A simple and novel method for tritium labeling of gangliosides and other sphingolipids

106

Biochimica et Biophysics 0 Elsevier/North-Holland

Acta, 529 (1978) 106-114 Biomedical Press

BBA 57161

A SIMPLE AND NOVEL METHOD FOR TRITIUM GANGLIOSIDES AND OTHER SPHINGOLIPIDS

LABELING

OF

GiiNTER SCHWARZMANN Physiologisch-Chemisches

Znstitu t Z, Philipps-Universitiit

Marburg, Marburg (G. F. R.)

(Received November 25th, 1977)

Summary A very simple method for introducing tritium specifically into the ceramide portion of gangliosides, neutral glycosphingolipids and sphingomyelin has been developed using potassium boro [ 3H] hydride and palladium as catalyst. In this way six different gangliosides, five different neutral glycosphingolipids and sphingomyelin with high specific radioactivity were prepared for the first time. This simple procedure which does not require sophisticated apparatuses is suitable for large scale preparation of tritium-labeled sphingolipids as well as for nanogram quantities of individual sphingolipids so as to serve for analytical purposes. During the course of the labeling procedure no degradation of even the most labile trisialosyl-gangliotetraosylceramide has been observed. The yield of tritiated compounds is almost quantitative. The specific radioactivity depends on the unsaturation of the ceramide moiety and the specific activity of the boro [ 3H] hydride employed.

Abbreviations: (from * Nomenclature according to Svennerholm. L. (1963) J. Neurochem. 10. 613-623: ** Nomenclature as outlined by Wiegandt, H. (1973) Hoppe-Seyler’s Z. Physiol. Chem. 354,1949-1956; * * * Designation as recofnmended by IUPAC-IUB Lipid Nomenclature Document. 1976. r Designation according to Sung. S.-S.J.. Essehnan. W.J. and Sweely. C.C. (1973) J. Biol. Chem. 246, 6528--6533; NeuAc. N-acetyhieuramic acid; GM~ * = GLaclNeuAc ** = II3 NeuAcLacCer * * * = NeuNAccvP --) 3G@l--* 4Glc@l--) 1Cer; Ghf2 * = GGtrilNeuAc * * = II3 NeuAcGgOse3Cer ** * = GaINA@ --+ 4GaI(3 + 2oNeuAc)pl-+ 4GlcPl+ 1Cer; GM~ * = GGtetlNeuAc ** = IISNeuAc-GgOseq-Cer *** = GaJ@l -f 3GaINAqYl-* 4GaI(3 + 2cuNeuAc)fll+ 4Glt$l--* 1Cer: GDla * = GGtet2aNeuAc ** = IV3NeuAc. IIJNeuAc-GgOseq-Cer *** = NeuAccr2 d 3GaIlPl -f 3GaINAc$l+ 4GaI(3 + 2aNeuAc)pl * 4Glc@l+ 1Cer; GDlb * = GGtet2hNeuAc ** = 113h 4GlcPl (NeuAc)2-GgOseq-Cer *** = GaBI1 -f 3GaINAcal+ 4GaB3 + 2oNeuAc8 + 2aNeuAc)pl *** = NeuAco2’ + 1Cer; GT1 * = GGtet3NeuAc ** = IV3NeuAc. 113(NeuAc)2-GgOse4-Cer + GL-la = GlcPl 1’Cer. 3G~@l--* 3GalNAc61 h 4Gai(3 + 2oNeuAc8 + 2aNeuAc)pl + 4GlcPl+ -_* 1Cer (plucosylceramine); GGlb = Gal--* 1Cer (galactosylceramide, cerebroside); GL-2 = 1’Cer GaI@l --, 4Glc61 + 1’Cer (Iactosylceramide); GL-3 = Gb3a *** = Gala1 -t 4GaI/Il -t 4Glcbl+ h 3GaIol -+ 4Gal --* 4Glc61 h 1’Cer = = globotriaosylceramide; GL-4 = Gb4a *** = GaINA& globotetraosylceramide.

107

The labeled neutral glycosphingolipids were analysed by autoradiography following thin-layer chromatography and by analyses of their hexose content. In addition, gangliosides were analysed by determination of their sialic acid content.

Introduction Glycosphingolipids have long been recognized as constituents of plasma membranes [ 1,2]. Although the structures of many of the gangliosides and neutral glycosphingolipids are known for quite some time it was not until recently that a variety of specific functions has been assigned to this interesting class of compounds. Among these the participation of glycosphingolipids in cell to cell contact [3,4] and malignant transformation of cells [5,6] have been discussed. Involvement of gangliosides in binding of viruses, toxins and hormones have also been shown by numerous investigators [7-111. Obtaining more insight in the biological role played by glycosphingolipids in general seems highly desirable. To be able to analyse even the smallest amounts of naturally occurring glycolipids as well as to trace minimal amounts of metabolic enzymes in a set of hereditary diseases like sphingolipidoses by highly labeled substrates it was necessary to develop a very simple and highly effective labeling procedure applicable to all kinds of sphingolipids. Also the use of the labeled glycosphingolipids would assist in investigation of their fate after being exogenously added to different cells. Quite recently, Veh et al. [12] have been able to extend the procedure of van Lenten and Ashwell [13] to gangliosides. In this method, 3H is introduced into the sialic acid moiety by modification of the latter using mild periodate oxidation followed by reduction with NaB3H4. While this method is very suitable to investigate the occurrence and characteristics of sialidases, it is not possible to apply this procedure to asialo-glycolipids. Other methods used in tritiation of glycosphingolipids are based on the Wilzbach 3Hz gas irradiation technique [14,15], the catalytic addition of 3Hz gas to double bonds [16-191 and on the galactose oxidase procedure [20-231 all of which require either sophisticated apparatuses and/or tedious work-up procedures. The latter method is only applicable to lipids having either a terminal galactose or N-acetylgalactosamine residue. The intention of this work was to develop a convenient and reliable labeling procedure without causing dramatic changes in the nature of the compound. In addition this procedure should be applicable to all kinds of sphingolipids. Materials and Methods Dowex ionexchange resins (Dowex 50-WX2 and Dowex l-X2; 200-400 ASTM) were obtained from Serva, Heidelberg, G.F.R. DEAE-cellulose was from Schleicher and Schiill, Dassel, G.F.R. Silica gel (Kieselgel 60; 230-400 ASTM), precoated thin-layer plates, orcinol, a-naphthol and resorcinol were analytical grade and purchased from E. Merck AG, Darmstadt, G.F.R. Potassium boro[‘H]hydride (170 mCi/mmol-13.3 Ci/mmol) was obtained from

108

Amersham Buchler GmbH, Braunschweig, G.F.R. D-Galactose dehydrogenase and NAD’ were purchased from Boehringer AG, Mannheim, G.F.R. All other reagents were from E. Merck AG and of the highest purity available. Solvents were analytical grade or distilled before use. Preparation of gangliosides. Crude ganglioside mixtures were obtained from lyophilized and acetone-extracted human brain powder by extraction into CHClJCH30H followed by a modified Folch partition and dialysis [24,25]. The crude ganglioside mixtures contained 9.5-11% sialic acid, corresponding to approx. 30% of mixed gangliosides. Pure gangliosides were obtained using the procedure described by Hakomori [26] followed by preparative thin-layer chromatography. Tay-Sachs ganglioside GMz was provided by Dr. K. Sandhoff, Max-Planck-Institut fiir Psychiatric, 8000 Miinchen, G.F.R. Preparation of sphingomyelin. Sphingomyelin was prepared from the lower phase after Folch partition of a CHCIJCHJOH extract of human brain. After mild alkaline hydrolysis sphingomyelin was purified by silica gel column chromatography using CHC13/CH30H mixtures of increasing polarity. Preparation of neutral glycosphingolipids and GM3. Total sphingolipids were extracted from acetone-dried powder of human spleen into CHC1JCH30H. The extract was evaporated in vacua whereby CHCIB and CH30H were replaced by several additions of toluene. From the resulting toluene dispersion of lipidic material, the precipitated hemoglobin was filtered off. Glycosphingolipids were then precipitated by stirring the toluene solution into 30 volumes dry acetone. The precipitate was collected by filtration and the glycosphingolipids separated from phospholipids and other material as described [26]. Neutral glycolipids were separated from GM3 and other gangliosides by the procedure of Yu and Ledeen [ 271. A final purification was achieved by silica gel column chromatography . Calorimetric assays. Ganglioside-bound sialic acid was determined either with the periodic acid-thiobarbituric acid procedure [28] following mild acid hydrolysis in 0.05 M HzS04 at 80°C for 1 h or directly by the method of Svennerholm [29] as modified by Miettinen and Takki-Luukkainen [ 301. In all cases 0~~-acid glycoprotein (with 12.6% NeuAc) served as reference standard. Neutral hexoses were determined with the orcinol-HzS04 procedure [31,32] using lactose and cerebroside as reference compounds. Galactose was determined using D-galactose dehydrogenase. 3H labeling of gangliosides. About 5-10 pmol water-soluble gangliosides were dissolved in 0.8 ml Hz0 in a Teflon-lined screw-capped vial. Following the addition of 0.1 ml 1 M NaOH, 50-100 pmol KB3H4 (2.7-5.4 mg) were added. After flushing with N?, 0.1 ml (14 pmol) of 25 mg/ml PdClz was carefully layered over the ganglioside solution. The vial was then immediately capped and the hydrogenation reaction allowed to proceed, with vigorous stirring, for at least 3 h at room temperature. By the addition of 2-3 ml methanol, palladium was precipitated from its colloidal suspension. The pH of the resulting solution was adjusted to 5 by dropwise addition of dilute acetic acid. Prior to evaporation the precipitated palladium was centrifuged off. To remove labile 3H, the residue was repeatedly taken up in water and evaporated in vacua. Na’ was removed by passing the aqueous solution of labeled ganglio-

sides over a small column containing 2 ml of a cation-exchange resin (Dowex 50.X2, H’). The filtrate and washings were evaporated to dryness in vacua with the aid of toluene. Boric acid was removed by several evaporations from CH30H solution in vacua. The 3H-labeled gangliosides were then analysed. 3H labeling of neutral glycosphingolipids, shingomyelin and GMs. In a typical experiment, up to 100 mg sphingolipid were dissolved in 3 ml tetrahydrofuran in a screw-capped vial at 50°C. To the solution were added 125 pmol each of KB3H4 and PdC12. After flushing with Nz, the hydrogenation was started by the addition of 0.25 ml 1 M NaOH and continued for several hours at room temperature . Following addition of CHC1&HJOH (2 : 1, v/v) and a few drops of dilute acetic acid, the palladium was filtered off. The 3H-labeled sphingolipids were then purified as follows: Ghls was treated as described under labeling of gangliosides. Neutral glycosphingolipids were purified by preparative thin-layer chromatography and sphingomyelin was freed of salts by Folch partition [24]. 3H labeling of sphingolipids in sub-milligram quantities. The procedure described for labeling of neutral glycolipids can be adapted to very small amounts of total sphingolipids which are derived from total extracts of cells corresponding to less than 2 mg of cellular protein (unpublished data). For easy handling of small amounts of KB3H4, the latter is dissolved in diethyleneglycoldimethylether. Aliquots are withdrawn for labeling sphingolipids under investigation. Thin-layer chromatography. Thin-layer chromatography was performed on precoated plates using CHC13/CH30H/Hz0 (60 : 35 : 8, v/v), containing 20 mg CaCl, per 100 ml solvent mixture [33] for development of gangliosides, and CHC13/CH30H/Hz0 (65 : 25 : 4, v/v) for separation of neutral glycosphingolipids and sphingomyelin. Spots of gangliosides were visualised by spraying with Ehrlich reagent [34]. Neutral glycosphingolipids were detected with the a-naphthol/HzS04 spray reagent [ 351. Phospholipids were made visible using the molybdenum blue reagent [ 361. Plates were also developed by autoradiography [ 371. Determination of specific radioactivity. Radioactivity of tritiated sphingolipids was measured in duplicate in aliquots of dissolved glycosphingolipids of known concentration. The specific radioactivity was determined in a Berthold BF 8000 liquid scintillation counter (Dr. Berthold, D-7547 Wildbad, F.R.G.) on line with a Hewlett-Packard 9815 A computer with quench correction using the channel ratio method. [ 3H]Toluene from Amersham-Buchler served as internal standard. The scintillation fluid (10 ml) Unisolve I (Roth, Karlsruhe, F.R.G.), was used. Partial acid hydrolysis of globoside. Treatment of GL-4 with 0.05 M HCl at 70°C for 12 h, yielded GL-3, GL-2 and GL-la which were separated by preparative thin-layer chromatography on precoated thin-layer plates. Results The labeling procedure yielded, almost quantitatively, 3H-labeled glycosphingolipids in a simple and convenient manner. Slight variations in yields from 90 to 97% occurred depending on the mode of purification. During tritiation of

110 TABLE I SPECIFIC RADIOACTIVITIES CONDITIONS

OF 3H-LABELED

SPHINGOLIPIDS PREPARED

UNDER DIFFERENT

Labeling was performed in tetrahydrofuran in case of neutral and in water in case of gangliosides unless otherwise stated. Sphingomyelin was tritiated in tetrahydrofuran. Whenever lipids have been treated equally or very similarly during the course of tritiation they have been assembled into groups. Sphingolipid

Specific activity (Ci/mol)

GM~ GMZ

1910 600

Group

437 385 265

A A A

GDla GDlb GT~

14 56 54

B B B

GL-2 GL-3 GL-4

12 63 62

C C C

GL-la ** GL-2 ** GL-3 **

100 93 121

D D D

Sphingomyelin

101

GM~ * GM~ GM~

GL-lb ***

24

* Labeling was carried out in tetrahydrofuran. ** These neutral gIycosphingoIipids were prepared by partial acid hydrolysis from globoside (GL-4) from human spleen. * * * Prepared by reduction of 3-keto-cerebroside with KB3 H4 (see Results).

gangliosides, no loss of sialic acid has been observed. The labeled substances were subjected to thin-layer chromatography as described under Materials and Methods. No degradation of the starting sphingolipids was detected. Specific radioactivity The results obtained upon treatment of various sphingolipids are listed in Table I. Using boro [ 3H]hydride of relatively high specific activities (12.4 or 13.3 Ci/mmol), gangliosides with specific radioactivities of about 2000 Ci/ mol were obtained, as shown in case of GM1. Accordingly using boro[3H]hydride of specific activities from 170 to 330 Ci/mmol produced [3H]sphingolipids with specific radioactivities ranging from 54 to 120 Ci/mmol. The comparison of gangliosides (group A, Table I) with respect to their specific activities is quite noteworthy. Although these gangliosides have been treated in a very similar way using boro [ 3H] hydride from the same stock, there is about a 1.5-fold increase of radioactivity observed in the two GM3 preparations as compared to the one of GM1. This difference in radioactivity very probably reflects the degree of unsaturation in the ceramide moiety. It is a well established fact that the ceramide residue of human brain gangliosides mainly consists of steraic acid besides CzO and Cl8 sphingosines [38]. On the other hand neutral glycosphingolipids and GMs from human red blood cells and platelets, e.g. contain about 30% of unsaturated fatty acids [39]. This naturally

111

leads to a higher uptake of radioactivity upon hydrogenation with tritium. The same observation holds comparing gangliosides (group B, Table I) with neutral glycosphingolipids (group C, Table I) which have been treated similarly with respect to the specific activity of the boro[3H]hydride employed. Within each individual group of glycolipids the results obtained are in fairly good agreement. Thus imposing that this method of tritiation is rather reproducible. The GL-lb with 24 Ci/mol has been obtained upon reduction of a 3-keto derivative of cerebroside according to Iwamori et al. [40]. In this case the double bond of the sphingosine moiety remains intact during reduction with borohydride as could be shown by ozonolysis of the resulting cerebroside (unpublished results). This method is unfortunately not applicable to gangliosides and higher glycosylated neutral sphingolipides as yet (unpublished results). Specificity of tritium incorporation

From the observation that apart from the analytical error the three glycolipids GL-la, GL-2 and GL-3 (group D, Table I) have about the same radioactivities the conclusion can be drawn that tritium specifically is introduced into the ceramide residue. In this case the latter is common for all three lipids due to their preparation by partial acid hydrolysis of GL-4. This view is supported by the fact that treatment of labeled GM, following incorporation into cells with Vibrio cholera sialidase released unlabeled sialic acid and yielded labeled GL-2 [411. Efficiency of tritium incorporation

Incorporation of tritium into glycolipids is dependent upon the amount of the tritiide employed. Using milligram quantities of boro[ ‘H] hydride a 1.2-fold molar excess of the latter ensures maximum incorporation of the label. On the other hand when only fractions of a milligram of the tritiide were employed a 60% decrease in tritium uptake was observed. In this case a little loss of tritium which escaped before the vial had been capped tightly and catalytic exchange reactions with water was naturally more noticeable. In general a 3-fold molar excess of the boro[3H]hydride is sufficient for maximum tritiation. Reproducibility of labeling

For incorporation into cell membranes and for measuring the abundance and activities of enzymes of lipid metabolism it is only relevant to obtain labeled sphingolipids with high specific radioactivities. On the other hand the application, for analytical purposes of the labeling procedure to evaluate the glycosphingolipid pattern of cells, e.g. demands highly consistent results. Therefore, amounts of 0.6 pmol of GM1 have been labeled in five parallel experiments using 3.7 pmol boro[3H]hydride (90 FCi). Within the analytical error these five samples of GM, had the same specific radioactivity, thus showing that the labeling procedure is rather reproducible.

112

Autoradiography (fluorography) of labeled gangliosides and neutral sphingolipids The radiopurity of the final products of labeling after purification was assessed by thin-layer chromatography followed by autoradiography. Single spots were obtained for gangliosides (Fig. 1) and all neutral glycosphingolipids but GL-2 and sphingomyelin (Fig. 2). Although GM1 was reinvestigated, following storage for 9 month without further purification one single spot was obtained. Thus showing that no noticeable production by autoradiolysis of radioactive impurities had occurred. According to the findings of Randerath [37] radioactive impurities of less than 1% of the glycolipids employed would have been detected easily using about 100 nCi of each of the lipids under investigation (Figs. 1 and 2). Due to differences in the ceramide portion GL-2 separates into a double bond as does sphingomyelin (Fig. 2). On the other hand the faster moving band of GL-2 is

Fig. 1. Thin-layer chromatography and autoradiograpny have been performed as described under Materials and Methods. The amount of radioactivity applied is given in parentheses. In the orfBklp1 radiograph the width of all bands is 1 cm. The figure shows GM3 (lane 1.101 nCi). GM2 (lane 2,114 nCi). GM1 (lane 3, 102 nCi). GD~~ (lane 4.112 nci), GDlb (lane 6.147 nCi), GTl (lane 6.144 nci), SPhiWlomYelin (tie 7. 138 nCi) and a mixture of the afore-mentioned ganghoslder. Without any further purification GM~ has been investigated after being stored for 9 month using 0.4 nmol(0.6 I.rg.256 Cilmol) of this ganghosfde. Fig. 2. Thin-layer chromatography and autoradiography have been performed as described under Materials and Methods. The amount of radioactivity applfed is given in parentheses. The figure shows GL-2 (lane 1. 113 nCi) as isolated from human spleen, GL-2 (lane 2. 94 nCi) as prepared from GL-4 by Partial acid hydrolysis, GL-2 (lane 3, 104 nCi) prepared from GM3 by treatment with vibrio cholera sialidase. GL-3 (lane 4, 106 nCi). GG4 (lane 6. 134 nCi). sphingomyelin (lane 6, 116 nCi) and a mixture of GL-2. GL-3 and GL-4 (lane 7). In the original radiograph the width of all bands is 1 cm.

113

obtained either by partial acid hydrolysis of GL-4 or by desialization both of which are extracted from the same organ (human spleen).

of GM3

Discussion The present method avoids dangerous or tedious biosynthetic techniques as well as the drawbacks of the afore-mentioned procedures [12,14,17,18,23]. During tritiation no degradation takes place. Therefore the purification procedure remains simple. High radioactive sphingolipids (about 2000 Ci/mol) with minimal structural alteration can be obtained easily without the need of sophisticated apparatuses. This method due to its efficiency, applicability, mildness and simplicity by far surpasses all other methods published to date. This procedure makes available a sensitive tool for the detection of traces of sphingolipids which might be overlooked easily by conventional techniques. This is important in-as-much as some biological effects of glycosphingolipids are highly dependent on their nature [9,41,42]. In addition the simple preparation of radio-labeled sphingolipids now makes possible the investigation of the fate of lipids exogenously added to cells [41] and assists in the studies of membrane glycolipid function. References

11

Klenk, E. and Fauenstein, K. (1961) Hoppe-Seyler’s Z. Physiol. Chem. 288.220-228 Yamakawa. T. and Suzuki, S. (1961) J. Biochem. Tokyo 38.199-212 Roseman, S. (1971) Chem. whys. Lipids 6,270-296 Hakomori, S.-I. (1970) Proc. Natl. Acad. Sci. U.S. 67.1741-1747 Hakomori. S.-I. (1973) Adv. Cancer Res. 18.266-287 Fishman, PH. and Brady, R.O. (1975) in Modifications of Lipid Metabolism (Perkins. E.G. and Witting. L.A.. eds.). p. 106. Academic Press, New York Haywood. A.M. (1974) J. Mol. Biol. 87.626-628 Van Heyningen, W.E. (1974) Nature 249.416-417 WoIIey, D.W. and Gommi. B.W. (1966) Proc. Natl. Acad. Sci. U.S. 63.969-963 MuBin, R.. Fishman. P.H.. Lee, G.. Aloj. SM.. Ledly. F.D.. Winand, R.S.. Kohn. L.D. and Brady, R.O. (1976) Proc. NatI. Acad. Sci. U.S. 73.842-346 Meldoles& M.F.. Fishman, P.H.. Aloj. S.M.. Kohn. L.D. and Brady, R.O. (1976) Proc. Natl. Acad. Sci.

12 13 14 16 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

U.S. 73.4060-4064 Veh, R.W.. Corfield. A.P., Sander, M. and Schauer. R. (1977) Biochim. Biophys. Acta 486,146-160 van Lenten, L. and AshweII. G. (1971) J. Biol. Chem. 246.1889-1894 Wilzbach. K.E. (1957) J. Am. Chem. Sot. 79.1013 Brady, R.O.. Gal. A.E.. Bradley. R.M. and Mlirtensson, E. (1967) J. Biol. Chem. 242.1021-1026 Crawhall, J.C. and Smyth, D.G. (1960) Biochem. J. 69,280-286 Gatt, S. and Rapport, M.M. (1966) Biochem. J. 101.680-686 Seyama. Y., Yamakawa. T. and Komai, T. (1968) J. Biochem. Tokyo 64.487493 DiCesare. J.L. and Rapport. MM. (1974) Chem. Phys. Lipids 13.447-462 Haira. A.K., Bowen, D.M.. Kishimoto. Y. and Radin. N.S. (1966) J. Lipid Res. 7.379-386 Bradley, RX and Kanfer, J.N. (1964) Biochim. Biophys. Acta 84.210-212 Suzuki. Y. and Suzuki, K. (1972) J. Lipid Res. 13,687-690 GhidonI, R.. Tettamanti. G. and Zambotti. V. (1974) Ital. J. Biochem. 23.320-328 Folch, J., Lees, M. and Sloane-StanIey, G.H. (1967) J. Biol. Chem. 226.497-609 Pure. K. (1970) Acta Chem. Stand. 24.13-22 Hakomori, S.-I. (1971) J. Lipid Res. 12. 267-259 Yu. R.K. and Ledeen. R.W. (1972) J. Lipid Res. 13.680-686 Warren, L. (1969) J. Biol. Chem. 234.1971-1976 Svemrerhobn. L. (1967) Biochim. Biophys. Acta 24.604-611 Miettinen. T. and Takki-Luukkainen. L-I. (1969) Acta Chem. Stand. 13.666-868 Brown, A.H. (1946) Arch. Biochem. 11.269-278 Svennerhohn. L. (1966) J. Neurochem. 1.42-63

1 2 3 4 6 6 7 8 9 10

114 33 34 35 36 37 38 39 40 41 42 43

van den Eiinden, D.H. (1971) Hoppe-Seybr’s Z. Physiol. Chem. 352.1601-1602 Patridge. S.M. (1948) Biochem. J. 42.238-253 Siakotos. A.N. and Rouser, G. (1966) J. Am. Chem. Sot. 42.913-919 Dittmer. J.C. and Lester, R.L. (1964) J. Lipid Res. 5,126-127 Randerath, K. (1970) Anal. Biochem. 34.188-206 Sambasivarao, K. and McLuer, R.H. (1964) J. Lipid Res. 5, 103-108 Tao, R.V.P., Sweely. C.C. and Jamieson. G.A. (1973) J. Lipid Res. 14. 16-25 Iwamori. M.. Moser, H.W. and Kishimoto. Y. (1975) J. Lipid Res. 16, 332-336 CaIIies, R., Schwarzmann. G., Radsak. K., Siege& R. and Wiegandt, H. (1977) Eur. J. Biochem. 80, 425-432 Cuatrecasas, P. (1973) Biochemistry 12,3647-3681 Keenan, T.W., Schmid. E., Franke. W.W. and Wiegandt. H. (1975) EXP. Cell. Res. 92, 269-270