VOL.
5,
NO.
2,
F L B R U A R Y
1966
Siliprandi, N. (1965), Biochim. Biophys. Acta 98, 207. Saito, K. and Hanahan, D. J. (1962), Biochemistry I , 521. Scherphof, G. L., and van Deenen, L. L. M. (1965), Biochim. Biophys. Acta 98, 204. Smith, H. (1951), The Kidney, London, Oxford Univ. Press. Spackman, D. H., Moore, S., and Stein, W. H. (1958), Anal. Chem. 30, 1190. Tattrie, N. H. (1959), J. Lipid Res. I , 60.
Tinker, D. O., Koch, A., and Hanahan, D. J. (1963), Arch. Biochem. Biophys. 103, 66. Tinker, D. O., Morgan, T. E., and Hanahan, D. J. (1964), Federation Proc. 23,221. Walsh, K., Kauffman, D. L., and Neurath, H. (1962), Biochemistry I , 893. Wheeldon, L. W., and Collins, F. C. (1958), Biochem. J. 70,43. Wurster, C. F., and Copenhaver, J. H. (1965), Biochim. Biophys. Acta 98, 351.
The Glycolipids of Dog Intestine* W. R. Vance, C. P. Shook, 111, and J.
M.McKibbint
ABSTRACT: Preparative silicic acid chro natography of whole lipid extracts of dog intestine gave three fractions which contained glycolipid. The Iargesi: fraction contained all of the ceramide oligoglycosides and these were separated from phospholipid and c ther impurities by solvent fractionation, Florisil chromiitography, and dialysis. The ceramide oligoglycosides were then separated into ganglioside and ceramide oligN3hexoside fractions by DEAE-cellulose chromatography. The latter was partially resolved by cold methanol fractionation followed by silicic acid chromatograpliy. The major component, a ceramide pentaglycoside, was recovered in pure form on the basis of column and thin layer chromatography and on analysis. This is a novel substance giving analytical data and hydrolytic cleavage products
S
tudies on the chemistry and metabolism of the glycolipids of vertebrate tissues have been largely confined to those of nervous tissues, erythrocyte stroma, spleen, and kidney. All glycolipids isolated from these sources appear to be glycosides of a ceramide with glucose, galactose, galactose sulfate ester, lactose, or oligosaccharides which may contain in addition Nacylhexosamine or sialic acid or both. The chemistry and metabolism of these substances haw: been reviewed (Law, 1960; Svennerholm, 1964; Carter et al., 1965). In this laboratory studies have been carried out on the * From the Department of Biochemistry, Iiniversity of Alabama Medical Center, Birmingham, Alabama. Received Augusf 8, 1965; revised November 3, 1965. This work as supported by a grant from the Life Insurance Medical Research Fund. A major portion of the data in this paper is taken from i t dissertation submitted by W. R. Vance to the University of Alabama in partial fulfillment of the requirements for the degree 3f Doctor of Philosophy in Biochemistry. A preliminary report of this work has been presented (McKibbin and Vance, 1964). f To whom inquiries should be addressed.
consistent with an N-acylgalactosaminyl-N-acylgalactosaminylgalactosylgalactosylglucosylceramide structure. It contains 3 0 x of the total intestine lipid sugar and most of the galactosamine. The ganglioside fraction was partially resolved on a silicic acid column, yielding a chromatographically homogeneous ganglioside as the major component. Analyses of this substance were consistent with a sialyldigalactosylglucosylceramide structure and with a minor glucosamine-containing impurity. The sequence of sugars and nature of the long-chain base are not known. Both the ganglioside and the pentaglycoside contain predominantly long-chain fatty acids with average molecular weights of 338 and 321, respectively.
composition of glycolipids of intestine and liver. These tissues contain some unique ceramide glycosides as well as other lipids containing fatty acyl primary amines. This paper describes the preparation of the ceramide oligoglycosidelr2 fraction of dog intestine and the isolation and composition of the major component. This substance is a ceramide pentaglycoside which contains two N-acylgalactosamines, two galactoses, and one glucose in that order on the ceramide. A description of the isolation and composition of another major glycolipid component, a ganglioside, is also included in this paper.
1 In this paper the following terminology is used: “ceramide oligoglycoside” is a ceramide glycoside containing more than three sugar residues. “Ganglioside” is a glycolipid which contains sialic acid. “Ceramide oligohexoside” is a ceramide oligoglycoside which does not contain sialic acid. The composition of all solvent mixtures is expressed on a volume per volume basis. 2 The following abbreviations are used in this paper: tlc, thin layer chromatography; CPG, ceramide pentaglycoside.
THE GLYCOLIPIDS OF DOG
43 5
I N T E S T I N E
BIOCHEMISTRY
Experimental Section Analytical Methods. Methods for the determination of phosphorus and primary amine have been described previously (McKibbin et al., 1961). Total N was determined by the method of Koch and McMeekin (1924) modified for lipid material (McKibbin and Taylor 1949b). Long-chain base nitrogen was determined by the method of McKibbin and Taylor (1949b) on aliquots hydrolyzed by refluxing for 3 hr with 2 N aqueous HCl. Chromic acid oxidations were carried out by the method of Bloor (1928), and ester groups by the method of Snyder and Stephens (1959). Sialic acid was determined by the method of Svennerholm (1957) as modified by Miettinen and Takki-Luukkainen (1959) without previous hydrolysis and using a commercial N-acetylneuraminic acid (Sigma Chemical Co.) as a standard. Hexosamine was determined by the method of Elson and Morgan (1933) as modified by Blix (1948) on an aliquot hydrolyzed by refluxing 3.5 hr with 2.5 or 3 N HC1. All anthrone-reacting material was determined by the method of Bailey (1958) using a galactose standard and expressed as galactose. Glucose was determined with glucose oxidase (Glucostat, Worthington Gorp*) on the neutralized prepared by refluxing the aliquot with 2 N HC1 for 3 hr. Since the molar color yield from the anthrone reaction with glucose is much higher than with galactose, total hexose was calculated from the galactose values corrected for the glucose content of the mixture. For ga1actose:glucose ratios of 2, as determined for the lipids considered here, multiplying the galactose values by 0.86 gave true values for total hexose. The glucose values were subtracted from total hexose and t h e remainder was expressed as “true galactose.” Formaldehyde was determined by the method of Frise11 and Mackenzie (1958). Total sulfur was determined by benzidine precipitation of sulfate from a Carius digest (Kahn and Leiboff, 1928) followed by determination of the benzidine with the naphthoquinone sulfonate reagent.
with 2 ml of solvent/g. The extracts in chloroform solution were purified by emulsification with 0.25 M aqueous MgClz (McKibbin et al., 1961). The purified lipid extracts in chloroform solution were then fractionated as described below. Isolation of the Ceramide Oligoglycoside Fraction. The entire fractionation procedure is shown in Figure 1. SILICIC ACID CHROMATOGRAPHY. Silicic acid (16.7 g) (Mallinckrodt No. 2844) and 16.7 g of Celite (Johns Manville, water washed) were mixed in a chloroform slurry and poured into a 17-mm id column fitted to a suction flask. The column was rinsed with an additional 50 ml of chloroform under slightly reduced pressure. The purified lipids, equivalent to 0.84 mrnole of lipid phosphorus, in 60 ml of chloroform solution were added to the column, followed by 30 ml of additional chloroform. The column was eluted with 100 ml of 2%, 200 ml of lo%, 200 ml of 17%, 200 ml of 50%, and 300 ml of 80 % methanol in chloroform, respectively. The recovery of lipids in these fractions (designated S2, S10, S17, S50, and S80, respectively) is given in Table I. The
Fractionation of Dog Intestine Lipids with SilicicAcid.a TABLE 1:
Elution Solvent (% MethFrac- anol in Volume tion CHC13) (ml) s2 SI0 S17 S50 S80
2 10
17 50 80
of Total Recovered
100 200 200 200 300
Lipid Phosphorus
Lipid Galactose
0.2 41 . O 18.1 22.8 12.6
0.3 20.6 16.0 52.5 5.7
~~
Silicic acid (16.7 g), 16.7 g of Celite, 17-mm id column, 0.843 mmole of lipid phosphorus, 91.1 pmoles of lipid galactose. a
Preparation ojthe Glycolipids
436 W.
R.
Extraction and Purification o j the Lipids. All of the intestine samples were taken from dogs immediately at sacrifice. The entire small intestine was slit longitudinally and washed with cold water, and the adhering mesenteric fat was removed. Initially the frozen tissue was powdered and refluxed with ethanol-ether (3 :l), and the insoluble residue was extracted with chloroform as described previously (McKibbin and Taylor, 1949a). The residue extraction was not necessary with the intestine. For large-scale preparations the tissue was prepared for reflux by homogenizing in ethanol-ether (3 :1) in an explosion-proof Waring Blendor fitted with a clamped top and a sheet Teflon gasket. The volume of extraction solvent was also reduced from 17 ml/g of fresh tissue to 8 ml/g by a two-stage extraction. The homogenate was refluxed initially for 45 min with solvent equivalent to 6 ml/g, then the extract was decanted and the residue was extracted again for 15 min
VANCE,
C.
P.
SHOOK,
111,
A N D
J.
M.
MCKIBBIN
ceramide mono-, di-, and trihexosides were contained in the S10 and S17 fractions along with most of the phospholipid. The composition of these glycolipids will be reported later. All of the ceramide oligoglycosides were contained in the S50 and S80 fractions and these were used for further fractionation. For large-scale preparations 41-mm id columns were used with the adsorbent, lipid load, and eluting solvent volumes proportional to cross-sectional area. The elution with 80 methanol in chloroform was omitted for economy. SOLVENT FRACTIONATION OF FRACTION S50. Fraction S50 qpivalent to 250 pmoles of lipid galactose was taken up in the solvent system : benzenepetroleum ether (bp 30-60”) (70 :30) YS. methanol-acetonewater (60 :20 :20). A five-plate countercurrent extraction was carried out with this system using separatory funnels
V O L . 5 , N O . 2, F E B R U A R Y 1 9 6 6
, Whole li id extract
Silicic acid chromatography
I
1
s10
s2
I
I
I
S17
S50
S80
\ /
Ceramide oligoglycosides Countercurrent extraction
PI
P2
~
P3
P4
P5
Ceramide oligoglycosides Florisil chromatography and dialysis
S50-P4-F75
S50-P5-F75 P-free ceramide oligoglycosides DEAE-cellulose chromatography
gangliosides and ceramide oligohexosides
\
Methanol
DEAE-cellulose chromatography
7
Soluble
Silicic acid chromatography
Insoluble
Silicic acid chromatography
A
FIGURE
B
C
