidney and spleen, have been examined furt,her for the presence of dolichol esters. The concentration of total dolichols in each tissue was approximat,ely 25 5% of that in liver. In kidney almost all of the prenol preparation was present as free alcohol whereas in spleen approximately one-fourth was present as ester. lteversed-phase partition thin-layer chromatograpby. shoned that t*hc proportions of t,he individual prenols in the mixt,ure of prenols derived from t’he pool of esters mere the same as from the pool of unesterified prenols. It was also clear that the chromatographic patterns for t’he spleen and kidney preparations were essentially the same as for the liver preparation. In earh case the Cs, alcohol provided L&40% of the mixture with the C&, and Cl,,0 alcohols each providing 20-25 ‘X3and t,he Cs5 and C105alcohols each acwunting for 5-12%‘. Only :l trace of the Cso isoprenolog was presenL Details of t,he analysis of the lipid of each cell fraction from liver are given in Tables II, III, and IV. Also included are the results of gravimctric detJerminations of the “sterol” content of each chromatographic fraction after saponification. These “sterol” figures should bc used as a guide of sterol distribution rather than as an absolute assay of sterol cwntent, of each fract)ion. It is reason-
506
BUTTERWORTH
TABLE II OF UBIQUINOKE, DOLICHOLS ANI) DISTRIIII.TION 'Y~TEROL" IN CHROMATOGRAPHIC FRKTIONS ok' LIPID FROM "CELL DEBRIS + NUCLEI" Fnx~r~os
AND
HEMMING
Ubipuye
Dolichols
0 1 2 2
0 0 0 0
0 <0.5 19.2 1.2
31 4 5 G 8 10 12 Ether
0
<0.5
0
rized. The distribution of ubiyuinone can bc taken as a guide to the efficiency of the disruption of the tissue and of the fractionation of the cell fractions so liberated. It is widely accepted that the major part of the ubiyuinone of the cells of rat liver and kidney occurs in the mitochondria (12, 13). IJbiyuinone can also be isolated from the other cell fractions but t,he results of Sastri et al. (IX), who also &died t,he distribution of succinoxidase, show that some of this extramitochondrial ubiquinone is associated with
0.70 0.95 0.14 0 0
1.4 3.3 <0.5 0 0
0 0 0 0 354.0
DISTRIBUTION OF UBIQUINOKE, DOLICHOLS AND "STEROL" IN CHROMATOGKAPHIC FRACTIONS or LIPID FROM SuPERNaTaNT FRACTION
Eluent (9% E in P)
In
TABLE
bfi)
m “sEY 0 13.0 19.5 0
III
DIS'I~RIBu'I~ION OF UBIQUINOXE, DOLICHOLS AND "STEROL" IN CHROMATOGRAPHIC FRICTIONS OF LWID FROM MITOCHONDRIAL FRACTION Eluent (% E in PI
Ubipu$on In
0 i\
0 0
0 7.5
2.0 8.0
21
0
2.8
0
3 4 :I
0 0.04 2.53
<0.5 <0.5 8.5
0 0 0
71
2.74
18.8
m D”?%‘s
‘ ‘S kml” (md
5.8
81 12j Ether
0
0
TABLE
Eluent (% E in P)
Ubiquinone ha
0 1 2 2 3 4 5 G 8 10 12 100
0 0 0 0 0.15
IV
m Do:icFY
“Sterol” (WT)
<0.5 3.6 <0.5 0 0 0
0 0 0 0 45.4 222.0
0.42 1.40 0.84 0.07 0 0
TABLE
V
~LECOVERY OF FREE AND ESTERIFIED DOLICHOLS AND OF UBIQUINONE FROM SUBCELLULAR FRACTIONS FROM 500 G I'IG LIVER
58.8
able to assume that the sterol recovered in the early chromatographic fractions (“petrol,” 1 and 2 % ether in ‘Lpetrol” fractions) was present in the original lipid as an ester. It should again be noted that in each cell fraction the elution pattern of the dolichol mixture was biphasic, similar to that of sterol but different from the monophasic elution pattern of ubiyuinone. The intracellular distribution of the lipid constituents is made more clear in Table V, where the detailed results of Tables II, III, and IV are brought together and summa-
Ubiquinone (mg) Free dolichols (mg) Esterified dolichols bd Ubiquinone (% t’otal Free dolichols (2 total) Esterified dolichols (70 total) “sterol” (‘s Free total) Esterified “sterol”
1.79 4.7 20.4
5.46 27.3 10.3
2.88 3.6 8.0
10.13 35.6 38.7
17.7 13.2
53.9 76.7
28.4 10.1
100 100
52.7
26.6
20.7
100
44
34
22
100
27
8
65
100
FI
iz?jl)
COMBINET:,
L)OLICI~OI,
succinoxidase activity and may, therefore, be of mitochondrial origin. The distribution patt,ern for ubiyuinone in rat liver (13) and pig liver (4) is recorded in Table VI, together w&h that obtained by the authors. The three sets of figures are in reasonably good agreement and indicate that the preparation of the cell fractions reported in this paper was satisfactory. One of the most striking results in Tables V and VII is the marked difference between the distribution of the free and esterified forms of dolichols. About, half of the dolichols was in the free form and of this three-fourths was present in t’he mitochondria, t,he rest being equally divided between the ot)her two fractions. On the ot’her hand only onefourth of t’he esterified alcohols was present, in the mitochondrial fraction, the major portion (one-half) being located in the “cell debris + nuclei” fraction. It, is not without significance that t,his distribution pattern is ver!’ similar to t,hat for the closely related hexnhydropolyprenols of Aspe~gillus furnigatus (14). The Aspergillus prenols are predominant~ly cz’s, ront,ain 90 t,o 120 carbon atoms, and differ from the dolic*hols principally in that the W- and + isoprene residues are sat,urated (15). The two sets of results are compared in Table VIII. The figures for the free dolichol indicate that, this compound is as well qualified as ubiquinone to be considered as (sonfined to the mitochondrial caompartment of the ~11. It may bc that such a situation is consistent, wit,11 a structural role. It) is also relevant that, rwently a cofactor role for polyisoprcnoid alcohols in bacterial biosynthesis of pcptidoglycan units and in the transfer of thcsc units across the bwterial cell memTABLE COMI>ARIS~N
OF INTR.WELLUIAR
UBIQUINONE
WITH THE
(ref 13) Pig (ref 4) Pig (this paper)
l~ISTRII3IJTIoK
TH.\.L’
OF
REPORTEI)
IN
Xtochondria
Sllpernatant
LITER.\T~,RE
Celld~xk
Rat,
VI
37.5q;, 27, GS;, 17.7’j;,
+
41 0”’/(’ 54,9(‘f,P 53.9’)‘;)
21.57:, 17.5”~/” 28.‘g]
IN CELL
307
Fl:ilCT10SS TABLE
COI\‘CENTRATIOS
OF
DOLICHOLS
VII
FREE
AND
OF
.\SD
I+XERIIYELI
UBIQUINONE
IN
SIJIWELIAJLAR FK.ICTIONS
____Uhiquinone (mg/g protein) Free dolichols (mg/ g protein) Esterified dolichols (mg/g protein)
0.095
0.360
0.070
0.24
1.82
0.09
1.06
0.G9
0.20
TABLE
VIII
ESTERIFIED POLYPRENOL IS CELL FRACTIONS OF PIG LIVEI~ .\SD A. ~umiyat7cs AS a PEKCESTAGE OF THE TOTAL IN EACH TISSUE
DISTRIMJ,PION
OF FREE
ANI)
.4. .f2lmigalas (ref. 14)
Pig liver
~.
Cell fraction Free
Nuclei and cell debris Mitochorldria Sttpernatant
Esterified
Free
Esterified
13.2
52.7
9.2
34.3
76.7
2G.G
77.0
24.1
10.1
20.7
13.8
41.5
brane was proposed (16, 17). The results reported in this paper raise the possibility of a relat)ed role for dolichols in particulate fractions of pig liver. It is interesting t,hat the distribution of the est,erified form of dolichol in no way resembles that found for stem1 esters. The results for sterol est,er are fundamentally t#he same as those obtained by Schotz et al. (10) when working with rat liver. In both casts by far t’he major port’ion of the ester is to be found in the supernatal& fraction. :I similar picture holds for vitamin X ester (IS) and it, has been suggested (17) that t,his is the st)orage form of t,he alcohol. Inasmuch as the distribution pattern of esterified dolichols is very different from that of sterol and vitamirl A esters it may be that t,his is not simpl! a storage form of dolicahols.
1. Pig liver (1 kg) yielded 69 mg of dolichols of which 63 ‘Z was in a wmbined form, presumably as :I fatty acid ester.
508
BUTTERWORTH
2. A sample of liver (500 g) from a different pig yielded 74.3 mg of dolichols of which 52 % was in a combined form. 3. Most (76.7%) of the free dolichols was located in the mitochondria, Lvhile the major part (52.7%)) of the combined form was associated with a “cell debris plus nuclei” fraction. 4. The subcellular distribution of ubiyuinone, free st)erol, and sterol ester \vas essentially the same as reported in the literature for rat liver. 5. The proportion of individual dolichols in the mixture of dolicholx was essentially the same whether the source was the mixture of esterified or free dolichols or whether the tissue was liver, kidney, or spleen. ACKNOWLEDGMENT8 The authors are grateful for useful discussions with Professor R. A. Morton and I)r. J. F. Pcnnock and also for the valllable technical assistance of Miss Brenda Williams in part of the work. REFERENCES 1. BURGOS,J., IIIS:MMING,F. W., PENNOCK,J. F., AND MORTON, R. A. Biochem, J., 88, -I70 (1963). 2. DCNPHY, P. J., KERR, J. I)., PENNOCK, J. F., WHITTLE, I<., >IND FEENEY, J., Biochim. Biophys. Ada, 136, 136 (1967). 3. FEENEY, J., .~ND HEMUING, F. W., Anal. Biochem., 20, 1 (1967).
AND
HEMMING
4. BURGOS,J., AND ~UORTON,R. A.,
Biochern.
J.,
82, 454 (1962). 5. SCHNEIDER, W. C., \ND HO~~EIJOOM,G. II., J. Biol. Chcm., 183, 123 (1950). 6.
7. CHANCE, II., AXU I~EDFEIRS,
IX. II.,
Hiochem.
J., 80, 632 (1961). A.” London and New 8. MOORE, T., “Vit,amin 1’ork, I+:lsevier (1957). 9. IIAXUAHAS, I). J., “Lipide Chemistry,” p. 201. Wiley, New \I-ork (1960). 10. SCHOTZ,~~.C., I
202,
227
(1953).
19. GLOVER, J., (;OODWN, T. W., END MORTON, 11. A. Biochem. J., 41, 97 (1947).