ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 198, No. 2, December, pp. 562-571, 1979
Characterization
of Rat Liver Cytosol
Retinyl
Ester Lipoprotein
Complex’
JORAM HELLER Jules Stein
Eye Institute,
UCLA School of Medicine,
Los Angeles, California
90024
Received February 5, 1979; revised July 30, 1979 A soluble high molecular weight lipoprotein complex containing retinyl esters and unesterified retinol was isolated from rat liver cytosol. This material accounts for about 10% of the total liver retinyl compounds, and its protein moiety accounts for about 0.1% of the protein of the liver homogenate and about 0.7% of the cytosol protein. The lipoprotein was purified by gel filtration and hydroxyapatite chromatography. The lipoprotein complex gave a single band by electrophoresis on cellulose acetate as judged by both lipid- and proteinspecific stains. The lipoprotein complex did not dissociate into smaller subunits in low ionic strength buffer (1 mM sodium phosphate, pH 7.7). The retinyl ester lipoprotein complex has an absorption spectrum with peaks at 328 nm (retinyl chromophore) and 258 nm. Retinyl compounds in the carrier lipoprotein complex do not show an increase of the quantum yield of fluorescence and do not show energy transfer when excited at either 258 or 280 nm. There is no induced optical activity of the retinyl chromophore absorption band. The lipoprotein complex contains about 3% (by weight) of retinyl compounds, 96% of which are retinyl esters and 4% of which are unesterified retinol. The lipoprotein complex consists of about 66% (by weight) lipids, about 30% protein, and some 4% carbohydrate. There are at least 15 polypeptide chains ranging in size from about 2 x lo4 to about 2.1 x lo5 M,. Retinyl compounds in the lipoprotein complex are stable for at least 3 months in 0.05 M phosphate buffer, pH 7.4, at 4°C. Stability was judged from the total amount of retinyl esters plus unesterified retinol. Retinyl compounds of the lipoprotein complex were unstable below pH 6.4 or in the presence of 1 M NaCl.
From 80 to 90% of stored retinol (vitamin A) in the mammalian body is found in the liver, and from 80 to 95% of this is found as retinyl esters (l-3). Although it was formerly thought that most of the retinyl compounds in the liver were found in the Kupffer cells (4), it was recently shown by Linder et al. (5), utilizing the technique of direct isolation of Kupffer cells, that over 96% of total liver retinol and retinyl ester is present in the hepatocytes with less than 4% of total retinyl compounds found in the Kupffer cells. Retinol stored in the liver is delivered to the target tissue via the blood. All the retinol in the blood is complexed with a specific protein, the serum retinol-binding protein
(RBP)” (6, 7). The retinol in serum RBP is found exclusively as unesterified retinol. In fact, serum RBP does not bind retinyl palmitate (8), the predominant retinyl ester in the liver. Serum RBP is synthesized in the liver and apparently is released from the liver into the circulation only as a complex with retinol (9). Only about 0.3% of total liver retinyl compounds are found as retinyl bound to intracellular RBP (2). Krishnamurthy et al. (10) reported in 1958 that retinyl esters and retinol in the soluble liver homogenate seemed to behave as if they were complexed with a protein. This was suggested by the precipitation of the retinyl ester and retinol by ammonium sulfate and other protein precipitating agents, and by
* This paper was supported by Grants EY 00704, EY 00331, and EY 00702 from the National Institutes of Health, USPHS.
2 Abbreviations used: RBP, serum retinol-binding protein; CRLP, cytosol retinyl ester lipoprotein complex; SDS, sodium dodecyl sulfate.
0003.9861/79/140562-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
562
LIVER RETINYL
ESTER LIPOPROTEIN
the inability to extract the retinyl chromophores with diethyl ether unless a protein denaturing agent was also used. In the intervening years, no further reports have elaborated on these interesting observations by Krishnamurthy et al. (10). As part of a continuing interest in intracellular retinol-binding proteins we have further investigated the liver cytosol retinyl ester and retinol-binding proteins. This paper describes the isolation and characterization of a soluble, high molecular weight lipoprotein complex from rat liver. This complex accounts for at least 10% of the total liver retinyl ester and unesterified retinol. EXPERIMENTAL
PROCEDURES
Preparation of rut liver cytosol. White laboratory rats (about 300 g each) were kept on a normal ad libitum diet (Purina laboratory chow for rats, vitamin A supplemented by the manufacturer). The animals were starved for 18 h before being sacrificed and then anesthetized with diethyl ether. The peritoneal cavity of each animal was opened and the liver was removed. Blood was washed away and the liver was chopped into 2- to 3-mm pieces with scissors. Two milliliters of cold sucrose buffer (0.25 M sucrose, 10 mM N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 50 mM KCl, 1 mM MgCl,, 2 mM ethylenediaminetetraacetic acid, and 0.1 mM dithiothreitol), adjusted to pH 7.4 with NaOH (density of buffer at 4°C was 1.036 g/ml), was added per gram of liver and the liver was then homogenized at top speed for 1 min at 4°C in a glass and Teflon pestle homogenizer with a 0.125 mm clearance. The pestle was driven by a Talboys Engineering Model 103 motor. The homogenate was filtered through two layers of cheesecloth. The homogenate was centrifuged for 1 h at 29,000g at 4°C (Sorvall SS-34 rotor). The clear middle layer between a thin white lipid float and a precipitate was carefully removed with a syringe and a No. 18 cannula. This clear supernatant was then centrifuged in a fixed angle rotor for 1 h at 105,OOOgat 4°C (Beckman type 65 rotor, 39,000 rpm). At the end of this centrifugation, there was a 2- to 3-mm-thick layer of white opaque material floating on top of a clear middle layer (cytosol). Carefully avoiding the float, the infranatant was removed with a syringe and cannula as before. The floats and precipitates from both centrifugations were discarded. Gel j2tration chromatography of cytosol. Fifty milliliters of rat liver cytosol was applied to an agarose column (5 x 122 cm; Bio-Gel A-5m, 100-200 mesh, Bio-Rad, Richmond, Calif.; nominal agarose content, 6%). The column was equilibrated with 50 mM potas-
COMPLEX CHARACTERIZATION
563
sium phosphate buffer, pH 7.4, containing 0.1 mM dithiothreitol and 0.1 mM sodium azide. Flow rate (pump) was 88 ml/h (4.5 ml/cmz) and the column was operated at 4°C. Fractions of 10 ml were collected. The effluent absorption at 280 nm was monitored with an LKB Uvicord II. Absorption and spectra of fractions were monitored with a Beckman doublebeam ratio recording spectrophotometer (Beckman ACTA MVI). Fluorescence, protein, and sugar were also determined as described below. Hydroxyapatite chromatography. The retinyl compound fraction obtained by gel filtration (Fig. 1, first peak) was further purified on a column of hydroxyapatite (5 x 3.5 cm; Bio-Gel HT, Bio-Rad, Richmond, Calif.). The column was equilibrated with 50 mM potassium phosphate buffer, pH 6.9, containing 0.1 mM dithiothreitol and 0.1 mM sodium azide. After applying the material, the column was eluted with increasing concentrations of potassium phosphate buffer, pH 6.9. The CRLP was eluted with 500 mM potassium phosphate, pH 6.9. Flow rate was about 50 ml/h and fractions of about 10 ml were collected. Protein determination. Protein was determined either by the dye-binding assay (11) or the Lowry assay (12) with crystalline bovine serum albumin (Sigma) serving as standard. Protein was quantitatively determined by amino acid analysis after hydrolysis with 3 N p-toluenesulfonic acid (13) or 6 N HCl, using the Beckman amino acid analyzer, Model 120 C. Carbohydrate determination. Total hexose sugar was determined by the phenol-sulfuric acid method (14) with mannose as standard. Sialic acid was determined according to the method of Warren (15). Spectroscopic measurements. Absorption spectra were measured with a double monochromotor, doublebeam ratio recording spectrophotometer (Beckman ACTA MVI). Circular dichroism and fluorescence were measured as previously described (16). Electrophoresis. Electrophoresis on cellulose acetate strips was performed with the Beckman Microzone system according to the manufacturer’s instructions. A barbital buffer, pH 8.6 and ionic strength 0.075, was used. All of the samples were dialyzed and concentrated in this buffer before electrophoresis (negative pressure dialysis-concentrator, Bio-Molecular Dynamics, Oregon). Electrophoresis was performed at 22°C for 20 min with a constant voltage of 250 V and a beginning current of 3 mA. The cellulose acetate strips were stained for lipoproteins with 0.04% oil red 0, according to Fletcher and Styliou (17) and for proteins with 0.2% Ponceau-S stain in 3% trichloroacetic acid and 3% sulfosalicylic acid in water. Electrophoresis on 4% crosslinked polyacrylamide slabs in the presence of 0.1% SDS was performed according to Weber et al. (18). The gels were stained with Coomassie blue. Lipid determination. Purified holo-CRLP was extracted with 3.75 vol of chloroform:methanol (2:1,
564
JORAM HELLER
v/v). The lower chloroform phase was recovered, evaporated to dryness with nitrogen, and weighed. An aliquot (about 50 mg, dissolved in chloroform) was applied to a glass column (0.6 x 13.5 cm) packed with 1 g of activated silica gel (lOO”C, 18 h, 200 mesh; J. T. Baker, Phillipsburg, N. J.), equilibrated with chloroform. The column was developed according to Ditmer and Wells (19) by eluting the “simple” lipids with 20 ml of chloroform followed by elution of “complex” lipids with 20 ml of methanol. The solvents were evaporated to dryness with nitrogen and the dried lipid fractions were weighed. The “simple” and “complex” lipid fractions were further analyzed by quantitative thin layer chromatography as described previously (20). Determination of retinyl ester and retinal. Retinyl ester and retinol which were extracted from holoCRLP during the preparation of delipidated CRLP were separated by chromatography on a column (0.6 x 13 cm) of aluminum oxide according to Futterman and Andrews (21). Retinol concentration was determined from its absorption and identified as retinol by its specific complex with human serum apo-RPB (8, 16). The retinyl ester fraction obtained by the alumina chromatography was measured by its absorbance. It was then saponified at 55°C for 90 min in 0.35 M ethanolic KOH (21). The liberated retinol was extracted with petroleum ether after the addition of 0.5 ml of water to the mixture, and was measured by absorption spectroscopy, and identified as above by the specific complex it forms with human serum apo-RBP (8, 16). Retinol and retinyl ester concentrations. Concentration of retinol and retinyl ester was determined from the absorption at the peak of the long-wavelength absorption band. The l sz8of all-truns retinol in ethanol was taken as 50,500 (16), while the l az8of all tram retinyl palmitate in ethanol was taken as 49,300 (22). Determination of RNA and DNA. RNA was determined by the orcinol method and DNA by the diphenylamine method according to Dische (23). RESULTS
Purijication
of CRLP
A typical distribution of retinyl compounds in fractions obtained by centrifugation of rat liver homogenate is shown in Table I. The average value of retinyl compounds in the rat liver homogenate was about 1.5 pmol/g (300 g rats). This value should be compared to a value of 0.44 + 0.5 pmollg (mean + SD) of retinyl compounds found in normal human liver (24). When rat liver cytosol was subjected to gel filtration chromatography, all material possessing specific retinol or retinyl ester
TABLE I DISTRIBUTION OF RETINYL COMPOUND IN VARIOUS RAT LIVER FRACTIONS, OBTAINED AS PEELED MATERIAL FROM FOUR RATS WEIGHING ABOUT 300 g EACH
Fraction Whole liver homogenate” Low speed precipitate0 High speed precipitate” CytosoP Total Purified holo-CRLP”
Retinyl compounds (WmoYgliver) 1.58 1.28 0.15 0.15 0.14
Percent 100 81 10 10 101 9
” Determined by extracting with chloroform:methanol (2:l). l zz8used was 49,300. * Determined by direct measurement of the absorption of holo-CRLP at 328 nm after gel filtration step.
absorption and fluorescence appeared in the initial peak (Fig. 1). About 5 to 10% of the retinyl compounds appeared in the tail end of the peak (elution volume 1.2 to 1.4 liters, Fig. 1); only the material appearing in the main peak was pooled (Fig. 1, bar). No other fractions that showed this specific absorption and fluorescence were detected in the rest of the column eluate. The retinyl compounds recovered in this peak accounted for over 95% of the total retinyl compounds in the rat liver cytosol. The protein moiety of purified CRLP accounted for about 0.1% of the total protein in the crude homogenate and for about 0.7% of the total protein in the cytosol. The fraction which contained the retinyl compounds had a characteristic absorption spectrum (Fig. 2) with a peak at about 328 nm and another peak at 258 nm. The material responsible for the absorption peak at 328 nm was identified as predominantly due to retinyl ester with about 4% due to retinol (see below). The fraction with the characteristic 328-nm absorption peak was therefore called cytosol retinyl ester lipoprotein complex or CRLP. Absorption chromatography on hydroxyapatite of CRLP which was purified by gel filtration (Fig. 1) showed two fractions with retinyl compounds. A fraction which was eluted with 250 mM potassium phosphate buffer accounted for about 10% of the
LIVER RETINYL
ESTER LIPOPROTEIN
COMPLEX CHARACTERIZATION
565
FIG. 1. Gel filtration chromatography of rat liver cytosol. Fifty milliliters of rat liver cytosol was applied to an agarose column (Bio-Rad, Bio-Gel A-5m, 100-200 mesh, 5 x 122 cm) which was equilibrated in 50 mM potassium phosphate buffer, pH 7.4, at 4°C. Flow rate was 88 ml/h and fractions of 10ml were collected. Absorbance at 410 nm was due to hemoglobin. Bar represents pooled fraction.
recovered retinyl compound while the fraction which eluted at 500 mM potassium phosphate accounted for about 90% of the recovered retinyl compounds (Fig. 3). The last fraction had the highest ratio of As3dAzG0. Elution with 100 and 175 KIM potassium phosphate removed glycoproteins (with a peak absorption at 258 nm) which remain to be characterized. The yield of purified CRLP after hydroxyapatite chromatography varied between 10 and 50% of the material applied. The variation was due to different behavior of hydroxyapatite preparation. We found that the best yields were obtained with certain hydroxyapatite
0.7 -0.6 -0.5
10.4 03 i 0.2
o.li , , , yq.-& 260
300
340
3.90
WAVELENGTH
420
i
460
(nm)
FIG. 2. Absorption spectra of holo-CRLP. Dashed line, after gel filtration; solid line; after hydroxyapatite (scale on left). Light path 10 mm.
batches obtained from Bio-Rad (lot Nos. 7526 and 17400). When rat liver cytosol was extensively dialyzed against 1 lllM sodium phosphate buffer, pH 7.7, at 4”C, and was then subjected to gel filtration on a 6% agarose column in the same buffer, the material still appeared as a single peak in the void volume of the column. The holo-CRLP in 1 IrIM buffer had the same spectral characterizations as that isolated in 50 mJ4 buffer used above. It was concluded from this experiment that low ionic strength buffers with a slightly alkaline pH do not dissociate the holo-CRLP lipoprotein complex into smaller subunits. Electrophoresis
Cellulose acetate electrophoresis of holoCRLP after purification by gel filtration revealed only one band that stained with a lipid-specific stain (Fig. 4). The holo-CRLP obtained from the hydroxyapatite column by elution with 500mM potassium phosphate buffer also showed only one band staining with lipid-specific stain with a somewhat slower mobility (Fig. 4). A parallel electrophoresis of rat liver cytosol also revealed a single band that stained with lipid-specific stain and which corresponded in mobility to the material obtained after gel filtration. When an identical electrophoretic run was stained with a protein-specific stain the holo-CRLP obtained by gel filtration and by hydroxyapatite chromatography showed a single band that corresponded to the lipid-specific
566
JORAM HELLER
staining band (Fig. 4) while the cytosol showed at least seven different bands (Fig. 4). Absorption Spectra Holo-CRLP had a distinct absorption peak at about 328 nm (Fig. 2). The retinyl chromophore absorption in CRLP was similar to that of retinyl palmitate in organic solvents with the characteristic shoulder in the 310 to 320 nm region. In addition, holo-CRLP had an absorption peak at about 258 nm (Fig. 2), when purified in 50 mM phosphate buffer. Holo-CRLP obtained from the hydroxyapatite column by elution with 500 InM potassium phosphate had lost some of the absorption at 258 nm and consequently the ratio of AS3dAzS8increased (Fig. 2). Holo-CRLP showed a small but clear peak at 410 nm (Fig. 2). The absorption band in this region was responsible for the slight yellow color seen in concentrated solutions of holo-CRLP. The chromophore responsible for this absorption band has not yet been identified. Fluorescence Holo-CRLP showed the typical retinol (retinyl) fluorescence with a peak excitation
I
2 LIPID STAIN
3
FIG. 3. Hydroxyapatite chromatography. HoloCRLP after gel filtration was applied to a 5 x 3.5cm column of hydroxyapatite (Bio-Gel HT, Bio-Rad, Richmond, Calif.) equilibrated in 50 mM potassium phosphate buffer, pH 6.9, at 4°C. After the material was applied, the column was eluted with increasing concentration of potassium phosphate buffer, pH 6.9. Fractions of about 10 ml were collected at a flow rate of about 80 ml/h. The ordinate represents total absorption in a given fraction.
at about 330 nm and a peak emission at about 465 nm. Unlike a retinol-serum RBP complex in which the retinol fluorescence is enhanced about lo-fold over that shown by free retinol in a nonpolar solvent (25), the retinyl ester in holo-CRLP does not show fluorescence enhancement. Since unesterified retinol is only 4% of the total retinyl
4
5
6
PROTEIN STAIN
FIG. 4. Electrophoresis on cellulose acetate. Electrophoresis was performed as described under Experimental Procedures for 20 min at 250 V. The lipid stain was 0.04% of oil red 0 and the protein stain was 0.2% Ponceau-S stain. (1, 4) Holo-CRLP after hydroxyapatite; (2, 5), holo-CRLP after gel filtration; (3, S), cytosol.
LIVER RETINYL
ESTER LIPOPROTEIN
567
COMPLEX CHARACTERIZATION
TABLE II COMPOSITION OF HOLO-CYTOSOL RETINYL ESTER LIPOPROTEIN COMPLEX”
Compound
pm01
Retinyl ester” Retinol (free)” Total lipid Simple lipid@ Complex lipids’ Protein’ Neutral sugar’
0.96 0.04 13.4’ 9.1’ 4.3’
Percentage of total
w 0.505 0.011 10.70 8.35 2.35 4.96 0.58
Percentage of total lipids 4.72 0.10
65.9 78 22 30.54 3.54
a The table was calculated by adjusting the total amount of retinyl ester plus retinol to 1.00 pmol. * Determined after extraction of total lipids and separation on aluminum oxide column (21). Retinyl ester taken as retinyl palmitate (M, 525; e = 49,300). r The “average” molecular weight of the lipids was taken as 800. d Determined after total lipid extraction and separation on silica gel column. c Determined by quantitative amino acid analysis. ‘Determined by phenol-sulfuric acid method with mannose as standard.
compounds in holo-CRLP, it is impossible to say whether there is enhancement of retinol fluorescence. The fluorescence intensity of retinyl ester in holo-CRLP was essentially the same as that shown by an equal amount of retinyl palmitate in a nonpolar solvent. There was no energy transfer between the protein (excitation at 280 nm) and the retinyl chromophore, as judged by the specific retinyl fluorescence emission at 470 nm. Circular
Dichroism
There was no induced optical activity of the retinyl chromophore in holo-CRLP. This would indicate that the retinyl compounds in holo-CRLP are in a different environment from that of serum RBP where induced optical activity is shown by all bound retinyl compounds (8). Retinyl
Ester and Retinol
When the chloroform:methanol extract of holo-CRLP was fractionated into retinyl ester and retinol on an aluminum oxide column, according to Futterman and Andrews (21), about 96% of the retinyl compounds were found as the ester while about 4% were free retinol (Table II). The unesterified retinol, which was obtained
after the aluminum oxide chromatography, was identified by its ability to form a characteristic complex with apo-serum RBP to yield retinol-RBP. The reconstituted retinol-RBP showed all the properties associated with native retinol-RBP, such as induced CD spectra, enhanced retinol fluorescence, and ability to bind to serum prealbumin (8). The retinyl ester fraction was characterized by its spectrum, fluorescence, and cochromatography with authentic retinyl palmitate on thin layer chromatography. The retinyl ester obtained from holo-CRLP was unable to form a complex (to bind) with apo-serum RBP, a characteristic of retinyl palmitate (8). The retinyl ester which was obtained from holo-CRLP and which was purified by column chromatography on aluminum oxide was saponified according to Futterman and Andrews (21). The saponified material was extracted with petroleum ether and rechromatographed on aluminum oxide as above. In the fraction that previously contained all the retinyl ester, there were no retinyl compounds. All the material was now present in the unesterified retinol fraction of the aluminum oxide column eluate. This free retinol was further identified as such by combining it with human
568
JORAM HELLER
apo-serum RBP. The chromophore did complex with apo-RBP to give the characteristic CD spectrum of retinol-serum RBP complex (8, 16). The distribution of retinyl compounds in holo-CRLP into approximately 96% retinyl esters and 4% retinol was found in more than 10 different preparations. The amount of unesterified retinol varied between 3.5 and 4.6% of the total retinyl compounds in different preparations of holo-CRLP. It should be emphasized that the measurements were made on fresh preparations of holo-CRLP, generally within 48 h of sacrificing the rats. Storage of holo-CRLP at 4°C results in appreciable change in the ratio of retinol to retinyl ester as will be discussed in detail in a forthcoming paper. Retinyl ester and retinol account for about 3% of the total weight of holo-CRLP or about 4.8% of the total weight of lipids in holoCRLP. Lipids
Lipids (including retinyl compounds) accounted for about 66% of the total weight of holo-CRLP (Table II). Retinyl compounds accounted for about 4.8% by weight of total lipids (Table II). About 78% by weight of the total lipids were “simple” lipids (retinyl compounds, free fatty acids, triglycerides, simple sterols, and sterol esters) with the balance due to “complex” lipids. The “simple” lipid fraction from CRLP contained about 10% each of cholesterol and cholesterol ester, about 50% of free fatty acids, about 25% of triglycerides, and about 5% of retinyl compounds. The balance of the lipids (some 22% by weight) was due to “complex” or phospholipids. About 60% of the phospholipids was phosphatidyl ethanolamine and some 40% was due to phosphatidyl serine. Phosphatidyl choline and sphingomyelin were present in trace amounts (about 1 to 2% each). Protein
About 30.5% by weight of the holo-CRLP molecule was due to protein (Table II). Acrylamide SDS-gel electrophoresis of delipidated CRLP protein revealed at least 15 polypeptide chains, most of which were
FIG. 5. SDS-polyacrylamide disc gel electrophoresis of delipidated CRLP. The gel on the right represents protein standards: (from top) muscle phosphorylase a, bovine serum albumin, ovalbumin, and myoglobin. Electrophoresis of chloroform:methanolextracted CRLP was performed on 4% acrylamide gels according to Weber et al. (18) and stained with Coomassie blue.
present in relatively low amounts as judged by staining intensity (Fig. 5). The polypeptide chains ranged in size from about 2.1 x lo5 to about 2 x lo4 M, with the three major polypeptides having apparent molecular weights of about 1.1 x 105, 8.2 x 104, and 5.6 x 104. The amino acid composition of delipidated CRLP is given in Table III. No tryptophan was found after hydrolysis with ptoluenesulfonic acid (13), an observation which explains the lack of a distinct absorption band at 280 nm in holo-CRLP (Fig. 2). Carbohydrate
Purified holo-CRLP contained carbohydrate (Tables II and III). The carbohydrate
LIVER RETINYL
ESTER LIPOPROTEIN
TABLE III AMINO ACID COMPOSITION OF CRLP
Number of amino acid residues per 1000 residues Lysine Histidine Arginine Aspartic acid Threonine” Serine” Glutamic acid Proline Glycine Alanine Half-cystine Valine” Methionine Isoleucine” Leucine* Tyrosine Phenylalanine Tryptophan Glucosamine’
72.9 26.6 58.6 89.0 61.2 66.6 115.0 49.7 69.4 72.9 10.9 65.8 17.8 46.2 104.9 30.2 42.7 None 2.8
” Extrapolated to zero-time hydrolysis. * Extrapolated to infinite hydrolysis time. c Determined after 6 and 12 h hydrolysis in 4 N HCl at 100°C.
was linked to the protein since SDS-treated, delipidated CRLP still contained the carbohydrate after extensive dialysis against SDS-containing buffers. Glucosamine was the only amino sugar found (Table III). No sialic acid was found. DNA and RNA
No DNA or RNA was found in purified holo-CRLP in tests in which at least 1.0 pm01 of retinyl compounds was used (about 0.5 mg of protein). Stability
COMPLEX CHARACTERIZATION
569
absorption spectra with a number of new absorption peaks in the far-uv region and loss of absorption at 328 nm. The halflifetime of free retinol in aqueous buffer at neutral pH is of the order of 20 to 30 min. Thus the retinyl compounds in holo-CRLP at low ionic strength and neutral pH are protected from the usual degradation in aqueous buffers. It should be noted that when the term “stability” is used in the context above, no distinction is made between retinyl ester and unesterified retinol. Since both ester and retinol have about the same molar absorptivity and show similar shaped spectra, it was not possible to use the spectroscopic properties to determine the relative proportion of retinyl ester and unesterified retinol in holo-CRLP. As will be described in the accompanying paper, storage of holo-CRLP at pH 7.4 in the cold leads to hydrolysis of the ester. Holo-CRLP is unstable at pH 6.4 with a half-lifetime of retinyl compounds of about 48 h. When holo-CRLP was prepared by gel filtration at pH 7.4, in 50 mM sodium phosphate buffer which contained, in addition, 1 M NaCl, the retinyl compounds had a halflifetime of about 1 week at 4°C as judged by the loss of absorption at 328 nm. The instability of the retinyl compounds could be arrested and prevented by dialyzing out the NaCl. Thus holo-CRLP which was purified by gel filtration chromatography in the presence of 1 M NaCl, and in which NaCl was then removed by dialysis against 50 mM sodium phosphate buffer, pH 7.4, was as stable as holo-CRLP which was purified in low salt buffers. Thus, in order to exert its deleterious effect on stability, the 1 M NaCl had to be present throughout. Removal of the salt restored the stability. DISCUSSION
The present study shows that about 10% Solutions of purified holo-CRLP in 50 mM of the total retinyl compounds in rat liver sodium phosphate buffer, pH 7.4, were can be obtained in the cytosol fraction. The stable for at least 3 months at 4°C. The retinyl compounds in the cytosol are part of stability of the holo-CRLP complex was a large soluble lipoprotein complex. Holojudged by the stability of its retinyl com- CRLP is a complex molecular aggregate pounds. Both free retinol and retinyl ester reminiscent of some of the serum lipoproare rapidly oxidized and degraded in aqueous teins. And similar to the serum lipoprotein, media. The oxidized Inroducts have different~. holo-CRLP, despite its size and composi-
570
JORAM
tional complexity, behaves as a unique entity. It can be chromatographed on hydroxyapatite (Fig. 3), and it gives a single band on cellulose acetate electrophoresis (Fig. 4). The apparent molecular weight of the lipoprotein complex is on the order of about 5 x lo6 (or above) since it appears in the void volume of a 6% agarose column. Because of lack of reliable molecular weight standard markers in this range with the added technical difficulties inherent in the molecular weight determination of lipoproteins, no attempt was made in this study to measure the molecular weight of CRLP with greater precision. Holo-CRLP is a complex molecular aggregate. The molecular aggregate contains several polypeptide chains of various sizes. A carbohydrate moiety is covalently linked to the polypeptide chain(s). About 66% (by weight) of the molecule is accounted for by lipids. About 80% of the lipids are “simple” lipids, predominantly triglycerides and free fatty acids. The balance of the lipids (about 20%) is due to phospholipids. About 3.5 to 4% of the total is due to retinyl compounds, of which 96% are retinyl ester compounds and about 4% is unesterified retinal. If one assumes one molecule of unesterified retinol per one holo-CRLP molecule, the minimal molecular weight of holoCRLP would then be about 5.7 x 105.Since the molecular weight is probably lo- to 20fold larger than the minimal molecular weight, there would be some 10 to 20 molecules of unesterified retinol and some 250 to 500 molecules of retinyl ester per holo-CRLP complex. Although holo-CRLP mimics a serum lipoprotein, it is not detected when serum alone is subjected to gel filtration and is only observed in preparations of liver homogenate cytosol. Ong and Chytil (26) have recently described the isolation of a soluble protein of rat liver that binds retinol. The protein has a molecular weight of 14,000 (thereby distinguishing it from serum RBP), and it binds one molecule of retinol. This low molecular weight retinol-binding protein accounts for some 0.02 to 0.03% of the total soluble protein. In our study we have not found the low molecular weight protein described by Ong and Chytil. Two reasons
HELLER
can be given for this result. First, we have not added retinol to our extract prior to purification as was done by Ong and Chytil. Second, we have not found any retinylspecific fluorescence in our gel filtration studies in the elution range of low molecular weight proteins (Fig. 1). It is noteworthy that in various preparations of CRLP, prepared from rats of different size, age, sex, the proportion of retinyl ester and free retinol was almost constant (Table II). The variations which were found are probably within the overall experimental accuracy of methodology. The molar ratio of unesterified retinol to retinyl ester was between 1:20 and 1:25. Glover (2) reported recently that in sheep, the ratio of retinol to retinyl ester was 1:17. Only 5.3% of the total unesteri)ied retinol was found as a complex with serum RBP in sheep liver (2). It is thus clear that not only is CRLP important in the storage of retinyl esters but the majority of unesterified retinol is also bound to CRLP. Since the retinyl compounds in CRLP represent at least 10% of the retinyl compounds of the liver, and since the ratio of unesterified retinol to retinyl ester is the same for holo-CRLP as for the whole liver homogenate, the holo-CRLP molecule accounts also for at least 10% of the unesterified retinol found in the liver. The function of holo-CRLP in liver vitamin A metabolism remains to be elucidated. It seems plausible that CRLP is important in storage and protection of the vitamin. We have recently found that holo-CRLP possessesa retinyl esterase activity toward its own retinyl ester complement. A detailed report of this activity is given in the following paper. ACKNOWLEDGMENTS I thank Drs. Joseph Horwitz and Chi-Ching Chen for many helpful discussions and suggestions. L. Ding, J. Takasugi, and A. Duberg have provided excellent technical help. I thank Dr. J. Mead for allowing me to use some of the facilities of his laboratory. REFERENCES 1. UNDERWOOD, B. A. (19’74) World Rev. Nub. Diet. 19, 123-1’72. 2. GLOVER, J., JAY, C., AND WHITE, G. H. (1974) Vitam. Homn. 32, 215-235.
LIVER
RETINYL
ESTER
LIPOPROTEIN
3. OLSON, J. A. (1968) Vitam. Harm. 26, l-63. 4. POPPER, H., AND GREENBERG, R. (1941) Arch. Pathol. 32, 11-32. 5. LINDER, M. C., ANDERSON, G. H., AND ASCARELLI, I. (1971)J. Biol. Chem. 246,5538-5540. 6. KANAI, M., RAZ, A., AND GOODMAN, DEW. S. (1968) J. Clin. Invest. 47, 2025-2044. 7. GLOVER, J. (1973) Vitam. HOWL. 31, l-42. 8. HORWITZ, J., AND HELLER, J. (1974) J. Biol. Chem. 249, 4712-4719. 9. MUTO, Y., SMITH, J. E., MILCH, P. O., AND GOODMAN, DEW. S. (1972) J. Biol. Chem. 247, 2542-2550. 10. KRISHNAMURTHY, S., MAHADEVAN, S., AND GANGULY, J. (1958)J. Biol. Chem. 233,32-36. 11. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. LIU, T. Y., AND CHANG, Y. H. (1971) J. Biol. Chem. 246, 2842-2848. 14. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 15. WARREN, L. (1959)J. Biol. Chem. 234,1971-1975.
COMPLEX
CHARACTERIZATION
571
16. HELLER, J., AND HORWITZ, J. (1973) J. Biol. Chem. 248, 6308-6316. 17. FLETCHER, M. J., AND STYLIOU, M. H. (1970) Clin. Chem. 16, 362-365. 18. WEBER, K., PRINGLE, J. R., AND OSBORN, M. (1972) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 26, Pt. C, pp. 3-27, Academic Press, New York. 19. DITTMER, J. C., AND WELLS, M. A. (1969) in Methods in Enzymology (San Pietro, A., ed.), Vol. 24, pp. 511-512, Academic Press, New York. 20. HELLER, J. (1976)J. Biol. Chem. 251,2952-2957. 21. FUTTERMAN, S., AND ANDREWS, J. S. (1964) J. Biol. Chem. 239, 81-84. 22. HARRIS, R. S. (1967) in The Vitamins (Sebrell, W. H., and Harris, R. S., eds.), p. 11, Academic Press, New York. 23. DISCHE, Z. (1955) Nucleic Acids 1, 285-305. 24. UNDERWOOD, B.A., SIEGEL, H., WEISELL, R. C., AND DOLINSKI, M. (1970) Amer. J. Clin. Nutr. 23, 1037-1042. 25. FUTTERMAN, S., AND HELLER, J. (1972) J. Biol. Chem. 247, 5168-5172. 26. ONG, D. E., AND CHYTIL, F. (1978)5. Biol. Chem. 253, 828-832.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 198, No. 2, December, pp. 562-571, 1979
Characterization
of Rat Liver Cytosol
Retinyl
Ester Lipoprotein
Complex’
JORAM HELLER Jules Stein
Eye Institute,
UCLA School of Medicine,
Los Angeles, California
90024
Received February 5, 1979; revised July 30, 1979 A soluble high molecular weight lipoprotein complex containing retinyl esters and unesterified retinol was isolated from rat liver cytosol. This material accounts for about 10% of the total liver retinyl compounds, and its protein moiety accounts for about 0.1% of the protein of the liver homogenate and about 0.7% of the cytosol protein. The lipoprotein was purified by gel filtration and hydroxyapatite chromatography. The lipoprotein complex gave a single band by electrophoresis on cellulose acetate as judged by both lipid- and proteinspecific stains. The lipoprotein complex did not dissociate into smaller subunits in low ionic strength buffer (1 mM sodium phosphate, pH 7.7). The retinyl ester lipoprotein complex has an absorption spectrum with peaks at 328 nm (retinyl chromophore) and 258 nm. Retinyl compounds in the carrier lipoprotein complex do not show an increase of the quantum yield of fluorescence and do not show energy transfer when excited at either 258 or 280 nm. There is no induced optical activity of the retinyl chromophore absorption band. The lipoprotein complex contains about 3% (by weight) of retinyl compounds, 96% of which are retinyl esters and 4% of which are unesterified retinol. The lipoprotein complex consists of about 66% (by weight) lipids, about 30% protein, and some 4% carbohydrate. There are at least 15 polypeptide chains ranging in size from about 2 x lo4 to about 2.1 x lo5 M,. Retinyl compounds in the lipoprotein complex are stable for at least 3 months in 0.05 M phosphate buffer, pH 7.4, at 4°C. Stability was judged from the total amount of retinyl esters plus unesterified retinol. Retinyl compounds of the lipoprotein complex were unstable below pH 6.4 or in the presence of 1 M NaCl.
From 80 to 90% of stored retinol (vitamin A) in the mammalian body is found in the liver, and from 80 to 95% of this is found as retinyl esters (l-3). Although it was formerly thought that most of the retinyl compounds in the liver were found in the Kupffer cells (4), it was recently shown by Linder et al. (5), utilizing the technique of direct isolation of Kupffer cells, that over 96% of total liver retinol and retinyl ester is present in the hepatocytes with less than 4% of total retinyl compounds found in the Kupffer cells. Retinol stored in the liver is delivered to the target tissue via the blood. All the retinol in the blood is complexed with a specific protein, the serum retinol-binding protein
(RBP)” (6, 7). The retinol in serum RBP is found exclusively as unesterified retinol. In fact, serum RBP does not bind retinyl palmitate (8), the predominant retinyl ester in the liver. Serum RBP is synthesized in the liver and apparently is released from the liver into the circulation only as a complex with retinol (9). Only about 0.3% of total liver retinyl compounds are found as retinyl bound to intracellular RBP (2). Krishnamurthy et al. (10) reported in 1958 that retinyl esters and retinol in the soluble liver homogenate seemed to behave as if they were complexed with a protein. This was suggested by the precipitation of the retinyl ester and retinol by ammonium sulfate and other protein precipitating agents, and by
* This paper was supported by Grants EY 00704, EY 00331, and EY 00702 from the National Institutes of Health, USPHS.
2 Abbreviations used: RBP, serum retinol-binding protein; CRLP, cytosol retinyl ester lipoprotein complex; SDS, sodium dodecyl sulfate.
0003.9861/79/140562-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
562
LIVER RETINYL
ESTER LIPOPROTEIN
the inability to extract the retinyl chromophores with diethyl ether unless a protein denaturing agent was also used. In the intervening years, no further reports have elaborated on these interesting observations by Krishnamurthy et al. (10). As part of a continuing interest in intracellular retinol-binding proteins we have further investigated the liver cytosol retinyl ester and retinol-binding proteins. This paper describes the isolation and characterization of a soluble, high molecular weight lipoprotein complex from rat liver. This complex accounts for at least 10% of the total liver retinyl ester and unesterified retinol. EXPERIMENTAL
PROCEDURES
Preparation of rut liver cytosol. White laboratory rats (about 300 g each) were kept on a normal ad libitum diet (Purina laboratory chow for rats, vitamin A supplemented by the manufacturer). The animals were starved for 18 h before being sacrificed and then anesthetized with diethyl ether. The peritoneal cavity of each animal was opened and the liver was removed. Blood was washed away and the liver was chopped into 2- to 3-mm pieces with scissors. Two milliliters of cold sucrose buffer (0.25 M sucrose, 10 mM N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 50 mM KCl, 1 mM MgCl,, 2 mM ethylenediaminetetraacetic acid, and 0.1 mM dithiothreitol), adjusted to pH 7.4 with NaOH (density of buffer at 4°C was 1.036 g/ml), was added per gram of liver and the liver was then homogenized at top speed for 1 min at 4°C in a glass and Teflon pestle homogenizer with a 0.125 mm clearance. The pestle was driven by a Talboys Engineering Model 103 motor. The homogenate was filtered through two layers of cheesecloth. The homogenate was centrifuged for 1 h at 29,000g at 4°C (Sorvall SS-34 rotor). The clear middle layer between a thin white lipid float and a precipitate was carefully removed with a syringe and a No. 18 cannula. This clear supernatant was then centrifuged in a fixed angle rotor for 1 h at 105,OOOgat 4°C (Beckman type 65 rotor, 39,000 rpm). At the end of this centrifugation, there was a 2- to 3-mm-thick layer of white opaque material floating on top of a clear middle layer (cytosol). Carefully avoiding the float, the infranatant was removed with a syringe and cannula as before. The floats and precipitates from both centrifugations were discarded. Gel j2tration chromatography of cytosol. Fifty milliliters of rat liver cytosol was applied to an agarose column (5 x 122 cm; Bio-Gel A-5m, 100-200 mesh, Bio-Rad, Richmond, Calif.; nominal agarose content, 6%). The column was equilibrated with 50 mM potas-
COMPLEX CHARACTERIZATION
563
sium phosphate buffer, pH 7.4, containing 0.1 mM dithiothreitol and 0.1 mM sodium azide. Flow rate (pump) was 88 ml/h (4.5 ml/cmz) and the column was operated at 4°C. Fractions of 10 ml were collected. The effluent absorption at 280 nm was monitored with an LKB Uvicord II. Absorption and spectra of fractions were monitored with a Beckman doublebeam ratio recording spectrophotometer (Beckman ACTA MVI). Fluorescence, protein, and sugar were also determined as described below. Hydroxyapatite chromatography. The retinyl compound fraction obtained by gel filtration (Fig. 1, first peak) was further purified on a column of hydroxyapatite (5 x 3.5 cm; Bio-Gel HT, Bio-Rad, Richmond, Calif.). The column was equilibrated with 50 mM potassium phosphate buffer, pH 6.9, containing 0.1 mM dithiothreitol and 0.1 mM sodium azide. After applying the material, the column was eluted with increasing concentrations of potassium phosphate buffer, pH 6.9. The CRLP was eluted with 500 mM potassium phosphate, pH 6.9. Flow rate was about 50 ml/h and fractions of about 10 ml were collected. Protein determination. Protein was determined either by the dye-binding assay (11) or the Lowry assay (12) with crystalline bovine serum albumin (Sigma) serving as standard. Protein was quantitatively determined by amino acid analysis after hydrolysis with 3 N p-toluenesulfonic acid (13) or 6 N HCl, using the Beckman amino acid analyzer, Model 120 C. Carbohydrate determination. Total hexose sugar was determined by the phenol-sulfuric acid method (14) with mannose as standard. Sialic acid was determined according to the method of Warren (15). Spectroscopic measurements. Absorption spectra were measured with a double monochromotor, doublebeam ratio recording spectrophotometer (Beckman ACTA MVI). Circular dichroism and fluorescence were measured as previously described (16). Electrophoresis. Electrophoresis on cellulose acetate strips was performed with the Beckman Microzone system according to the manufacturer’s instructions. A barbital buffer, pH 8.6 and ionic strength 0.075, was used. All of the samples were dialyzed and concentrated in this buffer before electrophoresis (negative pressure dialysis-concentrator, Bio-Molecular Dynamics, Oregon). Electrophoresis was performed at 22°C for 20 min with a constant voltage of 250 V and a beginning current of 3 mA. The cellulose acetate strips were stained for lipoproteins with 0.04% oil red 0, according to Fletcher and Styliou (17) and for proteins with 0.2% Ponceau-S stain in 3% trichloroacetic acid and 3% sulfosalicylic acid in water. Electrophoresis on 4% crosslinked polyacrylamide slabs in the presence of 0.1% SDS was performed according to Weber et al. (18). The gels were stained with Coomassie blue. Lipid determination. Purified holo-CRLP was extracted with 3.75 vol of chloroform:methanol (2:1,
564
JORAM HELLER
v/v). The lower chloroform phase was recovered, evaporated to dryness with nitrogen, and weighed. An aliquot (about 50 mg, dissolved in chloroform) was applied to a glass column (0.6 x 13.5 cm) packed with 1 g of activated silica gel (lOO”C, 18 h, 200 mesh; J. T. Baker, Phillipsburg, N. J.), equilibrated with chloroform. The column was developed according to Ditmer and Wells (19) by eluting the “simple” lipids with 20 ml of chloroform followed by elution of “complex” lipids with 20 ml of methanol. The solvents were evaporated to dryness with nitrogen and the dried lipid fractions were weighed. The “simple” and “complex” lipid fractions were further analyzed by quantitative thin layer chromatography as described previously (20). Determination of retinyl ester and retinal. Retinyl ester and retinol which were extracted from holoCRLP during the preparation of delipidated CRLP were separated by chromatography on a column (0.6 x 13 cm) of aluminum oxide according to Futterman and Andrews (21). Retinol concentration was determined from its absorption and identified as retinol by its specific complex with human serum apo-RPB (8, 16). The retinyl ester fraction obtained by the alumina chromatography was measured by its absorbance. It was then saponified at 55°C for 90 min in 0.35 M ethanolic KOH (21). The liberated retinol was extracted with petroleum ether after the addition of 0.5 ml of water to the mixture, and was measured by absorption spectroscopy, and identified as above by the specific complex it forms with human serum apo-RBP (8, 16). Retinol and retinyl ester concentrations. Concentration of retinol and retinyl ester was determined from the absorption at the peak of the long-wavelength absorption band. The l sz8of all-truns retinol in ethanol was taken as 50,500 (16), while the l az8of all tram retinyl palmitate in ethanol was taken as 49,300 (22). Determination of RNA and DNA. RNA was determined by the orcinol method and DNA by the diphenylamine method according to Dische (23). RESULTS
Purijication
of CRLP
A typical distribution of retinyl compounds in fractions obtained by centrifugation of rat liver homogenate is shown in Table I. The average value of retinyl compounds in the rat liver homogenate was about 1.5 pmol/g (300 g rats). This value should be compared to a value of 0.44 + 0.5 pmollg (mean + SD) of retinyl compounds found in normal human liver (24). When rat liver cytosol was subjected to gel filtration chromatography, all material possessing specific retinol or retinyl ester
TABLE I DISTRIBUTION OF RETINYL COMPOUND IN VARIOUS RAT LIVER FRACTIONS, OBTAINED AS PEELED MATERIAL FROM FOUR RATS WEIGHING ABOUT 300 g EACH
Fraction Whole liver homogenate” Low speed precipitate0 High speed precipitate” CytosoP Total Purified holo-CRLP”
Retinyl compounds (WmoYgliver) 1.58 1.28 0.15 0.15 0.14
Percent 100 81 10 10 101 9
” Determined by extracting with chloroform:methanol (2:l). l zz8used was 49,300. * Determined by direct measurement of the absorption of holo-CRLP at 328 nm after gel filtration step.
absorption and fluorescence appeared in the initial peak (Fig. 1). About 5 to 10% of the retinyl compounds appeared in the tail end of the peak (elution volume 1.2 to 1.4 liters, Fig. 1); only the material appearing in the main peak was pooled (Fig. 1, bar). No other fractions that showed this specific absorption and fluorescence were detected in the rest of the column eluate. The retinyl compounds recovered in this peak accounted for over 95% of the total retinyl compounds in the rat liver cytosol. The protein moiety of purified CRLP accounted for about 0.1% of the total protein in the crude homogenate and for about 0.7% of the total protein in the cytosol. The fraction which contained the retinyl compounds had a characteristic absorption spectrum (Fig. 2) with a peak at about 328 nm and another peak at 258 nm. The material responsible for the absorption peak at 328 nm was identified as predominantly due to retinyl ester with about 4% due to retinol (see below). The fraction with the characteristic 328-nm absorption peak was therefore called cytosol retinyl ester lipoprotein complex or CRLP. Absorption chromatography on hydroxyapatite of CRLP which was purified by gel filtration (Fig. 1) showed two fractions with retinyl compounds. A fraction which was eluted with 250 mM potassium phosphate buffer accounted for about 10% of the
LIVER RETINYL
ESTER LIPOPROTEIN
COMPLEX CHARACTERIZATION
565
FIG. 1. Gel filtration chromatography of rat liver cytosol. Fifty milliliters of rat liver cytosol was applied to an agarose column (Bio-Rad, Bio-Gel A-5m, 100-200 mesh, 5 x 122 cm) which was equilibrated in 50 mM potassium phosphate buffer, pH 7.4, at 4°C. Flow rate was 88 ml/h and fractions of 10ml were collected. Absorbance at 410 nm was due to hemoglobin. Bar represents pooled fraction.
recovered retinyl compound while the fraction which eluted at 500 mM potassium phosphate accounted for about 90% of the recovered retinyl compounds (Fig. 3). The last fraction had the highest ratio of As3dAzG0. Elution with 100 and 175 KIM potassium phosphate removed glycoproteins (with a peak absorption at 258 nm) which remain to be characterized. The yield of purified CRLP after hydroxyapatite chromatography varied between 10 and 50% of the material applied. The variation was due to different behavior of hydroxyapatite preparation. We found that the best yields were obtained with certain hydroxyapatite
0.7 -0.6 -0.5
10.4 03 i 0.2
o.li , , , yq.-& 260
300
340
3.90
WAVELENGTH
420
i
460
(nm)
FIG. 2. Absorption spectra of holo-CRLP. Dashed line, after gel filtration; solid line; after hydroxyapatite (scale on left). Light path 10 mm.
batches obtained from Bio-Rad (lot Nos. 7526 and 17400). When rat liver cytosol was extensively dialyzed against 1 lllM sodium phosphate buffer, pH 7.7, at 4”C, and was then subjected to gel filtration on a 6% agarose column in the same buffer, the material still appeared as a single peak in the void volume of the column. The holo-CRLP in 1 IrIM buffer had the same spectral characterizations as that isolated in 50 mJ4 buffer used above. It was concluded from this experiment that low ionic strength buffers with a slightly alkaline pH do not dissociate the holo-CRLP lipoprotein complex into smaller subunits. Electrophoresis
Cellulose acetate electrophoresis of holoCRLP after purification by gel filtration revealed only one band that stained with a lipid-specific stain (Fig. 4). The holo-CRLP obtained from the hydroxyapatite column by elution with 500mM potassium phosphate buffer also showed only one band staining with lipid-specific stain with a somewhat slower mobility (Fig. 4). A parallel electrophoresis of rat liver cytosol also revealed a single band that stained with lipid-specific stain and which corresponded in mobility to the material obtained after gel filtration. When an identical electrophoretic run was stained with a protein-specific stain the holo-CRLP obtained by gel filtration and by hydroxyapatite chromatography showed a single band that corresponded to the lipid-specific
566
JORAM HELLER
staining band (Fig. 4) while the cytosol showed at least seven different bands (Fig. 4). Absorption Spectra Holo-CRLP had a distinct absorption peak at about 328 nm (Fig. 2). The retinyl chromophore absorption in CRLP was similar to that of retinyl palmitate in organic solvents with the characteristic shoulder in the 310 to 320 nm region. In addition, holo-CRLP had an absorption peak at about 258 nm (Fig. 2), when purified in 50 mM phosphate buffer. Holo-CRLP obtained from the hydroxyapatite column by elution with 500 InM potassium phosphate had lost some of the absorption at 258 nm and consequently the ratio of AS3dAzS8increased (Fig. 2). Holo-CRLP showed a small but clear peak at 410 nm (Fig. 2). The absorption band in this region was responsible for the slight yellow color seen in concentrated solutions of holo-CRLP. The chromophore responsible for this absorption band has not yet been identified. Fluorescence Holo-CRLP showed the typical retinol (retinyl) fluorescence with a peak excitation
I
2 LIPID STAIN
3
FIG. 3. Hydroxyapatite chromatography. HoloCRLP after gel filtration was applied to a 5 x 3.5cm column of hydroxyapatite (Bio-Gel HT, Bio-Rad, Richmond, Calif.) equilibrated in 50 mM potassium phosphate buffer, pH 6.9, at 4°C. After the material was applied, the column was eluted with increasing concentration of potassium phosphate buffer, pH 6.9. Fractions of about 10 ml were collected at a flow rate of about 80 ml/h. The ordinate represents total absorption in a given fraction.
at about 330 nm and a peak emission at about 465 nm. Unlike a retinol-serum RBP complex in which the retinol fluorescence is enhanced about lo-fold over that shown by free retinol in a nonpolar solvent (25), the retinyl ester in holo-CRLP does not show fluorescence enhancement. Since unesterified retinol is only 4% of the total retinyl
4
5
6
PROTEIN STAIN
FIG. 4. Electrophoresis on cellulose acetate. Electrophoresis was performed as described under Experimental Procedures for 20 min at 250 V. The lipid stain was 0.04% of oil red 0 and the protein stain was 0.2% Ponceau-S stain. (1, 4) Holo-CRLP after hydroxyapatite; (2, 5), holo-CRLP after gel filtration; (3, S), cytosol.
LIVER RETINYL
ESTER LIPOPROTEIN
567
COMPLEX CHARACTERIZATION
TABLE II COMPOSITION OF HOLO-CYTOSOL RETINYL ESTER LIPOPROTEIN COMPLEX”
Compound
pm01
Retinyl ester” Retinol (free)” Total lipid Simple lipid@ Complex lipids’ Protein’ Neutral sugar’
0.96 0.04 13.4’ 9.1’ 4.3’
Percentage of total
w 0.505 0.011 10.70 8.35 2.35 4.96 0.58
Percentage of total lipids 4.72 0.10
65.9 78 22 30.54 3.54
a The table was calculated by adjusting the total amount of retinyl ester plus retinol to 1.00 pmol. * Determined after extraction of total lipids and separation on aluminum oxide column (21). Retinyl ester taken as retinyl palmitate (M, 525; e = 49,300). r The “average” molecular weight of the lipids was taken as 800. d Determined after total lipid extraction and separation on silica gel column. c Determined by quantitative amino acid analysis. ‘Determined by phenol-sulfuric acid method with mannose as standard.
compounds in holo-CRLP, it is impossible to say whether there is enhancement of retinol fluorescence. The fluorescence intensity of retinyl ester in holo-CRLP was essentially the same as that shown by an equal amount of retinyl palmitate in a nonpolar solvent. There was no energy transfer between the protein (excitation at 280 nm) and the retinyl chromophore, as judged by the specific retinyl fluorescence emission at 470 nm. Circular
Dichroism
There was no induced optical activity of the retinyl chromophore in holo-CRLP. This would indicate that the retinyl compounds in holo-CRLP are in a different environment from that of serum RBP where induced optical activity is shown by all bound retinyl compounds (8). Retinyl
Ester and Retinol
When the chloroform:methanol extract of holo-CRLP was fractionated into retinyl ester and retinol on an aluminum oxide column, according to Futterman and Andrews (21), about 96% of the retinyl compounds were found as the ester while about 4% were free retinol (Table II). The unesterified retinol, which was obtained
after the aluminum oxide chromatography, was identified by its ability to form a characteristic complex with apo-serum RBP to yield retinol-RBP. The reconstituted retinol-RBP showed all the properties associated with native retinol-RBP, such as induced CD spectra, enhanced retinol fluorescence, and ability to bind to serum prealbumin (8). The retinyl ester fraction was characterized by its spectrum, fluorescence, and cochromatography with authentic retinyl palmitate on thin layer chromatography. The retinyl ester obtained from holo-CRLP was unable to form a complex (to bind) with apo-serum RBP, a characteristic of retinyl palmitate (8). The retinyl ester which was obtained from holo-CRLP and which was purified by column chromatography on aluminum oxide was saponified according to Futterman and Andrews (21). The saponified material was extracted with petroleum ether and rechromatographed on aluminum oxide as above. In the fraction that previously contained all the retinyl ester, there were no retinyl compounds. All the material was now present in the unesterified retinol fraction of the aluminum oxide column eluate. This free retinol was further identified as such by combining it with human
568
JORAM HELLER
apo-serum RBP. The chromophore did complex with apo-RBP to give the characteristic CD spectrum of retinol-serum RBP complex (8, 16). The distribution of retinyl compounds in holo-CRLP into approximately 96% retinyl esters and 4% retinol was found in more than 10 different preparations. The amount of unesterified retinol varied between 3.5 and 4.6% of the total retinyl compounds in different preparations of holo-CRLP. It should be emphasized that the measurements were made on fresh preparations of holo-CRLP, generally within 48 h of sacrificing the rats. Storage of holo-CRLP at 4°C results in appreciable change in the ratio of retinol to retinyl ester as will be discussed in detail in a forthcoming paper. Retinyl ester and retinol account for about 3% of the total weight of holo-CRLP or about 4.8% of the total weight of lipids in holoCRLP. Lipids
Lipids (including retinyl compounds) accounted for about 66% of the total weight of holo-CRLP (Table II). Retinyl compounds accounted for about 4.8% by weight of total lipids (Table II). About 78% by weight of the total lipids were “simple” lipids (retinyl compounds, free fatty acids, triglycerides, simple sterols, and sterol esters) with the balance due to “complex” lipids. The “simple” lipid fraction from CRLP contained about 10% each of cholesterol and cholesterol ester, about 50% of free fatty acids, about 25% of triglycerides, and about 5% of retinyl compounds. The balance of the lipids (some 22% by weight) was due to “complex” or phospholipids. About 60% of the phospholipids was phosphatidyl ethanolamine and some 40% was due to phosphatidyl serine. Phosphatidyl choline and sphingomyelin were present in trace amounts (about 1 to 2% each). Protein
About 30.5% by weight of the holo-CRLP molecule was due to protein (Table II). Acrylamide SDS-gel electrophoresis of delipidated CRLP protein revealed at least 15 polypeptide chains, most of which were
FIG. 5. SDS-polyacrylamide disc gel electrophoresis of delipidated CRLP. The gel on the right represents protein standards: (from top) muscle phosphorylase a, bovine serum albumin, ovalbumin, and myoglobin. Electrophoresis of chloroform:methanolextracted CRLP was performed on 4% acrylamide gels according to Weber et al. (18) and stained with Coomassie blue.
present in relatively low amounts as judged by staining intensity (Fig. 5). The polypeptide chains ranged in size from about 2.1 x lo5 to about 2 x lo4 M, with the three major polypeptides having apparent molecular weights of about 1.1 x 105, 8.2 x 104, and 5.6 x 104. The amino acid composition of delipidated CRLP is given in Table III. No tryptophan was found after hydrolysis with ptoluenesulfonic acid (13), an observation which explains the lack of a distinct absorption band at 280 nm in holo-CRLP (Fig. 2). Carbohydrate
Purified holo-CRLP contained carbohydrate (Tables II and III). The carbohydrate
LIVER RETINYL
ESTER LIPOPROTEIN
TABLE III AMINO ACID COMPOSITION OF CRLP
Number of amino acid residues per 1000 residues Lysine Histidine Arginine Aspartic acid Threonine” Serine” Glutamic acid Proline Glycine Alanine Half-cystine Valine” Methionine Isoleucine” Leucine* Tyrosine Phenylalanine Tryptophan Glucosamine’
72.9 26.6 58.6 89.0 61.2 66.6 115.0 49.7 69.4 72.9 10.9 65.8 17.8 46.2 104.9 30.2 42.7 None 2.8
” Extrapolated to zero-time hydrolysis. * Extrapolated to infinite hydrolysis time. c Determined after 6 and 12 h hydrolysis in 4 N HCl at 100°C.
was linked to the protein since SDS-treated, delipidated CRLP still contained the carbohydrate after extensive dialysis against SDS-containing buffers. Glucosamine was the only amino sugar found (Table III). No sialic acid was found. DNA and RNA
No DNA or RNA was found in purified holo-CRLP in tests in which at least 1.0 pm01 of retinyl compounds was used (about 0.5 mg of protein). Stability
COMPLEX CHARACTERIZATION
569
absorption spectra with a number of new absorption peaks in the far-uv region and loss of absorption at 328 nm. The halflifetime of free retinol in aqueous buffer at neutral pH is of the order of 20 to 30 min. Thus the retinyl compounds in holo-CRLP at low ionic strength and neutral pH are protected from the usual degradation in aqueous buffers. It should be noted that when the term “stability” is used in the context above, no distinction is made between retinyl ester and unesterified retinol. Since both ester and retinol have about the same molar absorptivity and show similar shaped spectra, it was not possible to use the spectroscopic properties to determine the relative proportion of retinyl ester and unesterified retinol in holo-CRLP. As will be described in the accompanying paper, storage of holo-CRLP at pH 7.4 in the cold leads to hydrolysis of the ester. Holo-CRLP is unstable at pH 6.4 with a half-lifetime of retinyl compounds of about 48 h. When holo-CRLP was prepared by gel filtration at pH 7.4, in 50 mM sodium phosphate buffer which contained, in addition, 1 M NaCl, the retinyl compounds had a halflifetime of about 1 week at 4°C as judged by the loss of absorption at 328 nm. The instability of the retinyl compounds could be arrested and prevented by dialyzing out the NaCl. Thus holo-CRLP which was purified by gel filtration chromatography in the presence of 1 M NaCl, and in which NaCl was then removed by dialysis against 50 mM sodium phosphate buffer, pH 7.4, was as stable as holo-CRLP which was purified in low salt buffers. Thus, in order to exert its deleterious effect on stability, the 1 M NaCl had to be present throughout. Removal of the salt restored the stability. DISCUSSION
The present study shows that about 10% Solutions of purified holo-CRLP in 50 mM of the total retinyl compounds in rat liver sodium phosphate buffer, pH 7.4, were can be obtained in the cytosol fraction. The stable for at least 3 months at 4°C. The retinyl compounds in the cytosol are part of stability of the holo-CRLP complex was a large soluble lipoprotein complex. Holojudged by the stability of its retinyl com- CRLP is a complex molecular aggregate pounds. Both free retinol and retinyl ester reminiscent of some of the serum lipoproare rapidly oxidized and degraded in aqueous teins. And similar to the serum lipoprotein, media. The oxidized Inroducts have different~. holo-CRLP, despite its size and composi-
570
JORAM
tional complexity, behaves as a unique entity. It can be chromatographed on hydroxyapatite (Fig. 3), and it gives a single band on cellulose acetate electrophoresis (Fig. 4). The apparent molecular weight of the lipoprotein complex is on the order of about 5 x lo6 (or above) since it appears in the void volume of a 6% agarose column. Because of lack of reliable molecular weight standard markers in this range with the added technical difficulties inherent in the molecular weight determination of lipoproteins, no attempt was made in this study to measure the molecular weight of CRLP with greater precision. Holo-CRLP is a complex molecular aggregate. The molecular aggregate contains several polypeptide chains of various sizes. A carbohydrate moiety is covalently linked to the polypeptide chain(s). About 66% (by weight) of the molecule is accounted for by lipids. About 80% of the lipids are “simple” lipids, predominantly triglycerides and free fatty acids. The balance of the lipids (about 20%) is due to phospholipids. About 3.5 to 4% of the total is due to retinyl compounds, of which 96% are retinyl ester compounds and about 4% is unesterified retinal. If one assumes one molecule of unesterified retinol per one holo-CRLP molecule, the minimal molecular weight of holoCRLP would then be about 5.7 x 105.Since the molecular weight is probably lo- to 20fold larger than the minimal molecular weight, there would be some 10 to 20 molecules of unesterified retinol and some 250 to 500 molecules of retinyl ester per holo-CRLP complex. Although holo-CRLP mimics a serum lipoprotein, it is not detected when serum alone is subjected to gel filtration and is only observed in preparations of liver homogenate cytosol. Ong and Chytil (26) have recently described the isolation of a soluble protein of rat liver that binds retinol. The protein has a molecular weight of 14,000 (thereby distinguishing it from serum RBP), and it binds one molecule of retinol. This low molecular weight retinol-binding protein accounts for some 0.02 to 0.03% of the total soluble protein. In our study we have not found the low molecular weight protein described by Ong and Chytil. Two reasons
HELLER
can be given for this result. First, we have not added retinol to our extract prior to purification as was done by Ong and Chytil. Second, we have not found any retinylspecific fluorescence in our gel filtration studies in the elution range of low molecular weight proteins (Fig. 1). It is noteworthy that in various preparations of CRLP, prepared from rats of different size, age, sex, the proportion of retinyl ester and free retinol was almost constant (Table II). The variations which were found are probably within the overall experimental accuracy of methodology. The molar ratio of unesterified retinol to retinyl ester was between 1:20 and 1:25. Glover (2) reported recently that in sheep, the ratio of retinol to retinyl ester was 1:17. Only 5.3% of the total unesteri)ied retinol was found as a complex with serum RBP in sheep liver (2). It is thus clear that not only is CRLP important in the storage of retinyl esters but the majority of unesterified retinol is also bound to CRLP. Since the retinyl compounds in CRLP represent at least 10% of the retinyl compounds of the liver, and since the ratio of unesterified retinol to retinyl ester is the same for holo-CRLP as for the whole liver homogenate, the holo-CRLP molecule accounts also for at least 10% of the unesterified retinol found in the liver. The function of holo-CRLP in liver vitamin A metabolism remains to be elucidated. It seems plausible that CRLP is important in storage and protection of the vitamin. We have recently found that holo-CRLP possessesa retinyl esterase activity toward its own retinyl ester complement. A detailed report of this activity is given in the following paper. ACKNOWLEDGMENTS I thank Drs. Joseph Horwitz and Chi-Ching Chen for many helpful discussions and suggestions. L. Ding, J. Takasugi, and A. Duberg have provided excellent technical help. I thank Dr. J. Mead for allowing me to use some of the facilities of his laboratory. REFERENCES 1. UNDERWOOD, B. A. (19’74) World Rev. Nub. Diet. 19, 123-1’72. 2. GLOVER, J., JAY, C., AND WHITE, G. H. (1974) Vitam. Homn. 32, 215-235.
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3. OLSON, J. A. (1968) Vitam. Harm. 26, l-63. 4. POPPER, H., AND GREENBERG, R. (1941) Arch. Pathol. 32, 11-32. 5. LINDER, M. C., ANDERSON, G. H., AND ASCARELLI, I. (1971)J. Biol. Chem. 246,5538-5540. 6. KANAI, M., RAZ, A., AND GOODMAN, DEW. S. (1968) J. Clin. Invest. 47, 2025-2044. 7. GLOVER, J. (1973) Vitam. HOWL. 31, l-42. 8. HORWITZ, J., AND HELLER, J. (1974) J. Biol. Chem. 249, 4712-4719. 9. MUTO, Y., SMITH, J. E., MILCH, P. O., AND GOODMAN, DEW. S. (1972) J. Biol. Chem. 247, 2542-2550. 10. KRISHNAMURTHY, S., MAHADEVAN, S., AND GANGULY, J. (1958)J. Biol. Chem. 233,32-36. 11. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. LIU, T. Y., AND CHANG, Y. H. (1971) J. Biol. Chem. 246, 2842-2848. 14. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 15. WARREN, L. (1959)J. Biol. Chem. 234,1971-1975.
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16. HELLER, J., AND HORWITZ, J. (1973) J. Biol. Chem. 248, 6308-6316. 17. FLETCHER, M. J., AND STYLIOU, M. H. (1970) Clin. Chem. 16, 362-365. 18. WEBER, K., PRINGLE, J. R., AND OSBORN, M. (1972) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 26, Pt. C, pp. 3-27, Academic Press, New York. 19. DITTMER, J. C., AND WELLS, M. A. (1969) in Methods in Enzymology (San Pietro, A., ed.), Vol. 24, pp. 511-512, Academic Press, New York. 20. HELLER, J. (1976)J. Biol. Chem. 251,2952-2957. 21. FUTTERMAN, S., AND ANDREWS, J. S. (1964) J. Biol. Chem. 239, 81-84. 22. HARRIS, R. S. (1967) in The Vitamins (Sebrell, W. H., and Harris, R. S., eds.), p. 11, Academic Press, New York. 23. DISCHE, Z. (1955) Nucleic Acids 1, 285-305. 24. UNDERWOOD, B.A., SIEGEL, H., WEISELL, R. C., AND DOLINSKI, M. (1970) Amer. J. Clin. Nutr. 23, 1037-1042. 25. FUTTERMAN, S., AND HELLER, J. (1972) J. Biol. Chem. 247, 5168-5172. 26. ONG, D. E., AND CHYTIL, F. (1978)5. Biol. Chem. 253, 828-832.
