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The tissue and subcellular distribution of [3H]dolichol in the rat
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
The Tissue and Subcellular ROY W. KEENAN, Department
of Biochemistry,
179,
634-642
(1977)
Distribution
JOSEPH
B. FISCHER,
The University
of Texas
Received
AND
Health
August
of [3H]Dolichol
Science
MICHAEL Center,
in the Rat’ E. KRUCZEK
San Antonio,
Texas
78284
31, 1976
Following the intravenous injection of nanomolar amounts of [3H]dolichol into rats, the radioactivity rapidly appeared in the high-density lipoprotein fraction of the plasma and circulated with a half-life of about 9 h. A fraction of the injected activity was excreted in the feces, presumably through the bile, but evidence was obtained that little oxidative breakdown of dolichol occurred. All tissues assayed acquired radioactivity, but the liver attained the highest specific activity and the largest percentage of the total radioactive dolichol. Subcellular fractionation of the liver revealed that mitochondrial preparations contained the bulk of the labeled dolichol at all times tested up to 40 h after injection. Disruption of the mitochondrial structure by two different techniques permitted the isolation of inner and outer membrane fractions and it was found that the [3Hldolichol was concentrated in the outer membrane fraction. The significance of these findings is discussed.
Despite the fact that a large amount of interest has been shown in establishing the role of the polyisoprenols in transglycosylation reactions, very little is known concerning the metabolism of the polyprenol molecule (1). Dolichol, which is actually a generic term applied to a group of related polyprenols is of particular interest. These compounds, which contain 17 to 22 isoprene units including a saturated hydroxyterminal isoprene unit, are particularly abundant in the livers of higher animals where they are found principally as the free alcohol and the ester (2, 3). The only known function of dolichol is to serve as lipid intermediates in transglycosylation reactions, but all studies to date indicate that only a minute fraction of the total dolichol participates in these reactions at any given time. In addition, the subcellular localization of dolichol (2) (although there is very limited information available) does not correspond with the site where it presumably functions, since dolichol concentration is relatively low in the microsomal fraction, in which transglycosylation reactions involving lipid-linked 1 Supported by NIH Grant 1 R01 AM 17897 and by Grant AQ-448 from the Robert A. Welch Foundation.
sugars are believed to occur. For the reasons mentioned above, it seemed worthwhile to investigate the in uiuo metabolism of dolichol with the idea that these studies might suggest functions for this material in addition to its role in transglycosylation reactions. One aspect of these studies which is of particular interest is related to the fact that dolichol is an unusually long molecule (we estimate dolichol-19 to be 70 A in length) which may greatly restrict the ways in which it can be transported or oriented in membranes. Our recent synthesis of [l-3Hldolichol (4) provided us with a means with which to study dolichol metabolism. In this paper we describe a number of observations indicating how dolichol is transported, localized within the cell, and excreted. EXPERIMENTAL
PROCEDURES
Materials. The [l-3H1dolichol used in these experiments was a gift from New England Nuclear. It was synthesized using our recently described procedure (41 and had a specific activity of 14.8 Ci/mmol. This material consists of a mixture of isoprenologs, the principal compound being a C-95 polyprenol. Prior to use, the radioactive dolichol sample was purified by chromatography on silica gel G thin-layer plates (Analtech Inc.) which were developed with hexane:ether @0:20, v/v). The developed plates were
634 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
CELLULAR
DISTRIBUTION
sprayed with Omnifluor (New England Nuclear) placed in a film casset and stored in a dry ice chest. After approximately 30 min of exposure, the film was developed and the dolichol-containing area of the plate as revealed by autoradiography, was extracted with chloroform:methanol (2:l) and filtered. The filtrate was dried and the residue was dissolved in 2% ether in hexane. This extract was chromatographed on a short alumina column and the dolichol was eluted in 20% ether in hexane [see Keenan and Kruczek (4) for details]. This purification procedure was necessary to remove impurities which form in the highly labeled dolichol preparations. Aliquots of the purified radioactive dolichol were dried and suspended in the steroid suspending vehicle provided by the National Cancer Institute. The concentration of the dolichol suspension was 239 nmol (33.3 @XI ml. Glucose 6-phosphate, cytochrome c, bovine serum albumin, digitonin, and rotenone were obtained from Sigma Chemical Company. Benzylamine, a product of Aldrich Chemical Co., was purified by dissolving it in ether and passing HCl gas through the ether solution. The precipitated hydrochloride salt which was washed extensively with ether and dried was used as the substrate in monoamine oxidase assays. All other solvents and reagents were of reagent grade. Subcellular fractionation techniques. Young Sprague-Dawley female rats (100-150 g) were used in these studies. The animals were injected in the tail vein with approximately 100 ~1 of a dolichol suspension (3.3 &il prepared as described above. At the appropriate times, the animals were lightly anesthetized with ether and blood was obtained by heart puncture. The rats were killed by decapitation and various organs were removed, weighed, and frozen. The livers were immediately subjected to the subcellular fractionation techniques described by Fleischer and Kervina (5) essentially without modification, except as mentioned under Results. The fractions isolated were the nuclear, plasma membrane, mitochondria, heavy microsomes, light microsomes, Golgi, and supernatant. The purified mitochondria were subfractionated into the inner and outer membranes by treatment with digitonin as described by Schnaitman and Greenawalt (6) or by the large-amplitude swelling technique developed by Parsons et al. (7). The best results were obtained with the latter technique when the liver from the injected animal was combined with livers from three untreated animals of the same age and sex. Assays. Protein was determined by the method of Lowry et al. (8) using bovine serum albumin as a standard. Nuclei were dialyzed prior to the determination of protein in order to remove the large quantity of sucrose which interfered with the protein assay.
OF
635
DOLICHOL
Dolichol radioactivity was measured in various tissues by selecting what appeared to be a representative sample of the larger tissues, i.e., intestine, liver, etc., or the entire sample in the case of small organs. A 20% homogenate of the tissue was prepared in sucrose and aliquots of these homogenates were extracted by the procedure of Dole (9). A micromodification of this procedure was also used on small samples of plasma and tissues. This procedure was found to give quantitative recovery of 13Hldolichol from tissue and had the advantage of being somewhat more rapid and simpler than extraction with chloroform-methanol mixtures. The lower phases of the extracts were also checked for the presence of water-soluble radioactivity but they seldom contained significant radioactivity. When sufficient radioactivity was present, samples of the lipid extracts were assayed for the presence of dolichol esters (10). The degree of purification of the subcellular fractions from rat liver was estimated by measuring the specific activities of the marker enzymes. Cytochrome oxidase and monoamine oxidase were measured essentially as described by Schnaitman and Greenawalt (6) except that we found it necessary to purify the benzylamine as described above in order to obtain good rates in the monoamine oxidase assay. Rotenone-insensitive NADH-cytochrome c reductase was determined by the procedure of Sottocasa et al. (11). Glucose 6-phosphatase was measured as described by Swanson (12) and the liberated phosphate was estimated by the method of Bartlett (13). These assays showed good correlation of marker enzymes with the appropriate subcellular fractions; results of the mitochondrial assays will be discussed in the appropriate section. Radioactivity was assayed by counting aliquots of lipid extracts from the Dole extraction procedure in a toluene based scintillator as previously described (10) and/or by digesting aliquots of homogenized tissue or subcellular fractions overnight at 65°C in 1.0 ml of NCS tissue solubilizer (AmershamSearle) and counting the digested samples in 12 ml of Beckman Bio-Solv 3. Samples were counted in a Beckman LS-230 liquid scintillation system together with blanks which had been subjected to the same digestion procedure. Vials were usually stored in the dark before counting, but were always counted until constant values were obtained. Where it was required, representative samples were recounted with added internal standards to correct for differences in counting efficiencies. RESULTS
AND
DISCUSSION
Following the intravenous injection of [3Hldolichol in the quantities which we employed (ca. 20 nmol) almost all of the radioactivity was rapidly taken up in the
636
KEENAN,
FISCHER.
high-density lipoprotein fraction (141. Only a small percentage of the label was detected in association with other plasma fractions or the red cells. Figure 1 shows the rate at which dolichol disappeared from the plasma. The fact that the 3-h point does not fall on a straight line is probably due to an incomplete injection of the dolichol suspension into the tail vein of this animal. The half-life of dolichol in the plasma was estimated from Fig. 1 as approximately 9 h. This value is based on six points from different animals all of the same age and sex. This is in accord with a value of approximately 13 h which was obtained by taking blood at similar intervals from one animal which had been injected with [3H]dolichol. These values are good evidence that dolichol circulates for a relatively long period of time as compared to the half-lives of retinol, cholesterol, and other lipids which have been estimated. The only previous reference to the occurrence of dolichol in the circulation was that it was detected in trace amounts in blood (1). The influence of nutritional state, endogenous dolichol concentrations, and other factors which might be expected to effect the half-life of injected dolichol have yet to be established. Lipid extraction of the plasma and analysis by thinlayer chromatography revealed that essentially all of the radioactivity in the plasma was present as free dolichol rather than the ester or breakdown products. The radioactivity in the plasma declined with time and other tissues became progressively more labeled, particularly the liver and the spleen. The data on the tissue distribution of 13Hldolichol are given in Table I. The specific activity values in this table may be somewhat misleading for two reasons. First, the actual values for the early points are likely to be lower than shown since the tissues contained blood which had a relatively high specific activity. This source of error was minimized, but not eliminated, by rinsing and blotting the tissues prior to assay. A second and more serious source of error was due to the fact that it was not possible to ensure that all of the administered radioactive compound was introduced into the blood
AND
KRUCZEK
FIG. 1. The plasma levels of radioactivity at various times following the intravenous injection of [3Hldolichol. Approximately 20 nmol of [3H]dolichol was administered to each rat. Blood was obtained by heart puncture at the indicated time, and the radioactivity of plasma was determined by digesting aliquots of plasma in a protein solubilizer and measuring the radioactivity as described under Experimental Procedures.
stream when it was injected into the tail vein. This is almost certainly why the 3-h point in Fig. 1 does not fall on the line and why the specific activities for tissues from this point are lower than expected. The linearity of all the other points in Fig. 1 is evidence that the doses administered were similar in these cases and that slow absorption of dolichol which was not injected into the blood stream was not a significant problem. A more straightforward way to look at the data is to compare the amount of the total radioactivity in each tissue with the total recovered radioactivity as shown in Table I. It is clear from these data that initially most of the radioactivity was in the plasma and that the bulk of the labeled material was deposited in the liver. The intestines and their contents also acquired large amounts of radioactivity. Most of this radioactivity was present in the intestinal contents rather than in the intestinal tissue. Feces and urine were collected from those rats which were maintained 20 and
CELLULAR
DISTRIBUTION
OF
TABLE DISTRIBUTION
OF RADIOACTIVITY
IN
TISSUES
637
DOLICHOL
I
OF RATS
GIVEN
[3H]Do~~~~o~
AT VARIOUS
TIMES
AFTER
ADMINISTRATIONS Tissue
Hours
:I Blood plasma Liver Intestines contents Spleen Lung Heart Pancreas Kidney Brain
Re-
covered radioac tivity (5%)
+
specific activity
Re-
:overed adioac tivity (%o)
60.3
9980
43.9
18.4 13.4
2460 1940
30.1 17.2
0.86 2.7 0.96 0.43 2.2 0.48
1650 2150 1480 1280 1350 350
gpecific wtivitJ
-6
dose
Rempecific overed I a ctivitj adioac
Reoverec
ldioac tivity (%)
Ipecifh Rectivitj I C’overec ri ndioac tivity (%I
40.0
21.4
3630
15.7
1670
3960 875
6120 1210
67.7 4.67
6570 200
975 2630 725 900 1000 200
1320 2350 1100 1230 1000 300
9.4 0.63 0.06 1.05 0.72
tivity (I)
5280
1800 575 1380 750 400 1000 575 150
after
1.0 5.3 0.63 0.81
2.0 0.43
L
1
Ipecific ctivitj
Re-
ipecifk
ctivity
adioac tivity (%) 3.75 86.1 2.7 3.3 1.8 0.30 0.71 0.53 0.78
416 7660 116 4075 750 275 882 175 250
a Female rats averaging 125 g each were injected intravenously with 3.3 &i of a 13H1dolichol suspension. At the indicated times following isotope administration, the animals were sacrificed and representative samples from each tissue were taken. The tissue samples were rinsed off and blotted to remove blood and subjected to lipid extraction and the radioactivity in the organic phase was determined. Aliquots of blood plasma were digested in protein solubilizer and counted. Blood plasma was assumed to constitute 3% of the body weight. The specific activity is expressed in terms of counts/per minute per 100 milligrams wet weight. Details of the procedures used in the extractions and radioactivity measurements are given under Experimental Procedures.
40 h. The results were not quantitative, but it was clear that most of the excreted radioactivity was present in the feces. To determine the extent of dolichol excretion two rats were each injected with 0.1 pmol of 13Hldolichol (approximately five times as much as used in the experiment described in Table I) and the urine and feces were collected. In keeping with our previous results, only small quantities of radioactivity were found in the urine, but approximately 16% of the total injected dose could be accounted for in the feces after 90 h. The largest amount of radioactivity was excreted between 44 and 66 h. Almost all the radioactivity was lipid-soluble and cochromatographed with free dolichol. The evidence from these experiments indicates that dolichol is probably excreted in the bile, but it is not known if it is first converted to a glucuronide as in the case of retinol or excreted unchanged. The radioactivity of the various tissues was measured by extracting the tissues
with isopropanol-heptane as described under Experimental Procedures. It is of interest that very little radioactivity could be detected in the aqueous phase from these lipid extracts. Since the dolichol is U-3Hldolichol, any oxidative metabolism of this compound would result in the liberation of water-soluble counts. This observation is excellent evidence that the oxidation of dolichol does not represent a signiflcant metabolic pathway under these conditions. When sufficient counts were available, lipid extracts from the tissues were also assayed for the presence of radioactivity in compounds other than dolichol by thin-layer chromatography as previously described (10). Although the results were semiquantitative, it was clear that there was a small amount of dolichol ester formed which increased with time. Our recent finding of a dolichol esteriflcation system in liver (10) and the data of Butterworth and Hemming (2) make it seem likely that if a sufficiently long pe-
638
KEENAN.
FISCHER,
riod of time had elapsed, a sizable percentage of the radioactivity would have been found as the ester. The ideal way in which to investigate the tissue distribution of injected dolichol would be to use a large number of animals and carry out a sufficient number of assays to ensure statistically significant results. This is not practical at the present time because of the high cost and difficulty of purifying labeled dolichol as well as the large amount of work which would be involved in such studies. It is doubtful that the major findings of the present study would be affected by such a study. The most important findings were that the liver accumulated the dolichol initially present in the plasma and at all times had the highest specific activity of any tissue assayed. A fraction of the injected dose (ca. 16%) was excreted in the feces and the remainder of the radioactivity was present at low levels in various other tissues. The finding that the bulk of the labeled dolichol was present in the liver agreed with the work of Burgos et al. (3) and Butterworth and Hemming (Z), who found that pig liver was a rich source of these polyprenols although no data on the uptake, excretion, or relative levels of these compounds in other tissues were reported. Since our preliminary data indicated that most of the injected [3H]dolichol was taken up by the liver, it was of interest to determine if the radioactivity was localized in specific subcellular fractions. We TABLE SPECIFIC
ACTIVITIES
Subcellular
OF RAT LIVER
SUBCELLULAR
fraction
Nuclei Plasma membrane Whole mitochondria Outer membrane Inner membrane Heavy microsomes Light microsomes Supernatant
Specific
AND
KRUCZEK
used the procedure of Fleischer and Kervina (5) for the subcellular fractionation of the liver of rats which had been injected with [3H]dolichol. The mitochondrial fraction which contained the bulk of the radioactivity was subfractionated by the procedure of Schnaitman and Greenawalt (6). Details of the fractionation procedures and assays for their purity are given under Experimental Procedures. The values obtained for the 3-h point in Table II are lower than expected and, as noted before, are almost certainly due to a failure to inject all the dolichol into the tail vein. The specific activities shown in Table II are a function of the purity of the individual fractions. The specific activities given for the outer mitochondrial membrane fraction almost certainly represent minimal values, since this fraction has been shown to be difficult to free from contaminating material. What is significant and readily apparent is that at all times the mitochondria are more highly radioactivity than any other organelle and that this radioactivity is concentrated within the outer mitochondrial membrane. In addition to the fractions shown in Table II, Golgi apparatus was also purified. The amounts of protein and radioactivity in the purified Golgi fraction were too small to make an accurate assessment of the specific activity of this fraction. The data from Table II lead us to conclude that the injected dolichol is taken up to the largest extent by the mitochondrial II FRACTIONS activity
11/z h
3h
6h
97 41 369 668 69 100 79 145
18 36 350 409 57 97 53 71
46 73 589 1330 90 436 130 142
FOLLOWING (cpm/mg
[3HlDo~~~~o~ of protein)
11 h 55 278 1690 2800 240 665 230 127
INJECTION”
after 20 h
40 h
112 978 1290 192 425 256 72
247 154 1060 3360 261 245 190 62
a Livers from lOO- to 150-g female rats of the same age which had been injected intravenously &i of 13Hldolichol were subjected to subcellular fractionation as described under Experimental The mitochondrial membranes were disrupted by digitonin treatment and isolated as described man and Greenawalt (6).
with 3.3 Procedures. by Schnait-
CELLULAR
DISTRIBUTION
fraction. The activity appears to be localized in the outer membrane of this organelle. Other subcellular fractions show activity which increases with time, but relatively small differences between cell fractions, which may indicate nonspecific binding. The procedure of Schnaitman and Greenawalt (6) is based on the disruption of the mitochondrion with digitonin followed by isolation by differential centrifugation. This procedure was useful in that relatively small samples could be employed. Each fractionation in Table II was conducted on a single rat liver. Assays for marker enzymes indicated that the mitochondrial fractions, although enriched, were contaminated with other fractions. In order to have additional tissue to improve the purifications and to eliminate the possibility of artifacts due to the digitonin treatment, several of the experimental points were repeated using larger quantities of liver and separating the outer and inner membranes of the mitochondria by the procedure of Parsons et al. (7). In this case, membrane separation is brought about the large-amplitude swelling followed by isolation on discontinuous sucrose density gradients. The results of these experiments are shown in Table III. The microsomal fractions were obtained by centrifuging the postmitochondrial supernatant at 105,OOOg for 1 h and were not further purified as in the case of the earlier experiment. It is very likely that these preparations were contaminated with mitochondria, although this fraction does gradually acquire label relative to the other fractions. The low levels of radioactivity which were detected in the supernatant from the 105,OOOgspin were in keeping with our earlier finding that there was little dolichol present in the cytosol fraction. The marker enzyme distribution which we obtained is comparable with the results of others (6, 11) in that the inner mitochondrial membrane fraction contains only small amounts of outer membrane as judged by both the monoamine oxidase and the rotenone insensitive NADH-cytochrome c reductase assays. It is also clear
OF
DOLICHOL
639
that the outer membrane fraction contained considerable cytochrome oxidase. The reason why the levels of cytochrome oxidase were not much higher in the inner membrane preparations relative to the outer membrane is not fully understood. These values are very similar to those obtained by Schnaitman and Greenawalt (6) before treatment of inner membrane fractions with Lubrol to solubilize matrix protein. Although the procedures for subcellular fractionation are difficult to carry out with a high degree of precision, the results which were obtained in both experiments are so clear-cut that variation between samples does not affect the conclusions. There is little doubt that liver mitochondria acquired the bulk of the injected dolichol. It seemslikely that the specific activities reported for the outer mitochondrial membrane represent minimal values since the samples are contaminated with inner mitochondrial membrane as indicated by the presence of cytochrome oxidase in these preparations. It would appear that the digitonin treatment is more effective in disrupting the mitochondria, since the dolichol was found in much greater relative amounts in the outer membrane when this procedure was used. There is no doubt that in terms of both total and specific activity the preponderance of the dolichol in the cell must be associated with the outer mitochondrial membrane. There is no work in the literature directly comparable to the data presented here, since highly labeled dolichol has only recently become available. Only a few attempts have been made to determine the subcellular distribution of dolichol, probably because there is no good assay technique for determining these compounds. Useful data were obtained by Butterworth and Hemming (2) who carried out a partial subcellular fractionation on pig liver. Free dolichol was most abundant in the mitochondria, while esterified dolichol was present in the highest concentration in the nuclear and cell debris fraction. Rat liver was reported to contain somewhat less dolichol than pig liver and a slightly different mixture of polyprenols, the main component being a dolichol-18 rather than the
-
520
4.61
5.73
310
0.461
23.2
20
0.512
59.8
111
118c
4.41
0.496
18.1
40
142
4.13
0.379
10.4
10
862
2.92
0.395
29.8
20
“Fraction
38c
3.62
0.479
16.1
40
1265
0.656
6.78
347
10
4339
1.03
6.45
304
20
(h)
injection
Time
after
“Fraction
P’
B”
497
2.04
6.19
333
40
205
N.D.
1.94
N.D.
10 *
Crude
154
0.353
2.21
N.D.
20
microsomes
B Livers from rats injected with 3.3 &i of [OHJdolichol were combined with three livers from uninjected rats of the same age, sex, and weight and subjected to subcellular fractionation as previously describad. The mitochondria were broken by an amplitude-swelling procedure and the fractions were isolated as described by Parsons et al. (7). All other conditions and assays are as described under Experimental Procedures. b Not determined. c This value subject to variation due to low counts and poor recovery of protein.
Monoamine oxidase (nmol/min/mg of protein) NADH-cytochrome c reductase (rotenone insensitive) (~mollminl mg of protein) Cytochrome oxidase (fimol/min/mg of protein) Specific activity (cpm/mg/ protein)
10
Whole
TABLE
SPECIFXC ACTIVITIES OF RAT LIVER SUBCELLULAR FRACTIONSO mitochondria Inner membrane Outer membrane
189
0.260
1.95
N.D.
2 ix
i?
z
z
3 “?J
z
s 3 nj
x
CELLULAR
DISTRIBUTION
dolichol-19 found in pig liver (15). These differences are probably based on only one or two pooled samples and whether there are really significant differences in composition and concentration is not known. More recently, Martin and Thorne (16) found that following the injection of [4-S3Hlmevalonate into partially hepatectimized rats, dolichol isolated from the mitochondria and the cell debris fractions of liver possessed the highest specific activity. Dallner et al. (17) attempted to estimate the subcellular levels of dolichol phosphate indirectly by extracting various subcellular fractions and measuring their effectiveness in stimulating the formation of lipid-soluble radioactivity from UDP[‘4C]glucose incubated with liver microsomes. Extracts from the nuclear and Golgi fractions produced the greatest stimulation and presumably had the highest levels of dolichol phosphate, while extracts from whole mitochondria and inner and outer membranes from mitochondria were nearly the poorest subcellular source of stimulating material (dolichyl phosphate?). It seems likely that the observed distribution of 13H]dolichol was produced by incorporation of the molecule within the structure of the outer mitochondrial membrane rather than merely being dissolved in the lipid present in this structure, because one would predict other membranes would also be labeled if the compound was merely being held in a nonspecific hydrophobic region. If the cellular distribution of radioactivity is calculated from our data ssuming that mitochondria constitute 30-35% of the protein of the cell, approximately 70% or more of the injected dolichol was associated with this structure. The amount of administered dolichol was so small relative to the endogenous material (less than l/10,000 of that present in normal liver) and the uptake was so slow that it is reasonable to assume that the observed distribution was produced by equilibrium with endogeneous material and was not an artifact due to perturbation of the system. In unpublished experiments, evidence
OF
DOLICHOL
641
was obtained that 13Hldolichol palmitate was absorbed when administered by stomach tube in an olive oil solution. This indicates that at least part of the endogeneous dolichol could have a dietary origin and that the system being studied here may be a physiological one. A dietary source of dolichol might be important in young and rapidly growing animals. It is intriguing to speculate concerning the possible function of dolichol in the outer mitochondrial membrane. The only known role of these compounds is to function as lipid intermediates in glycosylation reactions. Although these reactions apparently do take place in liver mitochondria (16), almost all of the studies which have been reported indicate that the microsomal fraction is the principal site of the enzymes which catalyze reactions involving lipid-linked sugar intermediates. It is possible that the dolichol might have a role in membrane structure as well as being an intermediate in glycosylation reactions. One other aspect which is of interest when considering potential roles for dolichol in the mitochondria is the length of these molecules. Dolichols are the largest molecules in animal tissues containing only carbon to carbon bonds. We have constructed a CPK atomic model of dolichol19. This model suggests that the most stable conformer of dolichol-19 is extended to approximately 70 A in length with a helical structure in which every fourth methyl group within the chain has the same relative position (see Fig. 2). The length of this model is approximately equal to estimates of the thickness of outer mitochondrial membrane (18) and poses questions as to how dolichol molecules are likely to orient themselves within the membrane structure. It will also be of interest to study the means by which high-density lipoprotein fraction-bound dolichol in the plasma is transferred to the mitochondria and whether specific proteins exist for their transfer between organelles. ACKNOWLEDGMENTS The authors wish to thank Jody Woolever and Albert Milne for technical assistance and Dr. Arvin Modak for advice on whole animal experiments.
FIG.
2. A CPK
model
of dolichol-19.
REFERENCES 1. HEMMING, F. W. (1974) MTP Znt. Rev. Sci. Biothem.: Biochem. Lipids Ser. I 4, 39-98. 2. BUTTERWORTH, P. H. W., AND HEMMING, F. W. (1968) Arch. Biochem. Biophys. 128, 503-508. 3. BURGOS, J., HEMMING, F. W., PENNOCK, J. F., AND MORTON, R. A. (1963) Biochem. J. 88, 470-482. 4. KEENAN, F. W., AND KRIJCZEK, M. (1975) Anal. Biochem. 69, 504-509. 5. FLEISCHER, S., AND KERVINA, M. (1974) in Methods in Enzymology, Vol. 31, Part A, pp. 6-40, Academic Press, New York. 6. SCHNAITMAN, C., AND GREENAWALT, J. W. (1968) J. Cell Biol. 38, 158-171. 7. PARSONS, D. F., WILLIAMS, G. R., AND CHANCE, B. (1966) Ann. N. Y. Acad. Sci. 137, 643-666. 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. DOLE, J. P. (1956) J. Clin. Invest. 35, 150-154. 10. KEENAN, R. W., AND KRUCZEK, M. (1976) Bio-
The
scale represents
Angstrom
units.
chemistry 15, 1586-1591. 11. SOTTOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L., AND BERGSTRAND, A. (1967) in Methods in Enzymology, Vol. 10, pp. 448-463, Academic Press, New York. 12. SWANSON, M. A. (1955) in Methods in Enzymology, Vol. 2, p. 541, Academic Press, New York. 13. BARTLETT, G. R. (1959) J. Biol. Chem. 234, 466468. 14. KEENAN, R. W., KRUCZEK, M., AND FISCHER, J. B. (1977) Biochim. Biophys. Acta, in press. 15. GOUCH, D., AND HEMMING, F. W. (1970) Biothem. J. 118, 163-166. 16. MARTIN, H. G., AND THORNE, K. J. I. (1974) Biochem. J. 138, 277-280. 17. DALLNER, G., BEHRENS, N. H., PARODI, A. J., AND LELOIR, L. F. (1972) FEBS Lett. 24, 315317. 18. DEROBERTS, E. D. P., NOWINSKI, W. W., AND SAEZ, F. A. (1970) in Cell Biology, 5th ed. p. 204, W. B. Saunders Co., Philadelphia, Pa. 1970.
OF BIOCHEMISTRY
AND
BIOPHYSICS
The Tissue and Subcellular ROY W. KEENAN, Department
of Biochemistry,
179,
634-642
(1977)
Distribution
JOSEPH
B. FISCHER,
The University
of Texas
Received
AND
Health
August
of [3H]Dolichol
Science
MICHAEL Center,
in the Rat’ E. KRUCZEK
San Antonio,
Texas
78284
31, 1976
Following the intravenous injection of nanomolar amounts of [3H]dolichol into rats, the radioactivity rapidly appeared in the high-density lipoprotein fraction of the plasma and circulated with a half-life of about 9 h. A fraction of the injected activity was excreted in the feces, presumably through the bile, but evidence was obtained that little oxidative breakdown of dolichol occurred. All tissues assayed acquired radioactivity, but the liver attained the highest specific activity and the largest percentage of the total radioactive dolichol. Subcellular fractionation of the liver revealed that mitochondrial preparations contained the bulk of the labeled dolichol at all times tested up to 40 h after injection. Disruption of the mitochondrial structure by two different techniques permitted the isolation of inner and outer membrane fractions and it was found that the [3Hldolichol was concentrated in the outer membrane fraction. The significance of these findings is discussed.
Despite the fact that a large amount of interest has been shown in establishing the role of the polyisoprenols in transglycosylation reactions, very little is known concerning the metabolism of the polyprenol molecule (1). Dolichol, which is actually a generic term applied to a group of related polyprenols is of particular interest. These compounds, which contain 17 to 22 isoprene units including a saturated hydroxyterminal isoprene unit, are particularly abundant in the livers of higher animals where they are found principally as the free alcohol and the ester (2, 3). The only known function of dolichol is to serve as lipid intermediates in transglycosylation reactions, but all studies to date indicate that only a minute fraction of the total dolichol participates in these reactions at any given time. In addition, the subcellular localization of dolichol (2) (although there is very limited information available) does not correspond with the site where it presumably functions, since dolichol concentration is relatively low in the microsomal fraction, in which transglycosylation reactions involving lipid-linked 1 Supported by NIH Grant 1 R01 AM 17897 and by Grant AQ-448 from the Robert A. Welch Foundation.
sugars are believed to occur. For the reasons mentioned above, it seemed worthwhile to investigate the in uiuo metabolism of dolichol with the idea that these studies might suggest functions for this material in addition to its role in transglycosylation reactions. One aspect of these studies which is of particular interest is related to the fact that dolichol is an unusually long molecule (we estimate dolichol-19 to be 70 A in length) which may greatly restrict the ways in which it can be transported or oriented in membranes. Our recent synthesis of [l-3Hldolichol (4) provided us with a means with which to study dolichol metabolism. In this paper we describe a number of observations indicating how dolichol is transported, localized within the cell, and excreted. EXPERIMENTAL
PROCEDURES
Materials. The [l-3H1dolichol used in these experiments was a gift from New England Nuclear. It was synthesized using our recently described procedure (41 and had a specific activity of 14.8 Ci/mmol. This material consists of a mixture of isoprenologs, the principal compound being a C-95 polyprenol. Prior to use, the radioactive dolichol sample was purified by chromatography on silica gel G thin-layer plates (Analtech Inc.) which were developed with hexane:ether @0:20, v/v). The developed plates were
634 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
CELLULAR
DISTRIBUTION
sprayed with Omnifluor (New England Nuclear) placed in a film casset and stored in a dry ice chest. After approximately 30 min of exposure, the film was developed and the dolichol-containing area of the plate as revealed by autoradiography, was extracted with chloroform:methanol (2:l) and filtered. The filtrate was dried and the residue was dissolved in 2% ether in hexane. This extract was chromatographed on a short alumina column and the dolichol was eluted in 20% ether in hexane [see Keenan and Kruczek (4) for details]. This purification procedure was necessary to remove impurities which form in the highly labeled dolichol preparations. Aliquots of the purified radioactive dolichol were dried and suspended in the steroid suspending vehicle provided by the National Cancer Institute. The concentration of the dolichol suspension was 239 nmol (33.3 @XI ml. Glucose 6-phosphate, cytochrome c, bovine serum albumin, digitonin, and rotenone were obtained from Sigma Chemical Company. Benzylamine, a product of Aldrich Chemical Co., was purified by dissolving it in ether and passing HCl gas through the ether solution. The precipitated hydrochloride salt which was washed extensively with ether and dried was used as the substrate in monoamine oxidase assays. All other solvents and reagents were of reagent grade. Subcellular fractionation techniques. Young Sprague-Dawley female rats (100-150 g) were used in these studies. The animals were injected in the tail vein with approximately 100 ~1 of a dolichol suspension (3.3 &il prepared as described above. At the appropriate times, the animals were lightly anesthetized with ether and blood was obtained by heart puncture. The rats were killed by decapitation and various organs were removed, weighed, and frozen. The livers were immediately subjected to the subcellular fractionation techniques described by Fleischer and Kervina (5) essentially without modification, except as mentioned under Results. The fractions isolated were the nuclear, plasma membrane, mitochondria, heavy microsomes, light microsomes, Golgi, and supernatant. The purified mitochondria were subfractionated into the inner and outer membranes by treatment with digitonin as described by Schnaitman and Greenawalt (6) or by the large-amplitude swelling technique developed by Parsons et al. (7). The best results were obtained with the latter technique when the liver from the injected animal was combined with livers from three untreated animals of the same age and sex. Assays. Protein was determined by the method of Lowry et al. (8) using bovine serum albumin as a standard. Nuclei were dialyzed prior to the determination of protein in order to remove the large quantity of sucrose which interfered with the protein assay.
OF
635
DOLICHOL
Dolichol radioactivity was measured in various tissues by selecting what appeared to be a representative sample of the larger tissues, i.e., intestine, liver, etc., or the entire sample in the case of small organs. A 20% homogenate of the tissue was prepared in sucrose and aliquots of these homogenates were extracted by the procedure of Dole (9). A micromodification of this procedure was also used on small samples of plasma and tissues. This procedure was found to give quantitative recovery of 13Hldolichol from tissue and had the advantage of being somewhat more rapid and simpler than extraction with chloroform-methanol mixtures. The lower phases of the extracts were also checked for the presence of water-soluble radioactivity but they seldom contained significant radioactivity. When sufficient radioactivity was present, samples of the lipid extracts were assayed for the presence of dolichol esters (10). The degree of purification of the subcellular fractions from rat liver was estimated by measuring the specific activities of the marker enzymes. Cytochrome oxidase and monoamine oxidase were measured essentially as described by Schnaitman and Greenawalt (6) except that we found it necessary to purify the benzylamine as described above in order to obtain good rates in the monoamine oxidase assay. Rotenone-insensitive NADH-cytochrome c reductase was determined by the procedure of Sottocasa et al. (11). Glucose 6-phosphatase was measured as described by Swanson (12) and the liberated phosphate was estimated by the method of Bartlett (13). These assays showed good correlation of marker enzymes with the appropriate subcellular fractions; results of the mitochondrial assays will be discussed in the appropriate section. Radioactivity was assayed by counting aliquots of lipid extracts from the Dole extraction procedure in a toluene based scintillator as previously described (10) and/or by digesting aliquots of homogenized tissue or subcellular fractions overnight at 65°C in 1.0 ml of NCS tissue solubilizer (AmershamSearle) and counting the digested samples in 12 ml of Beckman Bio-Solv 3. Samples were counted in a Beckman LS-230 liquid scintillation system together with blanks which had been subjected to the same digestion procedure. Vials were usually stored in the dark before counting, but were always counted until constant values were obtained. Where it was required, representative samples were recounted with added internal standards to correct for differences in counting efficiencies. RESULTS
AND
DISCUSSION
Following the intravenous injection of [3Hldolichol in the quantities which we employed (ca. 20 nmol) almost all of the radioactivity was rapidly taken up in the
636
KEENAN,
FISCHER.
high-density lipoprotein fraction (141. Only a small percentage of the label was detected in association with other plasma fractions or the red cells. Figure 1 shows the rate at which dolichol disappeared from the plasma. The fact that the 3-h point does not fall on a straight line is probably due to an incomplete injection of the dolichol suspension into the tail vein of this animal. The half-life of dolichol in the plasma was estimated from Fig. 1 as approximately 9 h. This value is based on six points from different animals all of the same age and sex. This is in accord with a value of approximately 13 h which was obtained by taking blood at similar intervals from one animal which had been injected with [3H]dolichol. These values are good evidence that dolichol circulates for a relatively long period of time as compared to the half-lives of retinol, cholesterol, and other lipids which have been estimated. The only previous reference to the occurrence of dolichol in the circulation was that it was detected in trace amounts in blood (1). The influence of nutritional state, endogenous dolichol concentrations, and other factors which might be expected to effect the half-life of injected dolichol have yet to be established. Lipid extraction of the plasma and analysis by thinlayer chromatography revealed that essentially all of the radioactivity in the plasma was present as free dolichol rather than the ester or breakdown products. The radioactivity in the plasma declined with time and other tissues became progressively more labeled, particularly the liver and the spleen. The data on the tissue distribution of 13Hldolichol are given in Table I. The specific activity values in this table may be somewhat misleading for two reasons. First, the actual values for the early points are likely to be lower than shown since the tissues contained blood which had a relatively high specific activity. This source of error was minimized, but not eliminated, by rinsing and blotting the tissues prior to assay. A second and more serious source of error was due to the fact that it was not possible to ensure that all of the administered radioactive compound was introduced into the blood
AND
KRUCZEK
FIG. 1. The plasma levels of radioactivity at various times following the intravenous injection of [3Hldolichol. Approximately 20 nmol of [3H]dolichol was administered to each rat. Blood was obtained by heart puncture at the indicated time, and the radioactivity of plasma was determined by digesting aliquots of plasma in a protein solubilizer and measuring the radioactivity as described under Experimental Procedures.
stream when it was injected into the tail vein. This is almost certainly why the 3-h point in Fig. 1 does not fall on the line and why the specific activities for tissues from this point are lower than expected. The linearity of all the other points in Fig. 1 is evidence that the doses administered were similar in these cases and that slow absorption of dolichol which was not injected into the blood stream was not a significant problem. A more straightforward way to look at the data is to compare the amount of the total radioactivity in each tissue with the total recovered radioactivity as shown in Table I. It is clear from these data that initially most of the radioactivity was in the plasma and that the bulk of the labeled material was deposited in the liver. The intestines and their contents also acquired large amounts of radioactivity. Most of this radioactivity was present in the intestinal contents rather than in the intestinal tissue. Feces and urine were collected from those rats which were maintained 20 and
CELLULAR
DISTRIBUTION
OF
TABLE DISTRIBUTION
OF RADIOACTIVITY
IN
TISSUES
637
DOLICHOL
I
OF RATS
GIVEN
[3H]Do~~~~o~
AT VARIOUS
TIMES
AFTER
ADMINISTRATIONS Tissue
Hours
:I Blood plasma Liver Intestines contents Spleen Lung Heart Pancreas Kidney Brain
Re-
covered radioac tivity (5%)
+
specific activity
Re-
:overed adioac tivity (%o)
60.3
9980
43.9
18.4 13.4
2460 1940
30.1 17.2
0.86 2.7 0.96 0.43 2.2 0.48
1650 2150 1480 1280 1350 350
gpecific wtivitJ
-6
dose
Rempecific overed I a ctivitj adioac
Reoverec
ldioac tivity (%)
Ipecifh Rectivitj I C’overec ri ndioac tivity (%I
40.0
21.4
3630
15.7
1670
3960 875
6120 1210
67.7 4.67
6570 200
975 2630 725 900 1000 200
1320 2350 1100 1230 1000 300
9.4 0.63 0.06 1.05 0.72
tivity (I)
5280
1800 575 1380 750 400 1000 575 150
after
1.0 5.3 0.63 0.81
2.0 0.43
L
1
Ipecific ctivitj
Re-
ipecifk
ctivity
adioac tivity (%) 3.75 86.1 2.7 3.3 1.8 0.30 0.71 0.53 0.78
416 7660 116 4075 750 275 882 175 250
a Female rats averaging 125 g each were injected intravenously with 3.3 &i of a 13H1dolichol suspension. At the indicated times following isotope administration, the animals were sacrificed and representative samples from each tissue were taken. The tissue samples were rinsed off and blotted to remove blood and subjected to lipid extraction and the radioactivity in the organic phase was determined. Aliquots of blood plasma were digested in protein solubilizer and counted. Blood plasma was assumed to constitute 3% of the body weight. The specific activity is expressed in terms of counts/per minute per 100 milligrams wet weight. Details of the procedures used in the extractions and radioactivity measurements are given under Experimental Procedures.
40 h. The results were not quantitative, but it was clear that most of the excreted radioactivity was present in the feces. To determine the extent of dolichol excretion two rats were each injected with 0.1 pmol of 13Hldolichol (approximately five times as much as used in the experiment described in Table I) and the urine and feces were collected. In keeping with our previous results, only small quantities of radioactivity were found in the urine, but approximately 16% of the total injected dose could be accounted for in the feces after 90 h. The largest amount of radioactivity was excreted between 44 and 66 h. Almost all the radioactivity was lipid-soluble and cochromatographed with free dolichol. The evidence from these experiments indicates that dolichol is probably excreted in the bile, but it is not known if it is first converted to a glucuronide as in the case of retinol or excreted unchanged. The radioactivity of the various tissues was measured by extracting the tissues
with isopropanol-heptane as described under Experimental Procedures. It is of interest that very little radioactivity could be detected in the aqueous phase from these lipid extracts. Since the dolichol is U-3Hldolichol, any oxidative metabolism of this compound would result in the liberation of water-soluble counts. This observation is excellent evidence that the oxidation of dolichol does not represent a signiflcant metabolic pathway under these conditions. When sufficient counts were available, lipid extracts from the tissues were also assayed for the presence of radioactivity in compounds other than dolichol by thin-layer chromatography as previously described (10). Although the results were semiquantitative, it was clear that there was a small amount of dolichol ester formed which increased with time. Our recent finding of a dolichol esteriflcation system in liver (10) and the data of Butterworth and Hemming (2) make it seem likely that if a sufficiently long pe-
638
KEENAN.
FISCHER,
riod of time had elapsed, a sizable percentage of the radioactivity would have been found as the ester. The ideal way in which to investigate the tissue distribution of injected dolichol would be to use a large number of animals and carry out a sufficient number of assays to ensure statistically significant results. This is not practical at the present time because of the high cost and difficulty of purifying labeled dolichol as well as the large amount of work which would be involved in such studies. It is doubtful that the major findings of the present study would be affected by such a study. The most important findings were that the liver accumulated the dolichol initially present in the plasma and at all times had the highest specific activity of any tissue assayed. A fraction of the injected dose (ca. 16%) was excreted in the feces and the remainder of the radioactivity was present at low levels in various other tissues. The finding that the bulk of the labeled dolichol was present in the liver agreed with the work of Burgos et al. (3) and Butterworth and Hemming (Z), who found that pig liver was a rich source of these polyprenols although no data on the uptake, excretion, or relative levels of these compounds in other tissues were reported. Since our preliminary data indicated that most of the injected [3H]dolichol was taken up by the liver, it was of interest to determine if the radioactivity was localized in specific subcellular fractions. We TABLE SPECIFIC
ACTIVITIES
Subcellular
OF RAT LIVER
SUBCELLULAR
fraction
Nuclei Plasma membrane Whole mitochondria Outer membrane Inner membrane Heavy microsomes Light microsomes Supernatant
Specific
AND
KRUCZEK
used the procedure of Fleischer and Kervina (5) for the subcellular fractionation of the liver of rats which had been injected with [3H]dolichol. The mitochondrial fraction which contained the bulk of the radioactivity was subfractionated by the procedure of Schnaitman and Greenawalt (6). Details of the fractionation procedures and assays for their purity are given under Experimental Procedures. The values obtained for the 3-h point in Table II are lower than expected and, as noted before, are almost certainly due to a failure to inject all the dolichol into the tail vein. The specific activities shown in Table II are a function of the purity of the individual fractions. The specific activities given for the outer mitochondrial membrane fraction almost certainly represent minimal values, since this fraction has been shown to be difficult to free from contaminating material. What is significant and readily apparent is that at all times the mitochondria are more highly radioactivity than any other organelle and that this radioactivity is concentrated within the outer mitochondrial membrane. In addition to the fractions shown in Table II, Golgi apparatus was also purified. The amounts of protein and radioactivity in the purified Golgi fraction were too small to make an accurate assessment of the specific activity of this fraction. The data from Table II lead us to conclude that the injected dolichol is taken up to the largest extent by the mitochondrial II FRACTIONS activity
11/z h
3h
6h
97 41 369 668 69 100 79 145
18 36 350 409 57 97 53 71
46 73 589 1330 90 436 130 142
FOLLOWING (cpm/mg
[3HlDo~~~~o~ of protein)
11 h 55 278 1690 2800 240 665 230 127
INJECTION”
after 20 h
40 h
112 978 1290 192 425 256 72
247 154 1060 3360 261 245 190 62
a Livers from lOO- to 150-g female rats of the same age which had been injected intravenously &i of 13Hldolichol were subjected to subcellular fractionation as described under Experimental The mitochondrial membranes were disrupted by digitonin treatment and isolated as described man and Greenawalt (6).
with 3.3 Procedures. by Schnait-
CELLULAR
DISTRIBUTION
fraction. The activity appears to be localized in the outer membrane of this organelle. Other subcellular fractions show activity which increases with time, but relatively small differences between cell fractions, which may indicate nonspecific binding. The procedure of Schnaitman and Greenawalt (6) is based on the disruption of the mitochondrion with digitonin followed by isolation by differential centrifugation. This procedure was useful in that relatively small samples could be employed. Each fractionation in Table II was conducted on a single rat liver. Assays for marker enzymes indicated that the mitochondrial fractions, although enriched, were contaminated with other fractions. In order to have additional tissue to improve the purifications and to eliminate the possibility of artifacts due to the digitonin treatment, several of the experimental points were repeated using larger quantities of liver and separating the outer and inner membranes of the mitochondria by the procedure of Parsons et al. (7). In this case, membrane separation is brought about the large-amplitude swelling followed by isolation on discontinuous sucrose density gradients. The results of these experiments are shown in Table III. The microsomal fractions were obtained by centrifuging the postmitochondrial supernatant at 105,OOOg for 1 h and were not further purified as in the case of the earlier experiment. It is very likely that these preparations were contaminated with mitochondria, although this fraction does gradually acquire label relative to the other fractions. The low levels of radioactivity which were detected in the supernatant from the 105,OOOgspin were in keeping with our earlier finding that there was little dolichol present in the cytosol fraction. The marker enzyme distribution which we obtained is comparable with the results of others (6, 11) in that the inner mitochondrial membrane fraction contains only small amounts of outer membrane as judged by both the monoamine oxidase and the rotenone insensitive NADH-cytochrome c reductase assays. It is also clear
OF
DOLICHOL
639
that the outer membrane fraction contained considerable cytochrome oxidase. The reason why the levels of cytochrome oxidase were not much higher in the inner membrane preparations relative to the outer membrane is not fully understood. These values are very similar to those obtained by Schnaitman and Greenawalt (6) before treatment of inner membrane fractions with Lubrol to solubilize matrix protein. Although the procedures for subcellular fractionation are difficult to carry out with a high degree of precision, the results which were obtained in both experiments are so clear-cut that variation between samples does not affect the conclusions. There is little doubt that liver mitochondria acquired the bulk of the injected dolichol. It seemslikely that the specific activities reported for the outer mitochondrial membrane represent minimal values since the samples are contaminated with inner mitochondrial membrane as indicated by the presence of cytochrome oxidase in these preparations. It would appear that the digitonin treatment is more effective in disrupting the mitochondria, since the dolichol was found in much greater relative amounts in the outer membrane when this procedure was used. There is no doubt that in terms of both total and specific activity the preponderance of the dolichol in the cell must be associated with the outer mitochondrial membrane. There is no work in the literature directly comparable to the data presented here, since highly labeled dolichol has only recently become available. Only a few attempts have been made to determine the subcellular distribution of dolichol, probably because there is no good assay technique for determining these compounds. Useful data were obtained by Butterworth and Hemming (2) who carried out a partial subcellular fractionation on pig liver. Free dolichol was most abundant in the mitochondria, while esterified dolichol was present in the highest concentration in the nuclear and cell debris fraction. Rat liver was reported to contain somewhat less dolichol than pig liver and a slightly different mixture of polyprenols, the main component being a dolichol-18 rather than the
-
520
4.61
5.73
310
0.461
23.2
20
0.512
59.8
111
118c
4.41
0.496
18.1
40
142
4.13
0.379
10.4
10
862
2.92
0.395
29.8
20
“Fraction
38c
3.62
0.479
16.1
40
1265
0.656
6.78
347
10
4339
1.03
6.45
304
20
(h)
injection
Time
after
“Fraction
P’
B”
497
2.04
6.19
333
40
205
N.D.
1.94
N.D.
10 *
Crude
154
0.353
2.21
N.D.
20
microsomes
B Livers from rats injected with 3.3 &i of [OHJdolichol were combined with three livers from uninjected rats of the same age, sex, and weight and subjected to subcellular fractionation as previously describad. The mitochondria were broken by an amplitude-swelling procedure and the fractions were isolated as described by Parsons et al. (7). All other conditions and assays are as described under Experimental Procedures. b Not determined. c This value subject to variation due to low counts and poor recovery of protein.
Monoamine oxidase (nmol/min/mg of protein) NADH-cytochrome c reductase (rotenone insensitive) (~mollminl mg of protein) Cytochrome oxidase (fimol/min/mg of protein) Specific activity (cpm/mg/ protein)
10
Whole
TABLE
SPECIFXC ACTIVITIES OF RAT LIVER SUBCELLULAR FRACTIONSO mitochondria Inner membrane Outer membrane
189
0.260
1.95
N.D.
2 ix
i?
z
z
3 “?J
z
s 3 nj
x
CELLULAR
DISTRIBUTION
dolichol-19 found in pig liver (15). These differences are probably based on only one or two pooled samples and whether there are really significant differences in composition and concentration is not known. More recently, Martin and Thorne (16) found that following the injection of [4-S3Hlmevalonate into partially hepatectimized rats, dolichol isolated from the mitochondria and the cell debris fractions of liver possessed the highest specific activity. Dallner et al. (17) attempted to estimate the subcellular levels of dolichol phosphate indirectly by extracting various subcellular fractions and measuring their effectiveness in stimulating the formation of lipid-soluble radioactivity from UDP[‘4C]glucose incubated with liver microsomes. Extracts from the nuclear and Golgi fractions produced the greatest stimulation and presumably had the highest levels of dolichol phosphate, while extracts from whole mitochondria and inner and outer membranes from mitochondria were nearly the poorest subcellular source of stimulating material (dolichyl phosphate?). It seems likely that the observed distribution of 13H]dolichol was produced by incorporation of the molecule within the structure of the outer mitochondrial membrane rather than merely being dissolved in the lipid present in this structure, because one would predict other membranes would also be labeled if the compound was merely being held in a nonspecific hydrophobic region. If the cellular distribution of radioactivity is calculated from our data ssuming that mitochondria constitute 30-35% of the protein of the cell, approximately 70% or more of the injected dolichol was associated with this structure. The amount of administered dolichol was so small relative to the endogenous material (less than l/10,000 of that present in normal liver) and the uptake was so slow that it is reasonable to assume that the observed distribution was produced by equilibrium with endogeneous material and was not an artifact due to perturbation of the system. In unpublished experiments, evidence
OF
DOLICHOL
641
was obtained that 13Hldolichol palmitate was absorbed when administered by stomach tube in an olive oil solution. This indicates that at least part of the endogeneous dolichol could have a dietary origin and that the system being studied here may be a physiological one. A dietary source of dolichol might be important in young and rapidly growing animals. It is intriguing to speculate concerning the possible function of dolichol in the outer mitochondrial membrane. The only known role of these compounds is to function as lipid intermediates in glycosylation reactions. Although these reactions apparently do take place in liver mitochondria (16), almost all of the studies which have been reported indicate that the microsomal fraction is the principal site of the enzymes which catalyze reactions involving lipid-linked sugar intermediates. It is possible that the dolichol might have a role in membrane structure as well as being an intermediate in glycosylation reactions. One other aspect which is of interest when considering potential roles for dolichol in the mitochondria is the length of these molecules. Dolichols are the largest molecules in animal tissues containing only carbon to carbon bonds. We have constructed a CPK atomic model of dolichol19. This model suggests that the most stable conformer of dolichol-19 is extended to approximately 70 A in length with a helical structure in which every fourth methyl group within the chain has the same relative position (see Fig. 2). The length of this model is approximately equal to estimates of the thickness of outer mitochondrial membrane (18) and poses questions as to how dolichol molecules are likely to orient themselves within the membrane structure. It will also be of interest to study the means by which high-density lipoprotein fraction-bound dolichol in the plasma is transferred to the mitochondria and whether specific proteins exist for their transfer between organelles. ACKNOWLEDGMENTS The authors wish to thank Jody Woolever and Albert Milne for technical assistance and Dr. Arvin Modak for advice on whole animal experiments.
FIG.
2. A CPK
model
of dolichol-19.
REFERENCES 1. HEMMING, F. W. (1974) MTP Znt. Rev. Sci. Biothem.: Biochem. Lipids Ser. I 4, 39-98. 2. BUTTERWORTH, P. H. W., AND HEMMING, F. W. (1968) Arch. Biochem. Biophys. 128, 503-508. 3. BURGOS, J., HEMMING, F. W., PENNOCK, J. F., AND MORTON, R. A. (1963) Biochem. J. 88, 470-482. 4. KEENAN, F. W., AND KRIJCZEK, M. (1975) Anal. Biochem. 69, 504-509. 5. FLEISCHER, S., AND KERVINA, M. (1974) in Methods in Enzymology, Vol. 31, Part A, pp. 6-40, Academic Press, New York. 6. SCHNAITMAN, C., AND GREENAWALT, J. W. (1968) J. Cell Biol. 38, 158-171. 7. PARSONS, D. F., WILLIAMS, G. R., AND CHANCE, B. (1966) Ann. N. Y. Acad. Sci. 137, 643-666. 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. DOLE, J. P. (1956) J. Clin. Invest. 35, 150-154. 10. KEENAN, R. W., AND KRUCZEK, M. (1976) Bio-
The
scale represents
Angstrom
units.
chemistry 15, 1586-1591. 11. SOTTOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L., AND BERGSTRAND, A. (1967) in Methods in Enzymology, Vol. 10, pp. 448-463, Academic Press, New York. 12. SWANSON, M. A. (1955) in Methods in Enzymology, Vol. 2, p. 541, Academic Press, New York. 13. BARTLETT, G. R. (1959) J. Biol. Chem. 234, 466468. 14. KEENAN, R. W., KRUCZEK, M., AND FISCHER, J. B. (1977) Biochim. Biophys. Acta, in press. 15. GOUCH, D., AND HEMMING, F. W. (1970) Biothem. J. 118, 163-166. 16. MARTIN, H. G., AND THORNE, K. J. I. (1974) Biochem. J. 138, 277-280. 17. DALLNER, G., BEHRENS, N. H., PARODI, A. J., AND LELOIR, L. F. (1972) FEBS Lett. 24, 315317. 18. DEROBERTS, E. D. P., NOWINSKI, W. W., AND SAEZ, F. A. (1970) in Cell Biology, 5th ed. p. 204, W. B. Saunders Co., Philadelphia, Pa. 1970.