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In vivo incorporation of [2-14C]-mevalonate into dolichol of rabbit and pig liver
ARCHIVES
OF BIOCHEMISTRY
In Viva
AND
Incorporation
BIOPHYSICS
of [2J4C]-M and
P. H. W. BUTTERWORTH: Department
G46-653 (19GG)
113,
evalonate
into
Dolichol
Pig Liver’
H. H. DRAPER,3 F. W. HEMMING,
of Biochemistry,
The University
Received
of Rabbit
October
of Liverpool,
Lioerpool,
9ND
R. A. MORTON
England
11, 1965
In two experiments with pigs, administration of [2-14C] over 14 hours led to a level of incorporation into the poly cis-isoprenoid alcohol dolichol that was low but significant. The figures for total incorporation of radioactivity into dolichol were 15 and 470/d of those for ubiquinone, and 0.1 and 0.167, of those for the squalene-cholesterol system. A similar experiment with rabbits yielded corresponding figures of 3.5 and 0.2yG. Rigorous purification failed to change significantly the specific activity of the doichol. It is concluded that mammalian tissues are capable of synthesizing poly cisisoprenoids, but that in the case of dolichol in rabbit and pig liver the rate of synt)hesis is distinctly lower than that of poly-trans.isoprenoids.
Pig liver is a relatively rich source of dolichol (60-140 mg per kilogram), but rabbit liver cont’ains less (5-20 mg per kilogram t)issue). The alcohol has fifteen or sixteen of its eighteen internal isoprene units in t’he cis configuration (1). CHa CHa-
involved in normal isoprene polymerizat.ion. One would expect some st,ereochemical difference between t,he int’ermediates of t’he all-&s and all-trans pathways. Only recently, wit.h the use of [2-14C],4R-[4-3H]- and [2-14Cj,4S[4-3H]-mevalonic acid, has it been possible to
CHa
CHa
c: =CH-CHr-(-CHz&=CH-CH+-CHz-
4 H-CHs-CHzOH Dolichol
As a poly-cis isoprenoid compound it is related only to one ot#her group of compounds so far described, the nat’ural rubbers (2). &dies on the biosynthesis of rubber from mevalonate by the latex from Hevea brasiliensis have been reviewed recently by Bonner (2), and there is little doubt that in principle this follows the same pathway and involves t’he same int’ermediates as t’hose 1 This work was supported in part by grant AM 05282-02 from United States Public Health Service. 2 Holder of an Agricultural Research Council Research Studentship. Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin. a Present address: Division of Nutritional Biochemistry, Department of Animal Science, Universit,y of Illinois, Urbana, Illinois. 646
demonstrate this experimentally (3). It is clear that the c&form of t’he rubber isoprene unit is produced init#ially and that a trans-cis isomerase is not, involved. The other principal polyisoprenoid compounds in pig and rabbit liver are cholesterol, squalene, and ubiquinone. The biosynthesis of all trans-squalene from mevalonate is now almost fully worked out,, and recently it has been shown that the tyans configuration is formed by a process involving stereoselect.ive elimination of t*he hydrogen, which when replaced by a heavy hydrogen isotope would confer t,he 4S-configuration on the original mevalonate (4,5). The 4R-hydrogen remains intact. In t,he biosynt’hcsis of poly-cis rubber the reverse holds. Ubiquinone has an all-trans isoprenoid side chain, and it has been shown by a
INCORPORATION
OF MVA
number of workers to be derived from mevalonate (6). It is tacitly assumed that the steps from mevalonate to farnesyl pyrophosphate are common t.o the synthesis of both ubiquinone and squalene. Presumably chain lengthening of farnesyl pyrophosphate then continues until (in pig litier), a Cjo isoprenyl pyrophosphate is formed, and this can t,hen be condensed to the quinone nucleus of ubiquinone (7) or to a phenolic precursor thereof (S, 9). Thele is in fact surprisingly little direct’ evidence to substantiate t,his. Support for the idea is gained from the isolation (10) of a CsO polyisoprenoid alcohol from a tissue rich in ubiquinone10 and from the report t’hat solanesyl pyrophosphate will condense with ubiquinone-0 under the influence of a mitochondrial enzyme (7). Nevertheless, despite the lack of direct evidence, this seems to be the most logical route for the biosynthesis of the isoprenoid side chain of ubiquinone. Some knowledge of the relative rates of incorporation of mevalonate into the alltram isoprenoids squalene, cholesterol, and ubiquinone has already been gained, and it may be that this is to some extent under the contBrol (probably indirectly) of vitamin A (ll-13), although recent work by J. Green’s group, (14) throws some doubt on this. In this paper the authors have investigated the possible incorporation of [2-14C]mevalonate into t’he cis alcohol dolichol by pig and rabbit liver, and have made some observations on the rate of biosynthesis of this compound in relation t,o t,he rate of synthesis of the all-trans compounds. EXPERIMENTAL
Administration
uf Mevalonate
Rabbits. In experiment 1, 5 adult Columbian rabbits (2.6-3.2 kg each) were each given 16 flC of [2-‘%]mevalonate by intraperitoneal injection. The animals were killed at the following postinjection times: 0.5, 0.75, 1.0, 2.0, and 3.0 hours, respectively, and the livers were removed immediately for analysis. In experiment 2, 25 *C [2-‘4C] mevalonate was given to each of 3 New Zealand White rabbits (weighing about 2 kg each) in 5 equal intraperitoneal injections at a-hourly intervals over 12 hours. The animals were killed 2 hours after the last dose and the livers were removed and bulked for analysis.
INTO
LIVER
DOLICHOL
647
Pigs. In experiment 3, 175 PC [2-‘%]mevalonate was injected into a pig (36 kg) by four equal intramuscular doses at 4-hourly intervals over a period of 12 hours. The pig was killed after a further 2 hours and the liver was removed immediately and analyzed. In experiment 4, 100 PC of [2-‘%]mevalonate was given to a pig (23 kg) under the same conditions as for experiment 3.
Solvents and Materials Diethyl ether was dried with sodium wire and was distilled over reduced iron immediately before use. Petroleum ether (b.p. 4@60”) was also dried over sodium wire and distilled before use. These solvents will be referred to as ether (E) and petrol (P), respectively. Column chromatography was performed on alumina that was purchased from P. Spence & Co. Ltd., Widnes, England and then washed with hydrochloric acid, distilled water, and dried in an oven at 110”. Thin-layer chromatography was on silica gel G (Merck, E., A.G., Darmstadt, Germany).
Analysis of Tissues Tissues were digested with alkali and the ethersoluble material was retained as described previously (1). This unsaponifiable lipid was dissolved in petrol, and most of the sterol (“sterol A”) was removed by crystallization. This mother liquor was evaporated to dryness to yield “sterollow” unsaponifiable lipid.
Chromatography The chromatographic separations of the constituents of the sterol-low unsaponifiable lipid were carried out on Brockmann Grade III, acidwashed alumina. For each 100 mg of lipid, 10 gm adsorbent was used, and eluting solvents were successive lOC-ml fractions of petrol; 2, 4, 6, 8, 10, and 12y0 ether in petrol; and finally ether. After removing the eluting solvents, examination of the chromatographic fractions by infrared and ultraviolet absorption spectrophotometry indicated that the squalene was present in the petroleum ether fractions while the 6 and 8y0 E/P fractions contained ubiquinone. This compound was estimated by following spectrophotometrically the reduction by borohydride of an ethanolic solution (A& ?k = 142 at 275 mp). Dolichol ran a little behind ubiquinone on the column. St,erol appeared mainly in the ether fraction but some was also eluted by 12% E/P. This was referred to as “sterol B” to distinguish it from the sterols crystallized from petrol-sterol A.
648
BUTTERWORTH
Dolichyl Acetate In experiments 24, dolichol-containing fractions were bulked and acetylated, and the resulting mixtures were chromatographed on Brockman Grade III, acid-washed alumina. Dolichyl acetate was eluted by 2y0 E/P after passing petrol and 1% E/P through the column. Eight per cent E/P eluted the ubiquinone that was purified by crystallization. The purification of dolichyl acetate in experiment 1 is described in the Results section.
Purification
of Ubiguinone
The ubiquinone-containing fractions resulting from chromatography of the acetylated material were assayed spectrophotometrically (using NaBHI), and a known quantity of unlabelled ubiquinone-50 was added. The mixture was then crystallized from ethanol at -10” to constant specific activity. In experiment 1 ubiquinone obtained directly from chromatography of the unsaponifiable lipid was purified in this way.
Purification
of Dolichol
f+eliminary p&&&on. The dolichyl acetate fraction was saponified and the free alcohol was chromatographed on a column of alumina (acidwashed, Brockmann Grade III). It was eluted in the 6 and 8% E/P fractions, and infrared spectroscopy (1) showed the material to be almost pure. It was then weighed. Purification of dolichol from experiment 1 is described in the Results section. Rigorous puriJication. Preliminary purification of dolichol usually resulted in a sample containing only traces of highly labelled lanosterol. This was removed by careful thin-layer chromat.ography and by repeated crystallization from ethanol at -10”. Counting of paper chromatograms then showed the absence of any labelling other than that running as dolichol. In order to make quite sure that dolichol purified in this way was free of impurities, the sample of alcohol obtained in experiment 3 wras subjected to the following rigorous scheme of purification. After preliminary purification the dolichol fraction had a specific activity of 2081 dpm per milligram, but paper chromatography shorn-ed the presence of traces of highly labelled impurity running with the same R, as authentic lanosterol. Only 17$$ of the radioactivity ran with the same R/ as dolichol. The amount of lanost,erol present must have been very small, for it failed to give a stain with iodine (marker spots containing 10 Mg of lanosterol gave a good positive stain) and the infrared spectrum of the fraction was almost identical with that of pure dolichol.
ET AL. One half of this dolichol fraction was then crystallized from ethanol (see below), and the other half was chromat.ographed on t.hin layers of silica gel (1% methanol in benzene as developing solvent). Only the center of the dolichol band was removed from the plate for extraction, and although some dolichol was left on the plate, t,hat obtained was almost pure. The specific activity was 391 dpm per milligram, and practically all of the radioact.ivity now ran as dolichol when chromatographed on paper. After repeated crystallization from ethanol at -10” the specific activity of the alcohol was determined as 414 dpm per milligram. This material was again acetylated and the acetate was subjected to thin-layer chromatography in a manner similar to that described above in which 2L;70E/P was used as developing solvent. The specific activity of the dolichol moiety of this purified acetate was calculated t,o be 399 dpm per milligram. The half of the partially purified dolichol set aside for crystallization at -10” yielded, after the third crystallization, crystals with a specific activity at 404 dpm per milligram. After a fru%her two crystallizations the specific activity was 381 dpm per milligram. A portion of this sample was hydrogenated quantitatively (Tower’s microhydrogenation apparatus); Adam’s catalyst and a 1:l:l mixture of cyclohexane:glacial acetic acid: ethanol was used as solvent. The uptake of hydrogen (1.490 moles H&O0 gm at NTP) was in good agreement with that (1.489) of Burgos et al. (1963) for pure dolichol and using the same apparatus. The perhydrodolichol was acetylated and the acetate was chromatographed on thin layers of silica gel to yield a sample of pure perhydrodolichyl acetate with a specific activity of 364 dpm per milligram. This corresponds to a specific activity of the original dolichol of 384 dpm per milligram. It is interesting that the poor separation of dolichol (R, 0.46) and perhydrodolichol (R, 0.51) on thin layers of silica gel, using benzene as developing solvent, is vastly improved by separating the compolmds as the acetates. When 27, E/P is used as a developing solvent, perhydrodolichyl acetate has an R, of 0.92 and dolichyl acetate an RI of 0.43. The specific activity of dolichol quoted in Table I (391 dpm per milligram) is the mean of the final figures from the two different routes of purification (384 and 399 dpm per milligram). It is relevant that no significant change in specific activity could be brought about after the stage when the dolichol was subjected to thin-layer chromatography.
INCORPORATION
OF MVA
Measurement of Radioactivity The counting was done in a Packard Tri-carb scintillation counter (efficiency 55.67,), except in experiment 1 when a Lead Castle gas flow counter was used. Results were recorded as disintegrations per minute (dpm) and counts per minute (cpm), respectively.
Paper Chromatography During the purification of dolichol, samples were taken at different stages and subjected to reversed phase partition chromatography. Whatman 3MM paper was impregnated with liquid paraffin using a 37, solution of liquid paraffin in petrol; the mobile phase (ascending) was acetone. In this syst.em I11 values were 0.75 for lanosterol, 0.45 for dolichol, and 0.20 for dolichyl acetate. The developed chromatogram was cut into 2.cm wide strips parallel to the origin, and the ether extract of each was assayed for radioactivity. From the results it was possible to calculate the proportion of the total counts put on the paper that travelled alongside marker spots of dolichol (or in some cases dolichyl acetate). Marker spots were visualized as brown areas by spraying with a 3% solution of iodine in chloroform.
Pwification
of Xqualene
In experiment,s 2-4 the petrol fraction resulting from the chromatography of the unsaponifiable lipid was chromatographed as a line on thin layers of silica gel G; petrol was the developing solvent. Marker spots of pure squalene m-ere located as brown areas after staining with iodine, and the bands of adsorbent corresponding t,o these were removed and extracted with ether. By this time the sqllalene was quite pure.
Purijkation
oj Cholesterol
Cholesterol was isolated from the crystallized sterols (A) through the dibromide (15). RESULTS
Table I shows the results of experiment 1. Comment#s on these are reserved for the Discussion, except to point out that the “dolichol-containing fractions” were those obtained by chromatography of the sterollow unsaponifiable lipids on alumina. After the figures in Table I had been recorded, these dolichol-cont.aining fractions were acetylated, and thin-layer chromatography of a portion coupled with counting of regions removed from the chromatogram indicated t,hat practically all of the radioactivity was
INTO
LIVER
DOLICHOL
649
associated with lanosterol. The rest of the dolichyl acetate fraction was hydrolyzed, and carrier, unlabelled lanosterol was added. After three crystallizations, the specific activity of the lanosterol became constant. At this stage over 90 % of the radioactivity of the fractions was associated with lanosterol. The radioactivity remaining in the dolichol was very small, probably negligible. Since the incorporation of labelling into dolichol over a short period of time was not readily measurable, further experiments were carried out in which the administration of mevalonate was prolonged. In one of these experiments (experiment 2) 25 PC of [2-‘“Clmevalonate was given to each of three rabbits in five equal injections at 3-hourly intervals (Table II). On t’his occasion dolichol retained a small amount of radioactivity. The 14C remained associated with dolichol and (on acetylation) with dolichyl acetate when chromatographed (reversed phase, partition) on paper. The recovery of labelling in dolichol of the rabbit livers being very low, it was considered worthwhile to study t,he situation in the liver of the pig where concentration of dolichol is ten t,imes higher. In the third experiment a pig received four int#ramuscular doses of 43.75 PC of [2J4C]mevalonate at 4-hourly intervals. The liver was removed 2 hours after the last dose. Table III shows the results of this experiment. Since the level of incorporation of radioactivity into t’he dolichol was again low, the dolichol fraction was purified particularly rigorously to make quite sure that the radioactivity was not associated with impurity (see Experiment,al section). The stability of the specific activity of dolichol during t,he final stages of purification leaves no doubt that the labelling really was incorporated into the dolichol molecule. Table IV presents the results of experiment 4. This experiment was a repeat of experiment 3 except t,hat a total of 100 I.CCof mevalonate was given. DISCUSSION
The in vivo synthesis of the isoprenoid compounds cholesterol and squalene from the classic isoprenoid precursor mevalonic acid is well established, and the mechanism
650
BUTTERWORTH
ET AL
TABLE 1.
EXPERIMENT
~~SALYTICAL
n.4~.4
FOR
THE
I
LIVERS
OF
INJECTED JVI’I?H 16 PC OF
I~ABBITS
[2-14C-]MEV.4LoN.4TE Rabbit
number
Time after injection (hours) Liver weight (pm) Ubiquinone xv-eight (mg) Ubiquinone, specific activity“ (mg) Sterols (A), total activity” Cholesterol, specific activity(mg) “Squalene fraction,” total activity” “Dolichol-containing fraction,“*, c total tivity” a cpm X 1OV. * Chromatographic c Mainly lanosterol
0.5 iG 1.69 21.1 58.2 85.0 G71 347
ac-
fractions of the sterol-low (see text).
4
3
0.75 12“ 2.97 8.2 179.2 834 714 354
unsaponifiable
TABLE EXPERIVENT
2
1
1.0 83 1.83 22.1 245.2 832 100 293
2.0 55 1.4G 4.0 156.1 950 79 79
lipid
II
2. THE RECOVERY OF R.WIOM!T~VITY IN FRACTIONS OF THE UNSAPONIFIABLE RABBIT LIVER (214 GM) FROM THREE RABBITS AFTER INJECTING 75 pC OF
LIPID
OF
[2-‘4C-]&~EV.4LOIiATE
Weight (mg)
Fraction
Sterol (A)” Cholesterol from Sterol (B) b Squalene Ubiquinone Dolichol a Recrystallized * Fraction from
Sp. act. (dpm/mg)
37G
2,824 2,913 3,439 95,555 35,080 1,277
(A) 117.5 2.2 3.30 3.0
from unsaponifiable liquid. chromatography of sterol-low
unsaponifiable
TABLE EXPERIMENT
Total
act.
(dpm)
cG of radioactivity of unsaponifiable lipid
1.06 x 106
54.1
0.40 0.21 0.12 3.83
20.4 10.7 G.1 0.2
x x x x
106 10” 106 103
lipid.
III
3. THE RECOVERY OF RADIOACTIVITY IN FRACTIONS OF THE UNSAPONIFIABLE PIG LIVER (693 GM) AFTER INJECTING 175 pC OF [2-14C-]&~~v~~o~a~~ Fraction
Sterol (A)” Cholesterol from Sterol (B) * Squalene Ubiquinone Dolichol n See footnote
to Table
Weight
(mg)
2320 (A) 272 10.8 22.7 56.1
Sp. act.
(dpm/mg)
4944 5104 5025 38,280 1985 391
Total
act.
(dpm)
11.5 x 106 1.4 0.4 45.2 21.9
x x X x
106 106 lo3 103
LIPID
OF
% of radioactivity of unsaponifiable lipid
81.G 9.7 2.9 0.32 0.15
Il.
of these syntheses is more or less completely elucidated (4, 5). Similarly, the biosynthesis of the isoprenoid of the side chain of ubiquinone in mammalian tissues has been
shown repeatedly (6). Our results corroborate the previous observations as regards the biosynthesis of t,hese all-kans isoprenoid compounds.
INCORPORATION
OF MVA
INTO
TABLE EXPERIMENT
4. THE PIG
651
DOLICHOL
IV
RECOVERY OF RADIOJXTIVITY IN FRACTIONS OF THE UPISAPONIFIABLE (525 GM) AFTER ISJECTIXG 100 KC OF [%%]MEvALONATE LIVER
Fraction
Weight
Sterol (A)0 Sterol (B)” Squalene Ubiquinone Dolichol u See footnote
LIVER
to Table
(mg.)
Sp. act.
930 220 8.6 12.1 25.0
(dpm/mg)
5053 5050 7998 2943 236
Total
4.7 1.1 68.8 35.6 5.9
LIPID
act.
(dpm)
% of radioactivity of unsaponifiable lipid
x x x x x
106 106 103 103 103
72.5 17.0 1.06 0.55 0.09
OF
II.
We have been able to show that another isoprenoid compound, dolichol, is also synthesized in vivo by pig and rabbit liver. The significance of this observat,ion lies in the fact t,hat this is t’he first t,ime that mammalian tissues have been shown t,o synthesize a predominantly cis- isoprenoid molecule. In t’he initial experiment (experiment l), the metabolism of mevalonic acid was sbudied at, various times after the injection of equal amounts of labelled mevalonic acid into four rabbits. The results (Table I) show t,he rapid rise in the labelling of squalene derived from mevalonate and the transfer of label to the sterols after a short time lag. As discussed in t,he Results section, labelling in t’be “dolichol-containing” fraction was attributable mainly to the presence of lanosterol, and t’hese figures exhibit the expect#ed precursor-product relationship t)o sterol synthesis. Although t,he levels of ubiquinone per gram of liver for each of t,he four animals were relatively constant, no clear peak in the incorporat8ion of mevalonate was seen in t’he t.ime intervals sbudied. When studying the biosynt,hesis of ubiquinone in rat liver, Green’s group (14) experienced fluctuations in the incorporation of mevalonate of the same order. The factors which affect ubiquinone turnover are largely unknown, and it would seem t,hat statistical assays based on analyses of large numbers of animals may give rise to more reliable data. Dolichol isolat,ed from t’he bulked “dolicahol-containing” fractions of all four animals in this particular experiment retained negligible, if any, radioactivity. This would
seem to indicate that if dolichol synt.hesis had taken place at all then the rate of synt’hesis was very slow. By administ’ering mevalonic acid re peatedly over a longer period (experiment 2, Table II), it was shown that, in fact dolichol was synthesized by the animal t’o a small extent. However, it is open to question whether or not the labelled dolichol was derived from mevalonic acid by a pathway similar to that of ot’her isoprenoid compounds. The low level of incorporation and the long time elapsing between the first injection of labelled mevalonate and the removal of the liver from the animal would allow incorporation of radioactivity from metabolites of labelled compounds formed soon aft’er the first injection. However, it is clear that dolichol was definitely synthesised and, regardless of the precise pathway the biosynthesis of t,his cis isoprenoid, was much slower than that of the all truns compounds. It would be unwise to attribute too much significance t)o the absolute levels of radioact’ivity recovered in squalene, cholesterol, and ubiquinone in this experiment (Table II). The repeated administration of labelled mevalonic acid over a period of 12 hours and the analysis of results from single animals make the int,erpretat#ion from t’he point of view of the turnover of these compounds unreliable. For example, while it was noted that in experiment I (Table I) tissue concentrations of ubiquinone in t’he four rabbits were fairly constant (about 24 pg per gram), it should be not’ed t,hat in experiment 2 this figure had dropped to 15 pg per gram. Similarly, in the pig there was a variation in levels of ubiquinone from 23 to 33 pg per
652
BUTTERWORTH
gram. It is relevant that in rats, tissue levels of ubiquinone vary considerably, somtimes as much as 200 % (16). In the case of cholesterol, the extent of liver synthe&is is partially controlled by the level of this compound in the diet (17). However, the figures for the radioactivity recovered in t,hese compounds are quoted as evidence of the more active uptake of label from the mevalonic acid pool into the trans-isoprenoids than into the cis compound dolichol. This argument is pertinent to t’he interpretation of the data obtained from experiments 3 and 4. Thus it is clear that the level of labelling in dolichol purified from pig liver after repeated injection of radioactive mevalonate was again much lowe than in any of the other compounds studied (Tables III and IV). Because of the very low level of radioactivity in the dolichol, most stringent methods were employed to ensure t,he complete separation of the alcohol from any impurities. There can be no doubt that labelling was incorporated into t’he compound but, as in the rabbit, it is difficult to decide whether or not this originated by a direct route from mevalonic acid or by incorporation of a metabolite of some compound formed soon aft,er the first injection of labelled mevalonic acid. In trying to interpret these results, the possibility that the labelling was incorporated only into the trans-isoprene residues of dolichol had to be considered. No evidence is available yet concerning the precise positions of the trans residues in the molecule, and it was conceivable that the trans residues could be added, by the animal, to preformed poly-cis-isoprenoid molecules obtained from the diet. Presumably such hypothetical polycis-isoprenoid compounds would be taken by the animal in the dietary fat. It is relevant that the level of dolichol in livers of pigs raised on diets containing either no fat, or fat in the form of either beef tallow or corn oil, were the same as in livers of pigs raised on normal diets (18). It therefore seems unlikely that the formation of dolichol in pig liver is dependent upon the presence in the diet of a poly-cis-isoprenoid precursor. In fact the simplest and most likely interpretation is that labelling was incorporated
ET AL.
int’o bot’h trans- and cis-isoprene residues of dolichol. It seems most probable that, on the basis of our studies with the rabbit and the pig, mammalian systems synthesize both poly-cis- and poly-trans-isoprenoid compounds, but that the poly-cis synthetase system does not proceed at as rapid a rate as the poly-trans system. In this respect isoprenoid biosynthesis in rabbit and pig liver differs from that in at least one of the fungi, for in Asper~illus fumtiatus the incorporation of mevalonate into dolichol was almost t.wice that into ubiquinone (19). This st’udy of the metabolism of mevalonate by the two mammalian systems raises at least three important questions. It is inferred that the mammalian system contains a cis-isoprene polymerase, of which dolichol is the only known product. This conclusion tends to increase t,he significance of the presence of the cis alcohol in mammalian tissue, and t’he first problem is to try to understand what stereoselective system controls the format’ion of a cis or truns isoprene struct.ure. Second, there is a great diversity in the size of poly-isoprenoid compounds that appear to be ‘(end products” of the polymerase systems. n’evertheless within any one tissue the utilization of compounds that are wholly or partially isoprenoid in nature is rather specific as to size. The relationship between factor(s) controlling the length of isoprene chain and the further utilization of the chain is little understood. Third, several groups of important isoprenoid compounds (e.g., ubiquinones and vitamins K as well as the longchain alcohols) are known to have partially saturated chains. Very litt’le is known concerning the mechanism, control, or imporOance of the saturated isoprene units. Clearly, there is a need for much work in t,his field. REFERENCES 1. BCRGOS, J., HEMMING, F. W., PENNOCK, AND MORTON, R. A., Biochem. J.
J. F.,
88, 470
(1963). J., in “Biogenesis of Natural Compounds” (P. Bernfeld, ed.), pp. 727-737. Pergamon Press, London (1963). 3. ARCHER, B. L., BND BARNARD, D., Biochem. J. 96, 1P (1965). 2. BONNER,
INCORPORATION 4. CORNFORTH, NINGER,
OF MVA
J. W., C.,
CORNFORTH, R. H., Dox.4ND POPJAK, G. J., Proc. Roy.
Sot. (London), Ser. B 163, 492 (19%). 5. CORNFORTH, J. W., CORNFORTH, R. H., DONNINGER, C., POPJAK, G. J., RYBACH, G. AND SCHROEPFER, G. J. PTOC. Roy. Sot. (London), Ser. B 163, 436 (1966). 6. GLOVER, J., in “Biochemistry of Quinones” (R. A. Morton, ed.), pp. 207-260. Academic Press, New York (1965). ‘7. STOFFEL, W., AND Ma~x~us, C., Biochem. is. 333, 440 (1960). 8. PARSON, W. W., AND RUDNEY, H., Proe. Natl. Acad. Sci. U.S. 63, 599, (1965). 9. OLSEN, R. K., SMITH, J. L., DAVIES, G. D., MOORE, H. W., FOLKERS, K., PARSON, W. W., AND RUDXEY, H., J. Am. Chem. Sot. 87, 2298 (1965). F. W., MORTON, R.. A., AND PEX10. HEMMING, NOCE, J. F., Proc. Roy. Sot. (London), Ser. B 168, 291 (1963).
INTO
LIVER
DOLICHOL
653
11. GLOOR, U., AND WISS, O., Biochem. Biophys. Res. Commun. 1, 182 (1959). 12. GLOOR, U., AND WISS, O., Such. Biochem. Biophys. 83, 216 (1959). 13. PHILLIPS, W. E. J., Can. J. Biochem. Physiol. 39, 855 (1961). 14. DIPLOCK, A. T., GREEN, J., AND BI-XYAN, J., Biochem. J. 96, 138 (1965). 15. SCHWENK, E., AND WERTHESSES, W., Arch. Biochem. Biophys. 40, 334 (1952). 16. EDWIN, E. E., Bu~uaN, J., GREEN, J., AND -4. T., Brit. J. Nub. 16, 135 DIPLOCK, (1962). M. D., CHAIKOFF, I. L., FELTS, 17. MORRIS, J. M., ABRAHAM, S., AKD FANSAH, X. O., J. Biol. Chem. 224, 1039 (1957). P. H. W., DRAPER, H. H., 18. BUTTERWORTH, HEM~~ING, F. W., PENNOCK, J. F., AND MORTON, R. A., Biochem. J. 89, 32P (1963). P. H. W., HEM19. BURGOS, J., BUTTER~ORTH, MING, F. W., AND MORTON, R. A., Biochem. J. 91, 22P (1964).
OF BIOCHEMISTRY
In Viva
AND
Incorporation
BIOPHYSICS
of [2J4C]-M and
P. H. W. BUTTERWORTH: Department
G46-653 (19GG)
113,
evalonate
into
Dolichol
Pig Liver’
H. H. DRAPER,3 F. W. HEMMING,
of Biochemistry,
The University
Received
of Rabbit
October
of Liverpool,
Lioerpool,
9ND
R. A. MORTON
England
11, 1965
In two experiments with pigs, administration of [2-14C] over 14 hours led to a level of incorporation into the poly cis-isoprenoid alcohol dolichol that was low but significant. The figures for total incorporation of radioactivity into dolichol were 15 and 470/d of those for ubiquinone, and 0.1 and 0.167, of those for the squalene-cholesterol system. A similar experiment with rabbits yielded corresponding figures of 3.5 and 0.2yG. Rigorous purification failed to change significantly the specific activity of the doichol. It is concluded that mammalian tissues are capable of synthesizing poly cisisoprenoids, but that in the case of dolichol in rabbit and pig liver the rate of synt)hesis is distinctly lower than that of poly-trans.isoprenoids.
Pig liver is a relatively rich source of dolichol (60-140 mg per kilogram), but rabbit liver cont’ains less (5-20 mg per kilogram t)issue). The alcohol has fifteen or sixteen of its eighteen internal isoprene units in t’he cis configuration (1). CHa CHa-
involved in normal isoprene polymerizat.ion. One would expect some st,ereochemical difference between t,he int’ermediates of t’he all-&s and all-trans pathways. Only recently, wit.h the use of [2-14C],4R-[4-3H]- and [2-14Cj,4S[4-3H]-mevalonic acid, has it been possible to
CHa
CHa
c: =CH-CHr-(-CHz&=CH-CH+-CHz-
4 H-CHs-CHzOH Dolichol
As a poly-cis isoprenoid compound it is related only to one ot#her group of compounds so far described, the nat’ural rubbers (2). &dies on the biosynthesis of rubber from mevalonate by the latex from Hevea brasiliensis have been reviewed recently by Bonner (2), and there is little doubt that in principle this follows the same pathway and involves t’he same int’ermediates as t’hose 1 This work was supported in part by grant AM 05282-02 from United States Public Health Service. 2 Holder of an Agricultural Research Council Research Studentship. Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin. a Present address: Division of Nutritional Biochemistry, Department of Animal Science, Universit,y of Illinois, Urbana, Illinois. 646
demonstrate this experimentally (3). It is clear that the c&form of t’he rubber isoprene unit is produced init#ially and that a trans-cis isomerase is not, involved. The other principal polyisoprenoid compounds in pig and rabbit liver are cholesterol, squalene, and ubiquinone. The biosynthesis of all trans-squalene from mevalonate is now almost fully worked out,, and recently it has been shown that the tyans configuration is formed by a process involving stereoselect.ive elimination of t*he hydrogen, which when replaced by a heavy hydrogen isotope would confer t,he 4S-configuration on the original mevalonate (4,5). The 4R-hydrogen remains intact. In t,he biosynt’hcsis of poly-cis rubber the reverse holds. Ubiquinone has an all-trans isoprenoid side chain, and it has been shown by a
INCORPORATION
OF MVA
number of workers to be derived from mevalonate (6). It is tacitly assumed that the steps from mevalonate to farnesyl pyrophosphate are common t.o the synthesis of both ubiquinone and squalene. Presumably chain lengthening of farnesyl pyrophosphate then continues until (in pig litier), a Cjo isoprenyl pyrophosphate is formed, and this can t,hen be condensed to the quinone nucleus of ubiquinone (7) or to a phenolic precursor thereof (S, 9). Thele is in fact surprisingly little direct’ evidence to substantiate t,his. Support for the idea is gained from the isolation (10) of a CsO polyisoprenoid alcohol from a tissue rich in ubiquinone10 and from the report t’hat solanesyl pyrophosphate will condense with ubiquinone-0 under the influence of a mitochondrial enzyme (7). Nevertheless, despite the lack of direct evidence, this seems to be the most logical route for the biosynthesis of the isoprenoid side chain of ubiquinone. Some knowledge of the relative rates of incorporation of mevalonate into the alltram isoprenoids squalene, cholesterol, and ubiquinone has already been gained, and it may be that this is to some extent under the contBrol (probably indirectly) of vitamin A (ll-13), although recent work by J. Green’s group, (14) throws some doubt on this. In this paper the authors have investigated the possible incorporation of [2-14C]mevalonate into t’he cis alcohol dolichol by pig and rabbit liver, and have made some observations on the rate of biosynthesis of this compound in relation t,o t,he rate of synthesis of the all-trans compounds. EXPERIMENTAL
Administration
uf Mevalonate
Rabbits. In experiment 1, 5 adult Columbian rabbits (2.6-3.2 kg each) were each given 16 flC of [2-‘%]mevalonate by intraperitoneal injection. The animals were killed at the following postinjection times: 0.5, 0.75, 1.0, 2.0, and 3.0 hours, respectively, and the livers were removed immediately for analysis. In experiment 2, 25 *C [2-‘4C] mevalonate was given to each of 3 New Zealand White rabbits (weighing about 2 kg each) in 5 equal intraperitoneal injections at a-hourly intervals over 12 hours. The animals were killed 2 hours after the last dose and the livers were removed and bulked for analysis.
INTO
LIVER
DOLICHOL
647
Pigs. In experiment 3, 175 PC [2-‘%]mevalonate was injected into a pig (36 kg) by four equal intramuscular doses at 4-hourly intervals over a period of 12 hours. The pig was killed after a further 2 hours and the liver was removed immediately and analyzed. In experiment 4, 100 PC of [2-‘%]mevalonate was given to a pig (23 kg) under the same conditions as for experiment 3.
Solvents and Materials Diethyl ether was dried with sodium wire and was distilled over reduced iron immediately before use. Petroleum ether (b.p. 4@60”) was also dried over sodium wire and distilled before use. These solvents will be referred to as ether (E) and petrol (P), respectively. Column chromatography was performed on alumina that was purchased from P. Spence & Co. Ltd., Widnes, England and then washed with hydrochloric acid, distilled water, and dried in an oven at 110”. Thin-layer chromatography was on silica gel G (Merck, E., A.G., Darmstadt, Germany).
Analysis of Tissues Tissues were digested with alkali and the ethersoluble material was retained as described previously (1). This unsaponifiable lipid was dissolved in petrol, and most of the sterol (“sterol A”) was removed by crystallization. This mother liquor was evaporated to dryness to yield “sterollow” unsaponifiable lipid.
Chromatography The chromatographic separations of the constituents of the sterol-low unsaponifiable lipid were carried out on Brockmann Grade III, acidwashed alumina. For each 100 mg of lipid, 10 gm adsorbent was used, and eluting solvents were successive lOC-ml fractions of petrol; 2, 4, 6, 8, 10, and 12y0 ether in petrol; and finally ether. After removing the eluting solvents, examination of the chromatographic fractions by infrared and ultraviolet absorption spectrophotometry indicated that the squalene was present in the petroleum ether fractions while the 6 and 8y0 E/P fractions contained ubiquinone. This compound was estimated by following spectrophotometrically the reduction by borohydride of an ethanolic solution (A& ?k = 142 at 275 mp). Dolichol ran a little behind ubiquinone on the column. St,erol appeared mainly in the ether fraction but some was also eluted by 12% E/P. This was referred to as “sterol B” to distinguish it from the sterols crystallized from petrol-sterol A.
648
BUTTERWORTH
Dolichyl Acetate In experiments 24, dolichol-containing fractions were bulked and acetylated, and the resulting mixtures were chromatographed on Brockman Grade III, acid-washed alumina. Dolichyl acetate was eluted by 2y0 E/P after passing petrol and 1% E/P through the column. Eight per cent E/P eluted the ubiquinone that was purified by crystallization. The purification of dolichyl acetate in experiment 1 is described in the Results section.
Purification
of Ubiguinone
The ubiquinone-containing fractions resulting from chromatography of the acetylated material were assayed spectrophotometrically (using NaBHI), and a known quantity of unlabelled ubiquinone-50 was added. The mixture was then crystallized from ethanol at -10” to constant specific activity. In experiment 1 ubiquinone obtained directly from chromatography of the unsaponifiable lipid was purified in this way.
Purification
of Dolichol
f+eliminary p&&&on. The dolichyl acetate fraction was saponified and the free alcohol was chromatographed on a column of alumina (acidwashed, Brockmann Grade III). It was eluted in the 6 and 8% E/P fractions, and infrared spectroscopy (1) showed the material to be almost pure. It was then weighed. Purification of dolichol from experiment 1 is described in the Results section. Rigorous puriJication. Preliminary purification of dolichol usually resulted in a sample containing only traces of highly labelled lanosterol. This was removed by careful thin-layer chromat.ography and by repeated crystallization from ethanol at -10”. Counting of paper chromatograms then showed the absence of any labelling other than that running as dolichol. In order to make quite sure that dolichol purified in this way was free of impurities, the sample of alcohol obtained in experiment 3 wras subjected to the following rigorous scheme of purification. After preliminary purification the dolichol fraction had a specific activity of 2081 dpm per milligram, but paper chromatography shorn-ed the presence of traces of highly labelled impurity running with the same R, as authentic lanosterol. Only 17$$ of the radioactivity ran with the same R/ as dolichol. The amount of lanost,erol present must have been very small, for it failed to give a stain with iodine (marker spots containing 10 Mg of lanosterol gave a good positive stain) and the infrared spectrum of the fraction was almost identical with that of pure dolichol.
ET AL. One half of this dolichol fraction was then crystallized from ethanol (see below), and the other half was chromat.ographed on t.hin layers of silica gel (1% methanol in benzene as developing solvent). Only the center of the dolichol band was removed from the plate for extraction, and although some dolichol was left on the plate, t,hat obtained was almost pure. The specific activity was 391 dpm per milligram, and practically all of the radioact.ivity now ran as dolichol when chromatographed on paper. After repeated crystallization from ethanol at -10” the specific activity of the alcohol was determined as 414 dpm per milligram. This material was again acetylated and the acetate was subjected to thin-layer chromatography in a manner similar to that described above in which 2L;70E/P was used as developing solvent. The specific activity of the dolichol moiety of this purified acetate was calculated t,o be 399 dpm per milligram. The half of the partially purified dolichol set aside for crystallization at -10” yielded, after the third crystallization, crystals with a specific activity at 404 dpm per milligram. After a fru%her two crystallizations the specific activity was 381 dpm per milligram. A portion of this sample was hydrogenated quantitatively (Tower’s microhydrogenation apparatus); Adam’s catalyst and a 1:l:l mixture of cyclohexane:glacial acetic acid: ethanol was used as solvent. The uptake of hydrogen (1.490 moles H&O0 gm at NTP) was in good agreement with that (1.489) of Burgos et al. (1963) for pure dolichol and using the same apparatus. The perhydrodolichol was acetylated and the acetate was chromatographed on thin layers of silica gel to yield a sample of pure perhydrodolichyl acetate with a specific activity of 364 dpm per milligram. This corresponds to a specific activity of the original dolichol of 384 dpm per milligram. It is interesting that the poor separation of dolichol (R, 0.46) and perhydrodolichol (R, 0.51) on thin layers of silica gel, using benzene as developing solvent, is vastly improved by separating the compolmds as the acetates. When 27, E/P is used as a developing solvent, perhydrodolichyl acetate has an R, of 0.92 and dolichyl acetate an RI of 0.43. The specific activity of dolichol quoted in Table I (391 dpm per milligram) is the mean of the final figures from the two different routes of purification (384 and 399 dpm per milligram). It is relevant that no significant change in specific activity could be brought about after the stage when the dolichol was subjected to thin-layer chromatography.
INCORPORATION
OF MVA
Measurement of Radioactivity The counting was done in a Packard Tri-carb scintillation counter (efficiency 55.67,), except in experiment 1 when a Lead Castle gas flow counter was used. Results were recorded as disintegrations per minute (dpm) and counts per minute (cpm), respectively.
Paper Chromatography During the purification of dolichol, samples were taken at different stages and subjected to reversed phase partition chromatography. Whatman 3MM paper was impregnated with liquid paraffin using a 37, solution of liquid paraffin in petrol; the mobile phase (ascending) was acetone. In this syst.em I11 values were 0.75 for lanosterol, 0.45 for dolichol, and 0.20 for dolichyl acetate. The developed chromatogram was cut into 2.cm wide strips parallel to the origin, and the ether extract of each was assayed for radioactivity. From the results it was possible to calculate the proportion of the total counts put on the paper that travelled alongside marker spots of dolichol (or in some cases dolichyl acetate). Marker spots were visualized as brown areas by spraying with a 3% solution of iodine in chloroform.
Pwification
of Xqualene
In experiment,s 2-4 the petrol fraction resulting from the chromatography of the unsaponifiable lipid was chromatographed as a line on thin layers of silica gel G; petrol was the developing solvent. Marker spots of pure squalene m-ere located as brown areas after staining with iodine, and the bands of adsorbent corresponding t,o these were removed and extracted with ether. By this time the sqllalene was quite pure.
Purijkation
oj Cholesterol
Cholesterol was isolated from the crystallized sterols (A) through the dibromide (15). RESULTS
Table I shows the results of experiment 1. Comment#s on these are reserved for the Discussion, except to point out that the “dolichol-containing fractions” were those obtained by chromatography of the sterollow unsaponifiable lipids on alumina. After the figures in Table I had been recorded, these dolichol-cont.aining fractions were acetylated, and thin-layer chromatography of a portion coupled with counting of regions removed from the chromatogram indicated t,hat practically all of the radioactivity was
INTO
LIVER
DOLICHOL
649
associated with lanosterol. The rest of the dolichyl acetate fraction was hydrolyzed, and carrier, unlabelled lanosterol was added. After three crystallizations, the specific activity of the lanosterol became constant. At this stage over 90 % of the radioactivity of the fractions was associated with lanosterol. The radioactivity remaining in the dolichol was very small, probably negligible. Since the incorporation of labelling into dolichol over a short period of time was not readily measurable, further experiments were carried out in which the administration of mevalonate was prolonged. In one of these experiments (experiment 2) 25 PC of [2-‘“Clmevalonate was given to each of three rabbits in five equal injections at 3-hourly intervals (Table II). On t’his occasion dolichol retained a small amount of radioactivity. The 14C remained associated with dolichol and (on acetylation) with dolichyl acetate when chromatographed (reversed phase, partition) on paper. The recovery of labelling in dolichol of the rabbit livers being very low, it was considered worthwhile to study t,he situation in the liver of the pig where concentration of dolichol is ten t,imes higher. In the third experiment a pig received four int#ramuscular doses of 43.75 PC of [2J4C]mevalonate at 4-hourly intervals. The liver was removed 2 hours after the last dose. Table III shows the results of this experiment. Since the level of incorporation of radioactivity into t’he dolichol was again low, the dolichol fraction was purified particularly rigorously to make quite sure that the radioactivity was not associated with impurity (see Experiment,al section). The stability of the specific activity of dolichol during t,he final stages of purification leaves no doubt that the labelling really was incorporated into the dolichol molecule. Table IV presents the results of experiment 4. This experiment was a repeat of experiment 3 except t,hat a total of 100 I.CCof mevalonate was given. DISCUSSION
The in vivo synthesis of the isoprenoid compounds cholesterol and squalene from the classic isoprenoid precursor mevalonic acid is well established, and the mechanism
650
BUTTERWORTH
ET AL
TABLE 1.
EXPERIMENT
~~SALYTICAL
n.4~.4
FOR
THE
I
LIVERS
OF
INJECTED JVI’I?H 16 PC OF
I~ABBITS
[2-14C-]MEV.4LoN.4TE Rabbit
number
Time after injection (hours) Liver weight (pm) Ubiquinone xv-eight (mg) Ubiquinone, specific activity“ (mg) Sterols (A), total activity” Cholesterol, specific activity(mg) “Squalene fraction,” total activity” “Dolichol-containing fraction,“*, c total tivity” a cpm X 1OV. * Chromatographic c Mainly lanosterol
0.5 iG 1.69 21.1 58.2 85.0 G71 347
ac-
fractions of the sterol-low (see text).
4
3
0.75 12“ 2.97 8.2 179.2 834 714 354
unsaponifiable
TABLE EXPERIVENT
2
1
1.0 83 1.83 22.1 245.2 832 100 293
2.0 55 1.4G 4.0 156.1 950 79 79
lipid
II
2. THE RECOVERY OF R.WIOM!T~VITY IN FRACTIONS OF THE UNSAPONIFIABLE RABBIT LIVER (214 GM) FROM THREE RABBITS AFTER INJECTING 75 pC OF
LIPID
OF
[2-‘4C-]&~EV.4LOIiATE
Weight (mg)
Fraction
Sterol (A)” Cholesterol from Sterol (B) b Squalene Ubiquinone Dolichol a Recrystallized * Fraction from
Sp. act. (dpm/mg)
37G
2,824 2,913 3,439 95,555 35,080 1,277
(A) 117.5 2.2 3.30 3.0
from unsaponifiable liquid. chromatography of sterol-low
unsaponifiable
TABLE EXPERIMENT
Total
act.
(dpm)
cG of radioactivity of unsaponifiable lipid
1.06 x 106
54.1
0.40 0.21 0.12 3.83
20.4 10.7 G.1 0.2
x x x x
106 10” 106 103
lipid.
III
3. THE RECOVERY OF RADIOACTIVITY IN FRACTIONS OF THE UNSAPONIFIABLE PIG LIVER (693 GM) AFTER INJECTING 175 pC OF [2-14C-]&~~v~~o~a~~ Fraction
Sterol (A)” Cholesterol from Sterol (B) * Squalene Ubiquinone Dolichol n See footnote
to Table
Weight
(mg)
2320 (A) 272 10.8 22.7 56.1
Sp. act.
(dpm/mg)
4944 5104 5025 38,280 1985 391
Total
act.
(dpm)
11.5 x 106 1.4 0.4 45.2 21.9
x x X x
106 106 lo3 103
LIPID
OF
% of radioactivity of unsaponifiable lipid
81.G 9.7 2.9 0.32 0.15
Il.
of these syntheses is more or less completely elucidated (4, 5). Similarly, the biosynthesis of the isoprenoid of the side chain of ubiquinone in mammalian tissues has been
shown repeatedly (6). Our results corroborate the previous observations as regards the biosynthesis of t,hese all-kans isoprenoid compounds.
INCORPORATION
OF MVA
INTO
TABLE EXPERIMENT
4. THE PIG
651
DOLICHOL
IV
RECOVERY OF RADIOJXTIVITY IN FRACTIONS OF THE UPISAPONIFIABLE (525 GM) AFTER ISJECTIXG 100 KC OF [%%]MEvALONATE LIVER
Fraction
Weight
Sterol (A)0 Sterol (B)” Squalene Ubiquinone Dolichol u See footnote
LIVER
to Table
(mg.)
Sp. act.
930 220 8.6 12.1 25.0
(dpm/mg)
5053 5050 7998 2943 236
Total
4.7 1.1 68.8 35.6 5.9
LIPID
act.
(dpm)
% of radioactivity of unsaponifiable lipid
x x x x x
106 106 103 103 103
72.5 17.0 1.06 0.55 0.09
OF
II.
We have been able to show that another isoprenoid compound, dolichol, is also synthesized in vivo by pig and rabbit liver. The significance of this observat,ion lies in the fact t,hat this is t’he first t,ime that mammalian tissues have been shown t,o synthesize a predominantly cis- isoprenoid molecule. In t’he initial experiment (experiment l), the metabolism of mevalonic acid was sbudied at, various times after the injection of equal amounts of labelled mevalonic acid into four rabbits. The results (Table I) show t,he rapid rise in the labelling of squalene derived from mevalonate and the transfer of label to the sterols after a short time lag. As discussed in t,he Results section, labelling in t’be “dolichol-containing” fraction was attributable mainly to the presence of lanosterol, and t’hese figures exhibit the expect#ed precursor-product relationship t)o sterol synthesis. Although t,he levels of ubiquinone per gram of liver for each of t,he four animals were relatively constant, no clear peak in the incorporat8ion of mevalonate was seen in t’he t.ime intervals sbudied. When studying the biosynt,hesis of ubiquinone in rat liver, Green’s group (14) experienced fluctuations in the incorporation of mevalonate of the same order. The factors which affect ubiquinone turnover are largely unknown, and it would seem t,hat statistical assays based on analyses of large numbers of animals may give rise to more reliable data. Dolichol isolat,ed from t’he bulked “dolicahol-containing” fractions of all four animals in this particular experiment retained negligible, if any, radioactivity. This would
seem to indicate that if dolichol synt.hesis had taken place at all then the rate of synt’hesis was very slow. By administ’ering mevalonic acid re peatedly over a longer period (experiment 2, Table II), it was shown that, in fact dolichol was synthesized by the animal t’o a small extent. However, it is open to question whether or not the labelled dolichol was derived from mevalonic acid by a pathway similar to that of ot’her isoprenoid compounds. The low level of incorporation and the long time elapsing between the first injection of labelled mevalonate and the removal of the liver from the animal would allow incorporation of radioactivity from metabolites of labelled compounds formed soon aft’er the first injection. However, it is clear that dolichol was definitely synthesised and, regardless of the precise pathway the biosynthesis of t,his cis isoprenoid, was much slower than that of the all truns compounds. It would be unwise to attribute too much significance t)o the absolute levels of radioact’ivity recovered in squalene, cholesterol, and ubiquinone in this experiment (Table II). The repeated administration of labelled mevalonic acid over a period of 12 hours and the analysis of results from single animals make the int,erpretat#ion from t’he point of view of the turnover of these compounds unreliable. For example, while it was noted that in experiment I (Table I) tissue concentrations of ubiquinone in t’he four rabbits were fairly constant (about 24 pg per gram), it should be not’ed t,hat in experiment 2 this figure had dropped to 15 pg per gram. Similarly, in the pig there was a variation in levels of ubiquinone from 23 to 33 pg per
652
BUTTERWORTH
gram. It is relevant that in rats, tissue levels of ubiquinone vary considerably, somtimes as much as 200 % (16). In the case of cholesterol, the extent of liver synthe&is is partially controlled by the level of this compound in the diet (17). However, the figures for the radioactivity recovered in t,hese compounds are quoted as evidence of the more active uptake of label from the mevalonic acid pool into the trans-isoprenoids than into the cis compound dolichol. This argument is pertinent to t’he interpretation of the data obtained from experiments 3 and 4. Thus it is clear that the level of labelling in dolichol purified from pig liver after repeated injection of radioactive mevalonate was again much lowe than in any of the other compounds studied (Tables III and IV). Because of the very low level of radioactivity in the dolichol, most stringent methods were employed to ensure t,he complete separation of the alcohol from any impurities. There can be no doubt that labelling was incorporated into t’he compound but, as in the rabbit, it is difficult to decide whether or not this originated by a direct route from mevalonic acid or by incorporation of a metabolite of some compound formed soon aft,er the first injection of labelled mevalonic acid. In trying to interpret these results, the possibility that the labelling was incorporated only into the trans-isoprene residues of dolichol had to be considered. No evidence is available yet concerning the precise positions of the trans residues in the molecule, and it was conceivable that the trans residues could be added, by the animal, to preformed poly-cis-isoprenoid molecules obtained from the diet. Presumably such hypothetical polycis-isoprenoid compounds would be taken by the animal in the dietary fat. It is relevant that the level of dolichol in livers of pigs raised on diets containing either no fat, or fat in the form of either beef tallow or corn oil, were the same as in livers of pigs raised on normal diets (18). It therefore seems unlikely that the formation of dolichol in pig liver is dependent upon the presence in the diet of a poly-cis-isoprenoid precursor. In fact the simplest and most likely interpretation is that labelling was incorporated
ET AL.
int’o bot’h trans- and cis-isoprene residues of dolichol. It seems most probable that, on the basis of our studies with the rabbit and the pig, mammalian systems synthesize both poly-cis- and poly-trans-isoprenoid compounds, but that the poly-cis synthetase system does not proceed at as rapid a rate as the poly-trans system. In this respect isoprenoid biosynthesis in rabbit and pig liver differs from that in at least one of the fungi, for in Asper~illus fumtiatus the incorporation of mevalonate into dolichol was almost t.wice that into ubiquinone (19). This st’udy of the metabolism of mevalonate by the two mammalian systems raises at least three important questions. It is inferred that the mammalian system contains a cis-isoprene polymerase, of which dolichol is the only known product. This conclusion tends to increase t,he significance of the presence of the cis alcohol in mammalian tissue, and t’he first problem is to try to understand what stereoselective system controls the format’ion of a cis or truns isoprene struct.ure. Second, there is a great diversity in the size of poly-isoprenoid compounds that appear to be ‘(end products” of the polymerase systems. n’evertheless within any one tissue the utilization of compounds that are wholly or partially isoprenoid in nature is rather specific as to size. The relationship between factor(s) controlling the length of isoprene chain and the further utilization of the chain is little understood. Third, several groups of important isoprenoid compounds (e.g., ubiquinones and vitamins K as well as the longchain alcohols) are known to have partially saturated chains. Very litt’le is known concerning the mechanism, control, or imporOance of the saturated isoprene units. Clearly, there is a need for much work in t,his field. REFERENCES 1. BCRGOS, J., HEMMING, F. W., PENNOCK, AND MORTON, R. A., Biochem. J.
J. F.,
88, 470
(1963). J., in “Biogenesis of Natural Compounds” (P. Bernfeld, ed.), pp. 727-737. Pergamon Press, London (1963). 3. ARCHER, B. L., BND BARNARD, D., Biochem. J. 96, 1P (1965). 2. BONNER,
INCORPORATION 4. CORNFORTH, NINGER,
OF MVA
J. W., C.,
CORNFORTH, R. H., Dox.4ND POPJAK, G. J., Proc. Roy.
Sot. (London), Ser. B 163, 492 (19%). 5. CORNFORTH, J. W., CORNFORTH, R. H., DONNINGER, C., POPJAK, G. J., RYBACH, G. AND SCHROEPFER, G. J. PTOC. Roy. Sot. (London), Ser. B 163, 436 (1966). 6. GLOVER, J., in “Biochemistry of Quinones” (R. A. Morton, ed.), pp. 207-260. Academic Press, New York (1965). ‘7. STOFFEL, W., AND Ma~x~us, C., Biochem. is. 333, 440 (1960). 8. PARSON, W. W., AND RUDNEY, H., Proe. Natl. Acad. Sci. U.S. 63, 599, (1965). 9. OLSEN, R. K., SMITH, J. L., DAVIES, G. D., MOORE, H. W., FOLKERS, K., PARSON, W. W., AND RUDXEY, H., J. Am. Chem. Sot. 87, 2298 (1965). F. W., MORTON, R.. A., AND PEX10. HEMMING, NOCE, J. F., Proc. Roy. Sot. (London), Ser. B 168, 291 (1963).
INTO
LIVER
DOLICHOL
653
11. GLOOR, U., AND WISS, O., Biochem. Biophys. Res. Commun. 1, 182 (1959). 12. GLOOR, U., AND WISS, O., Such. Biochem. Biophys. 83, 216 (1959). 13. PHILLIPS, W. E. J., Can. J. Biochem. Physiol. 39, 855 (1961). 14. DIPLOCK, A. T., GREEN, J., AND BI-XYAN, J., Biochem. J. 96, 138 (1965). 15. SCHWENK, E., AND WERTHESSES, W., Arch. Biochem. Biophys. 40, 334 (1952). 16. EDWIN, E. E., Bu~uaN, J., GREEN, J., AND -4. T., Brit. J. Nub. 16, 135 DIPLOCK, (1962). M. D., CHAIKOFF, I. L., FELTS, 17. MORRIS, J. M., ABRAHAM, S., AKD FANSAH, X. O., J. Biol. Chem. 224, 1039 (1957). P. H. W., DRAPER, H. H., 18. BUTTERWORTH, HEM~~ING, F. W., PENNOCK, J. F., AND MORTON, R. A., Biochem. J. 89, 32P (1963). P. H. W., HEM19. BURGOS, J., BUTTER~ORTH, MING, F. W., AND MORTON, R. A., Biochem. J. 91, 22P (1964).