[17] Labeling complex carbohydrates of animal cells with monosaccharides

[17]

ANIMAL CELLS WITH MONOSACCHARIDES

175

excess hapten. For example, goat antibody directed against lacto-Ndifucohexaose I TM was purified as follows: The immunoglobulins of a goat serum containing per milliliter 1.1 mg of antibodies precipitable by antigen were concentrated 4-fold by precipitation with 40% (NH4)2SO4 followed by dialysis of the precipitated protein against PBS. Of the immunoglobulin concentrate, 2 ml was passed through a column of Sepharose 4B (0.8 cm x 3.0 cm) containing 10 t-tmol of covalently bound lacto-N-difucohexaose I derivative. The column was washed with PBS until the UV transmittance of the elute returned to base line. Antibody was then eluted from the column with a solution oflacto-N-difucohexaose I (2 mg/ml in PBS), concentrated by vacuum dialysis against PBS to 3 ml, and freed of hapten by passage through a column of Bio-Gel P-6 (2.5 cm × 35 cm) using PBS as eluent. Over 90% of the precipitable antibody in the original serum was recovered by this procedure. As an alternative to hapten elution, rabbit antibodies against lacto-Ntetraose ° have been purified by elution with 0.1 M acetic acid: 82% of hapten-binding activity present in the original serum was recovered from the eluate after neutralization and dialysis against PBS. Antibodies with wide thermal amplitude can be purifed by thermal elution. For example, a Waldenstr6m macroglobulin with cold agglutinin activity that binds N-acetylneuraminyl residues 11 was separated from other proteins by using a column containing Sephadex substituted with 3'-sialyllactose. The protein mixture in PBS was passed through the column at 0 °, and after washing with cold PBS the cold agglutinin was recovered from the column in 80% yield by elution with PBS at 37°. "~ For structures of oligosaccharides see this volume [14] and [22]. ii C.-M. Tsai, D. A. Zopf, R. K. Yu, R. Wistar, Jr., and V. Ginsburg, Proc. Natl. Acad. Sci. U.S.A. 74, 4591 (1977). As the cold agglutinin was also a cryoglobulin, it was first desialated, which rendered it soluble at 0°.

[17] L a b e l i n g C o m p l e x C a r b o h y d r a t e s o f A n i m a l Cells w i t h Monosaccharides

By

PETER D . YURCHENCO, COSTANTE CECCARINI,

and PAUL H. ATKINSON The biosynthesis of glycoproteins and glycolipids in intact cells can be conveniently studied by use of radioactive monosaccharide precursors. Our emphasis in this article is mainly on animal asparagine-linked or serine/threonine-linked oligosaccharides whose usual component mono-

176

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[ 17 ]

saccharides are N-acetylglucosamine, fucose, mannose, galactose, N-acetylgalactosamine, and sialic acids. '4C-Labeled monosaccharides have long been used for these purposes and increasingly in the last decade tritiated (31-I) sugars have been too (a review of the general applicability and commercial availability of 3H-labeled sugars has appeared. 1 Most studies, however, have been qualitative, and five major reviews that thoroughly cover the topic have appeared. 2-6 This paper presents a brief review of papers that have quantitative data and of methodology used in quantitation of the biosynthetic processes involved in complex carbohydrate synthesis. In general, where quantitative data are tabulated or qualitative results are recited we have mostly quoted from papers where labeled monosaccharides have been used in the study. We also have included key details of our application of one quantitative approach utilizing a radioactive monosaccharide. Such quantitation is experimentally fruitful, since it allows the measurement of flow rates, pools, and pool sizes along the pathways of the complex carbohydrates synthesis r's from initial sites of utilization to final sites of accumulation. Such knowledge then allows the utilization of radiosotopic precursors as measures of the absolute rate of synthesis of end products in comparative situations such as rapidly growing versus nongrowing cells, transformed versus normal cells, and during specific growth regulatory events. However, the problems of such quantitation are, in general, 2-fold. First the amounts of the sugars in the various pools along pathways of utilization are very low, requiring microdetermination in quantities of cells feasible for experimental manipulations; this point is made clear in Table I, where it is seen that total bound monosaccharide in various animal cells is in the nanomolar range per 106 cells. The pools preceding the macromolecular bound sugar, especially the nucleotide sugar pools, are even smaller in quantity under usual culturing conditions, i.e., with micromolar or smaller quantities of a particular hexose in the medium. The nucleotide-sugar pool in many different cell types is usually in the order of one tenth the size of the macromolecular pool (Tables I and II). Several studies have shown that this is the most significant soluble pool ' J. E. G. Barnett and D. L. Corina, Adv. Carbohydr. Chem. Biochem. 27, 127 (1972). 2 p. M. Kraemer, in "'Biomembmnes" (L. A. Manson, ed.), Vol. I. p. 67. Plenum, New York, 1971. 3 S. Roseman, Chem. Physics Lipids 5, 270 (1970). 4 L. Warren, in "Glycoproteins. Their Composition, Structure and Function, Part B" (A. Gottschalk, ed.), p. 1097. Elsevier, Amsterdam, 1972. V. Ginsburg, Adv. Enzymol. 26, 35 (1964). 6 L. F. Leloir, Biochem. J. 91, 1 (1964). r S. Kornfeld, R. Kornfeld, and V. Ginsburg, Arch. Biochem. Biophys. 110, 1 (1965). 8 p. D. Yurchenco and P. H. Atkinson, Biochemistry 16, 944 (1977).

TABLE I QUANTITIES OF TYPICAL MONOSACCHAR1DE COMPONENTS OF ANIMAL CELL

Sugar Glucose Galactose

N-Acetylglucosamine

N-Acetylgalactosamine

Mannose

Fucose

Sialic acid

Cell type H e L a $3 HeLa 65 H e L a $3 Human colonic mucosa L cells HeLa $3 Human colonic mucosa L cells 3T3 HeLa Sa Human colonic mucosa L cells 3T3 H e L a 65 HeLa $3 Human colonic mucosa L cells HeLa $3 HeLa $3 Human colonic mucosa L cells BHK21/Ct3 (growing) H e L a 65 HeLa Sa Human colonic mucosa L cells 3T3 BHK21/C13 (growing) BHK~l/C13 (transformed) Normal rat liver Morris hepatoma 7777

Nmoles per l0 e cells (or per mg protein*) 2.8 2.8 3.3 79.6* 3.3 4.0 a 86.2* 5.8 b 35.9* 1.1 a 59.7* 5.8 b 3.7* 4.8 2.3 32.0* 5.3 0.6-0.8 0.7 40.5* 0.6 0.79, 0.49 2.2 1.5 70.3* 0.9 7.6* 2.7 3.5 4.50 7.59

Reference c d c e f c e f g c e f g d c e f h c e f

i,k d c e f *g i k j j

a Determined as its amino sugar. o Total hexosamines. c L. Shen and V. Ginsburg, Arch. Biochem. Biophys. 122, 474 (1967). a S.-H. Tu, R. E. Nordquist, and M. J. Griffin, Biochim. Biophys. Acta 290, 92 (1972). e y . S. Kim and R. Isaacs, Cancer Res. 35, 2092 (1975). f M. C. Glick, C. Comstock, and L. Warren, Biochim. Biophys. Acta 219, 290 (1970). g J. Pouyssegur and I. Pastan, J. Biol. Chem. 252, 1639 (1977). h p. D. Yurchenco and P. H. Atkinson, Biochemistry 14, 3107 (1975). M. C. Glick and C. A. Buck, Biochemistry 12, 85 (1973). J E. Harms, W. Kreisel, H. P. Morris, and W. Reuter, Eur. J. Biochem. 32, 254 (1974). k C. A. Buck, M. C. Glick, and L. Warren, Biochemistry 9, 4567 (1970).

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FRACTION NUMBER FIG. 1. Fucose-soluble intermediates. HeLa cells were labeled with L-[1,5,6-aH]fucose for 24 hr. Cells were harvested, extracted with ethanol, and centrifuged to remove insoluble material. The extract (O) was mixed with authentic GDP-l-['4Clfucose (©), and chromatographed overnight on paper with solvent I. Strips (l cm) were assayed for radioactivity. The positions of fucose phosphate [H. S. Prihar and E. J. Behrman, Biochemistry 12, 997 (1973)], fuconic acid, and fucose were determined from average Rr values of several chromatograms, and their respective positions are marked with arrows from left to right. Reprinted with permission from P. D. Yurchenko and P. H. Atkinson, [Biochemistry 14, 3107 (1975)]. Copyright by the American Chemical Society. in g l u c o s a m i n e l a b e l i n g 9-1'a galactose l a b e l i n g 12-'5 f u c o s e l a b e l i n g 1~-2° ( F i g . 1), a l t h o u g h in m a n y c a s e s t r a c e s o f s u g a r a n d s u g a r p h o s p h a t e c a n b e d e t e c t e d , w h i c h a r e r a p i d l y e q u i l i b r a t e d ( w i t h i n 6 m i n ) in g l u c o s a m i n e G. A. Hayden, G. M. Crowley, and G. A. Jamieson, J. Biol. Chem. 245, 5827 (1970). ,0 j. Pouyssegur and I. Pastan, J. Biol. Chem. 252, 1639 (1977). '~ J. J. Kim and H. E. Conrad, J. Biol. Chem. 251, 6210 (1976). ,la j. G. Bekesi and R. J. Winzler, J. Biol. Chem. 244, 5663 (1969). ,2 H. M. Kalckar, D. Ullrey, S. Kijomoto, and S. Hakomori, Proc, Natl. Acad. Sci. U.S.A. 70, 839 (1973). ,3 C. W. Christopher, W. W. Colby, and D. Ullrey, J. Cell. Physiol. 89, 683 (1976). ~4H. M. Kalckar and D. Ulirey, Proc. Natl. Acad. Sci. U.S.A. 70, 2502 (1973). ,5 D. Ullrey, M. T. Gammon, and H. M. Kalckar, Arch. Bioehem. Biophys. 167,410 (1975). ,9 p. D. Yurchenco and P. H. Atkinson, Biochemistry 14, 3107 (1975). ,7 R. A. Novak and C. W. Abell, Tex. Rep. Biol. Med. 34, 199 (1976). ,8 A. Tenner, J. Zeig, and I. E. Scheffler, J. Cell. Physiol. 90, 145 (1977). ,9 R. L. Kaufman and V. Ginsburg, Exp. Cell Res. 50, 127 (1968). 20 j. G. Bekesi and R. J. Winzler, J. Biol. Chem. 242, 3873 (1967).

[17]

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labeling. 21 Glucosamine and mannose labeling are further complicated, since in several calf tissues incubated with radioactive monosaccharides (e.g., liver, kidney, thyroid), lipid-saccharide intermediates in glycoprotein biosynthesis have been observed, 22 although these compounds are usually macromolecular and do not fall under the above definition of "soluble" intermediates. Abbreviations: ATP, adenosine 5'-triphosphate; ADP, adenosine 5'-diphosphate; AMP, adenosine 5'-monophosphate; GTP, guanosine 5'-triphosphate; GDP, guanosine 5'-disphosphate; UDP, uridine 5'-diphosphate; Pi, inorganic phosphate; F-l-P, L-fucose 1-phosphate; GlcNAc, N-acetylglucosamine; GalNAc, N-acetyl-o-galactosamine; NANA, N-acetylneuraminic acid; MEM, Eagle's minimal essential medium, Joklik modified for spinner culture; TLC, thin-layer chromatography; HVPE, high-voltage paper electrophoresis. For abbreviations for monosaccharides, see Fig. 5. Handling Labeled Monosaccharide in Incorporation Studies

Tritiated monosaccharides are often used in the range of 1 to 20 ~Ci/ml of medium when labeling cultured cells, though up to 500 ~Ci/ml of 3H mannose have been used in some studies 22" with no apparent adverse effect. In general, because l~-labeled monosaccharides can be more efficiently counted in the scintillation spectrometer, less isotope need be used and a useful range is from 0.05-1.0 ~Ci/ml the exact concentration depending on the length of labeling time. If suspension cultures are used, additional efficiency of utilization can be obtained by concentrating cells above (5 to 10 times) their normal growth concentration. However, this procedure should be used only for brief labeling periods because synthetic processes (e.g., protein synthesis) may soon drop in rate under these conditions as essential nutrients (amino acids) are more rapidly used up. In our experience with HeLa $3 cells and human diploid fibroblasts, radioactive glucosamine, and mannose, and fucose can be utilized for periods of up to 24 hours with only minimal relocation of label into other compounds. However, the extent of conversion depends on the cell type (Table III). The labeled monosaccharides are best added to the experimental cultures in sterile saline and if possible should be ordered (for a small packaging surcharge) from the vendor that way. Adding these compounds in ethanol is not recommended because of the latter's toxic properties. Aliquots of the culture, sampled in order to ~1 j. F. McGarrahan and F. Maley, J. Biol. Chem. 237, 2458 (1962). 22 M. J. Spiro, R. G. Spiro, and V. B. Bhoyroo, J. Biol. Chem. 251, 6420 (1976). z2a B. M. Sefton, Cell 10,659 (1977).

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follow the course of uptake, should either be extensively washed in balanced salt solution prior to assay for macromolecular material or even homogenized to reduce experimental scatter and to obtain a zero incorporation at zero labeling time.

Effect on Incorporation Hexose Concentration in the External Medium Where it was possible in Table II, we have noted the glucose and labeling monosaccharide concentration in the medium since these quantities can affect both the rate of utilization of label into macromolecules and determine, in some cases, which monosaccharide in the macromolecular product becomes labeled (cf. Table III). In the case of utilization of radioactive glucosamine, it is the relative contributions of fructose-6-P and exogenous (radioactive) glucosamine to the glucosamine-6-P pool that determine the ultimate specific radioactivity of the UDP-N-acetyl (radioactive) hexosamines formed from the labeled glucosamine; these relative contributions are in part regulated by the relative concentrations of glucose and glucosamine (in the medium) so that the contribution of fructose-6-P to the glucosamine-6-P decreases as the level of glucose ifi the medium falls," even though the UDP-GIcNAc pool remains constant, as might be expected. 23 In the case of utilization of radioactivefucose it is relative contributions of fucose-l-P derived from fucose in the medium and GDP-mannose 24 that determine the ultimate specific radioactivity of the GDP-fucose; when the fucose is in micromolar concentration in the medium of HeLa cells the relative contributions are 1 part from F-1-P to 12 parts from the endogenous source, s,'6 GDP-mannose. z4 However, this ratio can change depending on the medium concentration of fucose TM (Table II), a point worth emphasizing since HeLa cells, at least, secrete about 3 times as much free fucose as they "scavenge" from the medium (see Fig. 7). It follows that in a closed system, such as a culture dish/bottle, an increasing concentration of fucose in the medium leads to an increased GDP-fucose pool--altering its equilibration rate with the labeled exogenous monosaccharide (Fig. 2). These several considerations show that the specific radioactivity of labeled monosaccharide can be diluted in several different ways prior to its incorporation into macromolecular product. Quantitation of the total flow of monosaccharide from the flow of isotope alone is thus not possible without knowing these dilutions. Radioactivity can also be converted to monosaccharides other than in 23 S. Kornfeld, R. Kornfeld. E. F. Neufeld, and P. J. O'Brien, Proc. Natl. Acad. Sci. U.S.A. 52, 371 (1964). ~4 D. W. Foster and V. Ginsburg, Biochim. Biophys. Acta 54, 376 (1961).

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LABELING TIME, HOURS FIG. 2. [3]Fucose label equilibration of fucosyl glycoprotein pool when growth medium is periodically renewed. HeLa cells resuspended at a density of 4.1 × 105/ml were labeled with 1/zCi of L-[3H]fucose per milliliter, and, at the times indicated, 1-ml aliquots were removed. About every 24 hr (indicated by arrows), the cells were pelleted by centrifugation and resuspended in fresh growth medium (from a single stock) labeled with L-[%I]fucose. Trichloroacetic acid-precipitable radioactivity per 107 cells is plotted as a function of labeling time. Dashed lines (- - -) indicate the expected equilibration behavior if growth medium were not renewed. Reprinted with permission from P. D. Yurchenco and P. H. Atkinson [Biochemistry 16, 944 (1977)]. Copyright by the American Chemical Society.

the input monosaccharide, as part of the carbon backbone is utilized in the synthesis of other monosaccharides, which finally become incorporated into complex carbohydrates (Table III). The extent of conversion varies greatly from sugar to sugar as well as from cell to cell, and the location of the input radioactivity should always be checked (by hydrolysis and chromatography, see below) to aid in interpretation of results. It is possible to manipulate labeling conditions so that the specific radioactivity of the nucleotide sugar pool is rapidly diluted or "chased" by excess unlabeled monosaccharide placed in the medium. In order to achieve this, it is necessary that the final concentration of monosaccharide should be above the Km for the process of uptake/utilization into macromolecules for those cells. This point is discussed in attempts to chase label in NIL, BHK, and 3T3 cells with [14C]sialic acid, 25 labeling of human diploid lung fibroblasts with [aH]fucose, 26 [14Clgalactose 23 C. B. Hirschberg, S. B. Goodman, and C. Green, Biochemistry 15, 3591 (1976). 26 C. Ceccarini and P. H. Atkinson, unpublished data; it was necessary to dilute 1 /.tM [3I-Ilfucose in the medium approximately a thousandfold before labeling ceased. The relationship between dilution of label (addition of fucose) and amount of labeling was a straight line on a Lineweaver-Burk plot.

186

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FIG. 3. Concentration of unlabeled fucose in the medium necessary to inhibit further incorporation of radioactive fucose. Cells at a density of 6 × 1P/ml were labeled with 10 p,Ci of [1,5,6-aH]fucose per milliliter (concentration approximately 2 /,d,/). At various intervals, aliquots (2 × 0.5 ml) were pipetted into 2.0 ml of ice-cold Earle's solution and centrifuged to a pellet. The cells were lysed by resuspension in 1.0 mi of H20, and 1.0 ml of 10% trichloroacetic acid was added. The percipitate was collected on Whatman glass filters (GFA), washed several times with ice cold 5% TCA and 95% ethanol, and the radioactivity was counted in a scintillation spectrometer. At 60 min of labeling (arrow) various portions of the culture were adjusted to 0.02 mM (A---A), 2 mM (O---©), or 40 mM (A---A) unlabeled fucose. One portion contained only radioactive fucose ( O - - Q , 2/zM). Reprinted with permission from P. H. Atkinson [J. Biol. Chem. 250, 2123 (1975)]. Copyright by the American Society of Biological Chemists, Inc.

labeling of B H K cells, z7 and [~I]fucose labeling of HeLa cells 28 (see Fig. 3) by addition of large quantities of unlabeled monosaccharide to media containing micromolar quantities of radioa.ctive precursor. The latter conclusion for fucose labeling is predictable from the work of Kaufman and Ginsburg, TM where it can be seen that adding up to millimolar concentrations of fucose to the culture medium results in continued and massive (over thousands-fold) expansion of the GPDfucose (Table II). The size of the GDP-fucose pool may be rate limiting for fucosylation of glycoproteins in HeLa cells, providing an explanation for the nonlinear relationship between the dilution of labeled sugar in the medium and the "dilution" of its incorporation into glycoprotein (for an example of this, see Fig. 3). Thus, it is necessary to dilute labeled fucose in the medium several thousandfold (i.e., into a millimolar range) before one observes cessation of labeling of glycoprotein--a situation that is 2r W. Deppert, H. Werchau, and G. Walter, Proc. Natl. Acad. Sci. U.S.A. 71, 3068 (1974). 28 p. H. Atkinson, J. Biol. Chem. 250, 2123 (1975).

[17]

ANIMAL CELLS WITH MONOSACCHARIDES

187

quite different for chasing amino acids, where a 10-fold dilution is usually sufficient. Effect on Incorporation by Inhibition o f Glycoprotein Biosynthesis

Inhibitors of protein synthesis (puromycin and cycloheximide) also block the incorporation of sugar precursors into glycoproteins; however, the time characteristics of the onset o f inhibition depends on the sugar used. Mannose and internal glucosamine are added while the polypeptide is still associated with the polyribosomes on the rough endoplasmic reticulum. 29-a° In general, addition of inhibitors of protein synthesis rapidly suppresses the incorporation of these two sugars. 3°'31"33'41 Other sugars which are normally associated with plasma-type glycoproteins, 42 such as distally located glucosamine, galactose (also terminally located fucose), and sialic acid appear to be added to the already completed polypeptides when these are processed through the smooth endoplasmic reticulum, the Golgi apparatus, and the associated endoplasmic reticulum. 29-31,33-37,40,42a'42b Inhibitors of protein synthesis do not immediately affect the incorporation of sugars located at sites more distal to the protein carbohydrate linkage, 3°'3a and therefore the appearance of newly labeled glycoproteins continues for considerable time and may continue to be transferred to their final cellular site of accumulation. 28'43 The total internal pool of glycosylated proteins is often large,~S'34'a5 though the pool of fucosylated s or sialylated 43 glycoproteins can be comparatively small. F r o m these collected data, we conclude that the size and rate of turnover of pools of acceptors and of donors for various monosaccharide precursors must be considered in interpretation of results obtained by use of inhibitors of synthesis of protein and carbohydrate chains: the rate of depletion of acceptor pools after inhibition will be different for different 29j. Molnar, G. B. Robinson, and R. J. Winzler, J. Biol. Chem. 240, 1882 (1965). 3oG. R. Lawford and H. S. Schachter, J. Biol. Chem. 241, 5408 (1966). 31j. Molnar and D. Sy, Biochemistry 6, 1941 (1967). 3zT. Hallinan, C. N. Murty, and J. H. Grant, Arch. Biochem. Biophys. 125, 715 (1968). 33p. Whur, A. Herscovics, and C. P. Leblond, J. Cell Biol. 43, 289 (1969). 34F. Melchers, Biochemistry 10, 653 (1971). 3.5y. S. Choi, P. M. Knopf, and E. S. Lennox, Biochemistry 10, 668 (1971). 36R. R. Wagner and M. A. Cynkin, J. Biol. Chem. 246, 143 (1971). 37C. M. Redman and M. G. Cherian, J. Cell Biol. 52, 231 (1972). 38D. Banerjee, C. P. Manning, and C. M. Redman, J. Biol. Chem. 251, 3887 (1976). 39M. L. Kiely, G. S. McKnight, and R. T. Schimke, J. Biol. Chem. 251, 5490 (1976). 40F. Melchers, Biochemistry, 12, 1471 (1973). 41j. M. Todd and M. H. Samli, Biochim. Biophys. Acta 297, 11 (1973). 42R. G. Spiro, Adv. Prot. Chem. 27, 349 (1973). 42, M. Neutra and C. P. Leblond, J. Cell Biol. 30, 137 (1966). 4~bM. A. Moscarello, L. Koshuba, and J. M. Sturgess, FEBS Lett. 26, 87 (1972). 43 p. M. Kraemer, J. Cell. Physiol. 69, 199 (1967).

188

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[17]

labeled monosaccharides; the rate of depletion of the rate-limiting pool donating labeled monosaccharide will also be different for different monosaccharides and may also depend on the composition of the medium of the cell milieu (Table II). Several nonmetabolizable sugars are known to interfere with the addition of sugars to polypeptides. 2-Deoxy-D-glucose (DOG) at concentrations ranging from 0.5 to 20 raM, has been shown to interfere with the incorporation of radioactive sugars into viral glycoproteins, 44-47 the expression of viral function 45'46'48"4aand the intracellular translocation, but not the secretion, of IgG molecules from tumor plasma cells. 4° Reports have been published indicating the DOG can be directly incorporated into cellular oligosaccharides. 5°'~' Washing the cells free of DOG or adding mannose (0.5-5 mM) will, in several cases, reverse the inhibitory action of 2-deoxyglucose. 46-48 However, other sugars, glucose, fructose, or fucose, do not relieve the inhibition. 45 Fluoro-sugar analogs also suppress in vivo glycoprotein biosynthesis without affecting amino acid uptake and cell viability. ~22-Deoxy-2-fluoro-D-glucose has been shown to inhibit the conversion of N-acetylglucosamine and N-acetylmannosamine to anabolic intermediates in vitro cell extracts. 53 Acetylated sugar analogs have been recently tested and appear to inhibit both glucosamine and leucine incorporation into glycoproteins; however, some proved to be toxic to P-288 murine lymphoma cells.~2 The antibiotic tunicamycin za is another glycoprotein inhibitor which appears in vitro, ~5'5~and possibly in vivo, 37to block the assembly of sugarlinked polyisoprenol intermediates. ~6 Thus, tunicamycin will probably prove to be a very useful specific oligosaccharide assembly inhibitor when it becomes commercially available. Finally, the release of serum glycoprotein 38 and biosynthesis of viral glycoproteins s8 are inhibited by 44 H. D. Klenk, C. Schoetissek, and R. Rott, Virology 49, 723 (1972). ~5 S. S. Gandhi, P. Stanley, J. M. Taylor, and D. D. White, Microbios 5, 41 (1972). 4o R. W. Knowles and S. Person, J. Virol. 18, 644 (1976). 47 M. F. G. Schmidt, R, T. Schwarz, and C. Scholtissek, Eur. J. Biochem. 70, 55 (1976). 49 E. A. Havell, J. Vilcek, E. Falcoff, and B. Berman, Virology 63, 475 (1975). 49 G. Kaluza, C. Scholtissek, and R. Rott, J. Gen. Virol. 14, 251 (1972). 5o M. R. Steiner, K. Somers, and S. Steiner, Biochem. Biophys. Res. Commun. "61, 795 (1974). 5, M. W. Myers and A. C. Sartorelli, Biochem. Biophys. Res. Commun. 63, 164 (1975). .~2R. J. Bernacki, M. Sharma, N. K. Porter, Y. Rustum, and W, Korytnyk, J. Supramol. Struct. 7, in press (1977). 53 A. M. Schultz and P. T. Mora, Carbohydr. Res. 40, 119 (1975). 54 A. Takatsuki and G. Tamura, J. Antibiot. 24, 785 (1971). ~ A. Takatsuki, K. Kohno, and G. Tamura, Agric. Biol. Chem. 39, 2089 (1975). oo j. S. Tkacz and J. O. Lampen, Biochem. Biophys. Res. Commun. 65, 248 (1975). 57 S.-C. Kuo and J. O. Lampen, Arch. Biochem. Biophys. 172, 574 (1976). s~ R. D. Dix and R..1. Courtney, Virology 70, 127 (1976).

[17]

ANIMAL CELLS WITH MONOSACCHARIDES

189

drugs, colchicine and cytochalasin B, that disrupt microtubules and microfilaments. The uptake of labeled monosaccharides appears to be inhibited by these drugs. 59

Effect of pH on the Incorporation of Labeled Monosaccharides The pH of the growth medium determines the growth behavior of cells in tissue culture 6°-nz and the incorporation of some radioactive sugars into macromolecular material has been studied at various pH levels. Fucose incorporation was maximum at the optimum pH for growth in a human diploid fibroblast cell, 6a and galactose incorporation in normal human foreskin fibroblasts showed two optimum peaks, pH 7.0 and 8.5. Galactosemic human cells, incorporated galactose optimally, albeit at lower rates, at pH 8.0 64Fucose incorporation was suppressed in cellular, but not in released, glycopeptides in cultures whose pH was maintained with organic buffers. 65 To our knowledge there are no studies on the effect of the external pH on the endogenous nucleotide-sugar pools in animal cell systems in tissue culture.

Utilization, Growth Effects, and Toxicity of Naturally Occurring Monosaccharides In consideration of the various experimental approaches, such as chasing labeled monosaccharides (see above) or labeling in the presence of millimolar quantities of monosaccharide 66 rather than the more usual micromolar quantities, the physiologic effects of the various concentrations should be considered. D-Glucose is a necessary growth factor for cultured cells; 67 and, although other sugars can be substituted, 68-73 59 C. W. Christopher, D. Ullrey, W. Colby, and H. M. Kalckar, Proc. Natl. Acad. Sci. U.S.A. 73, 2429 (1976). 60 C. Ceccarini and H. Eagle, Proc. Natl. Acad. Sci. U.S.A. 68,229 (1971). 61 H. Rubin, J. Ceil Biol. 51, 686 (1971). 62 H. Eagle, J. Cell. Physiol. 82, 11 (1973). 63 C. Ceccarini, In Vitro 11, 78 (1975). 64 H. Z. Hill, J. Cell Physiol. 86, 313 (1976). 6~ p. F. Daniel and G. Wolf, In Vitro 11,347 (1975). 6n M. J. Spiro, R. G. Spiro, and V. D. Bhoyroo, J. Biol. Chem. 251, 6400 (1976). 67 H. Eagle, Science 122, 501 (1955). ~8 R. S. Chang and R. P. Geyer, Proc. Soc. Exp. Biol. Med. 96, 135 (1957). n9 H. Eagle, S. Barban, M. Levy, and H. D. Schulze, J. Biol. Chem, 233,551 (1958). 7oj. Paul, in "Cells and Tissues in Culture" (E. N. Willmer, ed.), Vol. 1, p. 239. Academic Press, New York, 1965. 71 j. G. Rheinwald and H. Green, Cell 2, 287 (1974). 72 R. L. Bums, P. G. Rosenberger, and R. J. Klebe, J. Cell. Physiol. 88, 307 (1976). 7z G. S. Johnson and J. P. Schwartz, Exp. Cell Res. 97,281 (1976).

190

PREPARATIONS

[17]

several are toxic or inhibitory, both in vivo and in vitro. 19,73--77L-Glucose is inhibitory at 5.5 mM, and D - t a g a t o s e , a stereoisomer of D - g l u c o s e , is toxic. 72D-Galactose can replace glucose for some cell lines,'2'68'73 and the presence of pyruvate stimulates its utilization, but not for all cells. 69"72"78 The human fibroblast cell (WI38) can utilize galactose if the pH of the growth medium is lowered below pH 7. 79 In the presence of glucose, galactose (5.5 mM) induced alterations of cell surface morphology 'z's° as well as accessibility to surface macromolecules. 81 In general, amino sugars are either inhibitory and/or toxic to animal cells. D-Glucosamine was first shown to inhibit the growth of sarcoma 27 tumors in mice; TM HeLa cells begin to lyse after 24--48 hr in the presence of 2 mM glucosamine; 7~and Ehrlich ascites tumor cells were killed after exposure to 0.7 mM glucosamine for only 4 hr; 77 cell fusion in herpes simplexinfected cells is inhibited at 10 mM. 46 At relatively low concentrations, 0.35 mM, glucosamine can inhibit the incorporation of radioactive precursors in proteins and nucleic acids in ascites cells, s~ However, mouse fibroblast cells require higher levels of this sugar to inhibit these cellular processes.S3 This amino sugar also inhibits interferon production, viral development, and infectivity.44'4~'a8 Other studies have shown that glucosamine strikingly decreased the uridine nucleotide pool 77,83 and increased the UDP-N-acetylhexosamine pool."a'75"83It would appear that these effects are more pronounced in tumor cells than in normal cells. Other amino sugars, D-mannosamine and D-galactosamine, have been reported to be toxic to neoplastic cells and to inhibit protein and nucleic acid synthesis at concentrations of 0.7 raM. 77,82D-Mannose can substitute for glucose in numerous cells. 88-7°,72'84 However, at high concentrations, 69.4 mM, mannose was found to inhibit the growth of BSC-I monkey cells.76In contrast, Ehrlich ascites cells lose their viability and do not form tumors at relatively low doses of mannose (0.7 mM), and in these cells it also inhibited protein and nucleic acid biosynthesis. 77,83 Mannose at 5 raM, and in the presence of glucose, induced surface alterations, in L cells, leading to a loss of microvilli TM without interfering with cell growth. L-Fucose was found to be toxic to at least 5 mammalian cells. 7~However, r4 j. H. Quastel and A. Cantero, Nature (London) 171,252 (1953). 75 S. Kornfeld and V. Ginsburg, Exp. Cell Res. 41,592 (1966). 76 R. P. Cox and B. M. Gessner, CancerRes. 28, 1162 (1968). 77 j. G. Bekesi, Z. Molnar, and R. J. Winzler, Cancer Res. 29, 353 (1969). 78 C. L. Baugh, J. M. Fitzgerald, and A. A. Tytell, J. Cell. Physiol. 69, 259 (1967). 79 C. L. Baugh, J. M. Fitzgerald, and A. A. Tytell, J. Cell. Physiol. 70, 225 (1968). 8o H. Amos, M. Leventhal, L. Chu, and M. J, Karnovsky, Cell 7, 97 (1976). 8, C. G. Gahmberg and S.-I. Hakomori, Proc. Natl. Acad. Sci. U.S.A. 70, 3329 (1973). 82 j. G. Bekesi, E. Bekesi, and R. J. Winzler, J. Biol. Chem. 244, 3766 (1969). 83 H. B. Bosmann, Biochim. Biophys. Acta 240, 74 (1971). 84 p. Faik and M. J. Morgan, Biochem. Soc. Trans. 4, 1043 (1976).

[17]

A N I M A L CELLS W I T H

MONOSACCHARIDES

191

other workers have found fucose to be growth inhibitory, but only at relatively high doses. ,9.76.85 Some of the inhibitory and/or toxic sugars have been used as radioactive sugar precursors for glycoprotein biosynthesis. Addition of large quantities of these sugars to chase radioactively labeled glycoproteins must be controlled to ensure that the disturbance of cell growth, but rather by a decrease of the specific activity of the internal pool, as labeled molecules are diluted by newly synthesized molecules.

Quantitation of Biosynthesis of Complex Carbohydrates We had mentioned previously that to obtain absolute pool size data and flow rates microdetermination methods are necessary (Table IV). We have published a method that is well suited to obtaining quantitative flow data of fucose containing molecules, and the following is a description of some of the practical details employed in its use. It was necessary to use 3H-labeled sugar in these studies for reasons of cost when high concentrations were needed in short-term labeling studies and also because of difficulties in distinguishing 32p radiation and '4C radiation if the latter isotope was used to label fucose. In this regard [6-3H]fucose was the best label, both because of the high specific activity when obtained from New England Nuclear Corporation and also because tritium in the 1 position was unstable in the presence of a pig liver fucose kinase preparation.

Principle of Microassay of Fucose- and [3HWucose-ContainingPools GDP-fucose is extracted and purified from cells labeled with [3H]fucose and then hydrolyzed to yield fucose, which is further purified through Sephadex-gel filtration and chromatographic steps. The remaining insoluble cell material is hydrolyzed to release fucose from macromolecular material and is then purified from other contaminating sugars. It is necessary to quantitate the GDP-fucose pool by purifying the monosaccharide because we were unable to effectively separate GDP-fucose from other neutral sugar nucleotides. L-Fucose kinase is used to transfer a terminal radioactive phosphate from [y-'~2P]ATP to [aH]fucose according to the equation: ['~H]fucose + [ y - 32P]ATP

fucose kinase ) [3H]fucose l - ['~zP]phosphate + ADP

Since the specific activity of the [32P]ATP is known, the quantity of fucose converted can be determined. From this, the specific radioactivity (cpm/mol) of [3H]fucose-labeled GDP-fucose and glycoprotein fucose pools in the cells may be calculated. The recovery of [3H]fucose is 85 p. H. Atkinson, Methods Cell Biol. 7, 157 0973).

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194

PREPARATIONS

[ 17]

determined by the recovery of a known quantity of [14C]fucose. This, in conjunction with specific radioactivity measurement, is used to determine the pool size. This assay, and its uses, we have described in detail previously, s,16

Chromatography and High-Voltage Paper Electrophoresis Paper Chromatography. Descending paper (Whatman, 3 MM) chromatography was carried out with: solvent I: 95% ethanol : 1.0 M ammonium acetate, 7: 386; solvent II: isobutyric acid : ammonium hydroxide (28-30%), 5 : 3; solvent III: pyridine : ethyl acetate : water, 1.0 : 3.6: 1.15, upper organic phase. 87 High-voltage paper electrophoresis was carried out on 4 cm-wide strips of Whatman 3 MM paper in solvent IV: pyridine : acetic acid : water, 1 : I0 : 89, pH 3.6. Air-dried paper strips from either chromatography or HVPE, when analyzed for radioactivity, were cut into 1-cm or 0.25-inch fractions and eluted with 1.0 ml of water in a tube; aliquots (10-50/zl) were counted. Material pooled for further analysis was passed through a Swinnex 0.22 /~m (Millipore, Massachusetts) filter to remove suspended matter. Some of the characteristics of the paper chromatography systems we found useful in our approach are tabulated below. All paper systems used Whatman 3 MM paper (57 cm long) by descending migration. Rsuco,evalues are averaged values from several runs. Unlabeled nucleotides and nucleotide sugars are located on paper chromatograms by UV absorption. Inorganic phosphate and pyrophosphate are located by an ammonium molybdate spray reagent 88 which is prepared by dissolving 5 g of ammonium molybdate in 100 ml of 2 N H2SO4. After spraying, the paper strip is dried and further sprayed with a 5% aqueous solution of sodium sulfite. Phosphate appears with a blue color. After a day, however, the paper strip weakens and crumbles owing to the corrosive action of HzSO4; 1 #mol of phosphate marker is generally used. Unlabeled fucose phosphate (this compound is not commercially available and was a generous gift of Dr. H. S. Prihar, Department of Biochemistry, Ohio State University, Columbus, Ohio) is detected by spraying the horizontally hung chromatogram with a solution of alkaline phosphatase (Worthington, New Jersey, 0.02 mg/ml, 34 units/mg, in 20 mM Tris-HCl, pH 8.2) and incubating the paper horizontally in a humidified tissue culture incubator at 37° for 25 min. The paper is then airdried and stained with AgNO3 reagent as described above. 86 A. C. Paladini and L. F. Leloir, Biochem. J. 51,426 (1952). 8r p. Colombo, D. Corbetta, A. Pirotta, G. Ruflini, and A. Sartori, J. Chromatogr. 3, 343

(1%0). s8 B. Kakfi¢, in "Paper Chromatography" (I. M. Hais and K. Maeek, eds.), p. 772. Publ. House Czech. Aead. Sci., Prague, 1963.

[17]

193

ANIMAL CELLS WITH MONOSACCHARIDES

1. Solvent I:

95% ethanol: 1.0Mammonium acetate, 7: 388; development time: 20-23 hr Comments: Useful for separation of GDP-L-fucose, L-fucose 1phosphate, L-fuconic acid, and L-fucose. Adenine compounds are separated also. Fucose migrates about 38 cm. Substance

GDP-L-fucose L-Fucose I-phosphate L-Fuconic acid L-Fucose

R sucose

0.29 0.45 0.77 1.0

Substance

ATP ADP AMP

Inorganic phosphate

R sucose

0.07 0.08 0.18 0.30

2. Solvent II: isobutyric acid:ammonium hydroxide (28-30%), 5:32°; development time: 17 hr Comments: Does not efficiently separate nucleotide sugars from each other, but serves well to separate the free sugar from the nucleotide sugar. Substance

L-Fucose GDP-L-fucose

R fueo~e

0.56 0.26

Substance

R ruco~e

GDP-o-mannose GDP-D-glucose

0.20 0.22

3. Solvent III: pyridine:ethyl acetate:water, 1.0:3.6:1.15, upper organic phaseST; development time: 18-22 hr Comments: Useful for separating monosaccharides in relatively pure mixtures; sensitive to presence of salt; of sugars tested, only xylose migrates similar to fucose. Fucose migrates about 38 cm. Substance

L-Fucose

o-Mannose D-Glucose

L-Rhamnose o-Gal ac tose D-Ribose DL-Arabinose D-Fructose

N-Acetyl-D-glucosamine

R fucose

Substance

1.0 0.72 0.51 1.5 0.46 1.4 0.83 0.77 0.82

N-Acetyl-o-galactosamine N-Acetylneuraminic acid D-Glucosamine

D-Galactosamine D-Xylose L-Fucose l-phosphate ATP, ADP, AMP

Inorganic phosphate

R

fucose

0.72 0.58 0.29 0.22 1.03 0 0 0

196

PREPARATIONS

[ 17]

Thin-Layer Chromatography. Ascending thin-layer chromatography is carried out as follows: Kieselguhr G (Merck, Germany) is mixed for 1.5 min with 2.5 volumes of 0.15 M-NaH2PO4 and carefully spread 300/xm thick onto acid- and detergent-cleaned 20 x 20-cm glass plates on a flat surface. The spreader (DESAGA, Brinkman, New York) is moved across 4-5 plates in a single motion. The plates are allowed to dry undisturbed for several hours and then are further dried in horizontally placed racks and stored in a cool place in a vacuum desiccator. Samples are banded 10 /xl at a time 1-1.5 cm from one edge of the glass. The plates are then developed with ethyl acetate : methanol : 1-butanol : water, 16 : 3 : 3 : 2 (TLC System 189) until the solvent front ascends to ,the opposite end (60-70 min). Commercially spread Silica Gel G plates, 250 ~m thick (Analtech, Pittsburg, Pennsylvania)are impregnated with 0.03 M H3BO3 by ascending chromatography and allowed to dry as described above. These plates are activated in a 100° oven for 1-h prior to use and, after banding of samples, are developed with I-butanol : acetone : water, 4 : 5 : 1 (TLC System 119°). Air-dried plates are scraped in 3 cm × 0.25-inch (the latter in the direction of solvent flow) fractions and eluted with 1.0 ml water; 10-50-/xl aliquots are counted. For further analysis, remaining pooled fractions are passed through a Swinnex filter (Millipore) and desalted on Amberlite MB-1. The remaining scrapings are eluted with another 0.5 ml of water and passed through the same filter and Amberlite, and both eluents are combined. Sugar standards on paper strips are stained by the AgNO3 method 9' and are located on TLC plates either by spraying with AgNO3 reagent or with naphthoresorcinol. 9° For spraying with AgNO3 reagent, the three solutions [(A) 1 ml of saturated AgNO3 in 200 ml of acetone; (B) 10 ml of 10 N NaOH 190 ml of 95% ethanol; (C) 6 N NH4OH] were sprayed in sequence onto the dry plates with a few minutes of developing time between A and B. The position of marker sugars (25-50 tzg each) should be noted shortly after spraying, as a background blackening occurred within a few hours. Naphthoresorcinol reagent was prepared immediately prior to use (100 mg of naphthoresorcinol in 100 ml of 95% ethanol containing 1 ml of 36 N H2SO4). After spraying, the plates are incubated at 100° for about 10-15 min to develop the blue or violet color. Plates are photographed or otherwise recorded within 12 hr since a pink background developed over the course of several days. Ion-exchange chromatography for desalting is performed in a Pasteur pipette (8.7 cm × 0.5 cm) plugged at the bottom with glass wool and filled 89 M. Q.-K. Talukder, J. Chromatogr. 57, 391 (1971). 9o M. Lato, B. Brunelli, and G. Cuiffini, J. Chromatogr. 34, 26 (1968). 91 W. E. Trevelyan, D. P. Frocter, and J. S. Harrison, Nature (London) 166, 444 (1950).

[17]

ANIMAL CELLS WITH MONOSACCHARIDES

197

with dry Amberlite MB-1 mixed ion-exchange resin (1.7 ml bed volume) equilibrated with distilled water. For large volumes, a 1.5 × 20-cm column is used. Samples are passed through the column and eluted with 2.5 ml of water when a small column is used, or with up to 50 ml with the large column. Conditions for Sephadex chromatography are (a) Sephadex G-25, fine (0.9 x 142 cm) equilibrated and eluted with 50 mM ammonium acetate (b) Sephadex G-10 (1.5 × 92 cm) equilibrated and eluted with distilled water. In both cases, 1.25-ml fractions are collected and radioactivity is assayed in small aliquots. Where appropriate after chromatography or electrophoresis, samples (pH not lower than 5) are concentrated or taken to dryness either by vacuum evaporation at 30° or by lyophilization.

Quantities and Labeling of Cells As intimated above, determination of flow pathways is limited by technical considerations in cell fractionation (smallest convenient quantity of cells to produce a useful plasma membrane preparation) and also the limits of quantitation of monosaccharides (in general 1-100 nmoles, Table IV). We have found it convenient to work with 1 x 108 cells per time point when a plasma membrane purification is involved; if, in addition, a whole (unfractionated) cell sample is to be analyzed the number is 2 x 108 cells per time point. Hence, in one published experiment where 3 time points and 3 fractions were assayed (GDP-fucose, whole-cell glycoprotein-fucose, and plasma membrane glycoprotein-fucose), we started with cells concentrated to about 10 times their normal growth density (3 x 106 cells/ml) and labeled them, per milliliter, with 19/zCi of [3H-6]fucose (1.67 /xM in the medium) for 1, 2, and 4 hr. At these time points, aliquots of 2 x 10s cells were removed and processed for fucose; the purification is detailed below. In another experiment, it was necessary to compare the specific radioactivity (cpm/nmol) of GDP-fucose; unfractionated cell glycoprotein-fucose; plasma membrane glycoproteinfucose, purified by one cycle of zonal centrifugation; and plasma membrane glycoprotein-fucose further purified after the zonal centrifugation by banding in sucrose at their isopycnic density (1.16 g/ml). Because the recovery of plasma membranes is only 10-20% of the initial number of cell surfaces after one rate zonal centrifugation and 5-10% after the subsequent isopycnic banding it was necessary to start with 5 x 108 cells for the plasma membrane preparations for determination of glycoproteinfucose and 1 x 108 cells for the unfractionated cells glycoprotein-fucose and GDP-fucose. In all these experiments, it was necessary to use relatively high concentrations of labeled high-specific activity (25 Ci/mM)

198

PREPARATIONS

[I

7]

[3H]fucose (20 or more /zCi/ml) to obtain sufficient radioactivity to interpret data in very short-term labeling. A caution should be added that such high radioactivities may lead to perturbations of the physiology of cells, not normally envisaged in "tracer" studies of intermediary biochemistry; however, cell processes do go on at least in such circumstances as virus infection, where viruses continue to bud into the medium and there are found infections. 92 Fucose, derived from the various sources described above may then be quantitated in order to obtain specific radioactivity data.

Purification of Fucose from HeLa Cell GDP-Fucose and Glycoprotein-Fucose Pools Ethanol-extracted soluble and macromolecular material from cells radioactively labeled with [3H]fucose are prepared as outlined (see flow chart, Fig. 4). Briefly, cells, after washing with Earle's balanced salts, are extracted twice with 5-6 volumes of 60% ethanol in a boiling water bath for 5 min each.19The combined extracts are centrifuged at 900g for 5 min, HeLa Cells labeledwith pH] fucose EtOH extraction I

I

I

supn

pellet (60%)

in water

hydrolysis

TCA sulpn

I

pellet (28%)

I

supn

I pellet

Sephodex G - 25

/

GDP- [3H]fucose

excluded (12%)

SephadexG- I0 hydrolysis

TLC

I

[SH]fucose

desalting SephadexG-IO TLC [3H[] fucose

FIG. 4. Flow chart for isolation and purification of GDP-fucose and glycoprotein-fucose pools. Prior to assay with fucose kinase, [3H]fucose was isolated and purified from HeLa cell soluble and macromolecular pools as described in the text. Reprinted with permission from E D. Yurchenco and E H. Atkinson [Biochemistry 14, 3107 (1975)]. Copyright by the American Chemical Society.

~z p. H. Atkinson, S. G. Moyer, and D. F. Summers, J. Mol. Biol. 102, 613 (1976).

[17]

A N I M A L CELLS W I T H M O N O S A C C H A R I D E S

199

and the pellet is used for determination of macromolecular fucose. The supernatant is recentrifuged at 33,000 g for 20 min, evaporated to dryness under vacuum at 30°, redissolved in 1 ml of water, and centrifuged at 33,000 g for 20 min to remove ethanol-soluble but water-insoluble material (glycoprotein). GDP-fucose, on Sephadex G-25, eluted anomalously, and advantageously, after free fucose, since it is seen to be substantially separated from earlier eluting material including macromolecular fucose, fucose 1-phosphate, and free sugars. The peak containing GDP-fucose is pooled, concentrated, and further chromatographed on Sephadex G-10, where it is excluded, thus separating it from remaining free sugars. Material excluded on G-10 cochromatographed (97%) with GDP-fucose in solvent II and thus the labeled material is essentially radiochemically pure at this step. This material is pooled and hydrolyzed (I N HC1, 60 min, 100°), deacylating N-acetyl sugars, and GDP-fucose was hydrolyzed (100%), releasing free fucose. The hydrolyzed labeled material is eluted from a column of Amberlite MB-1 (removing hexosamines and salts), concentrated, and chromatographed on TLC system I (Fig. 5A). This step separates fucose from glucose, fructose, galactose, mannose, remaining amino sugars, arabinose, rhamnose, xylose, and most of ribose. Fucosecontaining fractions are pooled, desalted on Amberlite, reduced in volume, and chromatographed on TLC system II removing any remaining ribose (Fig. 5B). This purified fucose representing the GDP-fucose pool is saved for assay with fucose kinase. [:~H]Fucose was released by mild acid hydrolysis (0.1 N HCI, 100°, 45 min) from the macromolecular material left after ethanol extraction of the soluble pool (Fig. 4) and also from the purified plasma membranes. In small samples--for example, in the plasma membranes derived from 1 x 10a cells--this results in greater than 90% release of the fucose, indicating its terminal position. Sucrose from the preparative sucrose gradients must be removed from the purified plasma membranes by dialysis or repeated washing, since otherwise excess glucose would be present after hydrolysis, making impossible the microdetermination of other monosaccharides. The viscous material obtained after hydrolysis is mixed with an equal volume of 10% cold trichloroacetic acid. The precipitate that forms is removed by centrifugation at 33,000 g for 30 min, and this supernatant is desalted on a large Amberlite column. The eluate is reduced to a small volume and chromatographed on a Sephadex G-10 column, and the 3H radioactivity is chromatographed homogeneously as free sugar. About a 10% aliquot of this material is reduced in volume and chromatographed in TLC solvents I and II (Fig. 5A,B). The radioactive material in all cases chromatographed as did authentic fucose. This

200

PREPARATIONS

[17]

l 2ooor 50010

I0

20

RHA FUC RIB ARA MAN W.¢ @AL MIX

FIG. 5. Separation of sugars from GDP-fucose hydrolyzate by TLC systems I and II. FIG. 5A. Appropriate pooled sample and control fractions from TLCI were desalted as chromatographed on TLCII. Sample, control, and unlabeled markers were analyzed as described in (B). The sample fractions were pooled, desalted, concentrated, and analyzed by the fucose kinase assay. S, sample, from cellular GDP-[3H]fucose, [aH] (@ O); C, control, contains [14C]fucose (© O), derived from authentic GDP-[14C]fucose and [SH]fucose (0 e), from S. Rha, L-rhamnose; Fuc, L-fucose; Rib, o-ribose; Ara, DL-arabinose; Man, o-mannose; Glc, D-glucose; Gal, o-galactose; Xyl, o-xylose; Fruc, D-fructose; Mix, mixture of sugars shown on plate A. purified fucose, representing the macromolecular pool (Fig. 4), may be utilized for quantitation and determination of specific radioactivity.

Assay of Fucose Derived from the Various Pools As we have previously detailed s,~6 we would now assay our radiochemically pure and near chemically pure fucose by the fucose kinase assay: Ten nanomoles of a standard [3H]fucose solution are phosphorylated with [32]ATP in the presence of fucose kinase (this e n z y m e is not commercially available and must be prepared by published procedures),

[17]

201

ANIMAL CELLS WITH MONOSACCHARIDES

•

•

ZOO0

0

I0

20

28

RHA FUC

GLC MAN

6AL RIB

XYL FRU FUC÷RIB

FIG. 5B. Purified GDP-fucose was hydrolyzed with 1 N HC1 and prepared for TLCI chromatography as described in the text. Radioactive samples and standard sugars were applied as narrow l-cm bands, 5/,d at a time, 3 cm apart. After developing with the solvent and drying, areas 1 cm long by 3 cm wide were scraped, then eluted with 1 ml of H20, and the radioactivity was monitored. Naphthoresorcinol spray reagent was used to locate authentic sugars.

and the products are separated by HVPE using solvent IV. The 31-1and 32p double-labeled fucose 1-phosphate (F-l-P) is well separated from fucose and phosphates. This procedure defines the specific a2p radioactivity of fucose 1-phosphate which must also equal the specific radioactivity of the [y-32P]ATP used. The [aI-I]fucose purified from the cell GDP-fucose and macromolecular fucose can then be phosphorylated with [a2p]ATP and the products separated by HVPE. The specific tritium radioactivity of a fucose sample of unknown quantity is then determined from the defined specific activity of the [3zP]ATP. Since the total tritium fucose cpm in initial and incubation samples is known, the molar amount of fucose in these samples can easily be determined without necessitating complete conversion of substrate to product. Utilizing the procedure diagrammed in Fig. 4, we were able to purify fucose with the exception of one hexose contaminant, which is also phosphorylated in the fucose kinase reaction. Extended HVPE serves to separate this hexose phosphate from fucose 1-phosphate (Fig. 6B). The [ff-I]specific radioactivity of a fucose sample

202

PREPARATIONS

[17]

36,000

32,000 28,ooo

24,000 n 0 n ~4 ro

20,000 16,000

-r

12,000 8000

4000

),,o 4

8

12

16

20

24

28

32

36

40

44

48

52

FRACTION NUMBER

FIG. 6A. Double-label fucose kinase assay: 10 nmol of "standard," HVPE profile. An example of a high-voltage paper electrophoresis profile after 10 nmol of L-[6-3H]fucose was incubated with [3,-32P]ATP and fucose kinase preparation for ! .5 hr as described under Experimental Procedures (electrophoresis was at 37.5 V/cm for 3 hr); l-cm strips were assayed for radioactivity. The anode is to the right. The 3H peaks ((3) (as determined in other electrophoregrams of authentic standards) from left to right are (a) fucose and (b) fucose phosphate. The 32p peaks (O) from left to right are: (a) unknown (small peak just ahead of fucose), (b) probably AMP and ADP, since both have been shown to migrate at about the same rate as this peak (data not shown), (c) fucose phosphate, (d) ATP, and (e) inorganic phosphate. Pyrophosphate has been shown to migrate ahead of inorganic phosphate toward the anode. Fuconic acid, although not present, is known to migrate midway between fucose and fucose phosphate with an Rr-~-p of 0.53. Only fucose phosphate appears as a double-labeled peak. Reprinted with permission from P. D. Yurchenco and P. H. Atkinson [Biochemistry 14, 3107 (1975)]. Copyright by the American Chemical Society. may be calculated reproducibly from several assay tubes, and furtherm o r e this c a l c u l a t e d v a l u e is f o u n d to b e i n d e p e n d e n t o f the a m o u n t o f s a m p l e i n c u b a t e d w i t h e n z y m e . F r o m t h e s e d a t a , i n c l u d i n g specific r a d i o a c t i v i t i e s ( c p m / n m o l ) a n d a b s o l u t e p o o l s i z e s in G D P - f u c o s e , w h o l e cell g l y c o p r o t e i n - f u c o s e , a n d p l a s m a m e m b r a n e g l y c o p r o t e i n - f u c o s e , as well as t h e rate a t w h i c h l a b e l e d f u c o s e is u t i l i z e d , w e w e r e a b l e t o c o n s t r u c t t h e d i a g r a m s h o w n in Fig. 7. W e n o t e t h a t , a l t h o u g h o u r e s t i m a t i o n s w e r e m a d e w i t h f u c o s e k i n a s e , [a2P-y]ATP in t h e d e t e r m i n a tion o f f u c o s e , the m e t h o d s h o u l d in p r i n c i p l e w o r k f o r t h e flow p a t h w a y s

t.) 40OO

2000 Iooo IOO

IIO

12o

FRACTION NUMBER

FiG. 6B. Double-label fucose kinase assay of fucose derived from HeLa GDP-fucose. Sample was incubated with [y-32P]ATP and fucose kinase; the products were separated by electrophoresis at 37 V/cm for 7 hr, and 0.05-inch strips were analyzed for radioactivity. The slower moving single-labeled '~2Ppeak is a contaminant with a n Rr-i-p value of 0.95, the same as for hexose phosphates. The double-labeled peak separated from hexose phosphate is fucose phosphate. This figure shows the HVPE profile only in the region of fucose phosphate: (C)) zI-I cpm; (e) '~P cpm. Reprinted with permission from P. D. Yurchenco and P. H. Atkinson [Biochemistry 14, 3107 (1975)]. Copyright by the American Chemical Society. 107 HeLa CELLS ~ ' ~

/8tl

GDP. . . . . . . .

1,o2 °~,.s/2a,

Fucose

|~h

~

t'O-',m..,

44 n moles

,

.8°~ ~

free ft. fucose

12|i~omo,es,2

~, ~ ~ 8 6 n moles/23h J~ I" 09 n moles/23h ~---~----I"~11.1 INTERNAL |~/I I nn~bs/ POOLS /~i J~ Fucose~F-I-P ~ Ir.n~_~ I. . . . . . . . L 23h (<2~.totol) /Z { minor L ~, pathway 2 5 n moles/23h Fuconlc Acid ......

42 n moles/ 23h

" ~•

fuc, fucosyl-glycoprotein ~ucosy,"t~lycoproteln

J

CO2 FXC. 7. Summary diagram. The flow rate of label into GDP-fucose and into glycoprotein pools can be directly measured by counting samples at various labeling times. Since the GDP-fucose specific activity is known, the flow rates into glycoprotein pools can be determined [ 1]. 1 nmo123 h r - ' 007 cells)- 1]. From the observed flow rate of label into wholecell glycoprotein, it can be determined that actually 8.6 nmol of glycoprotein fucose flow into the cells per generation; 2.5 nmol 23 h r - ' (107 cells)-' is the flow rate of the glycoprotein released from a small internal pool (B) directly to the medium without mixing with the hulk cell fucosyl glycoprotein. The cells contain 4.4 nmol/107 cells of glycoprotein fucose, as determined by direct measurement. The measurably significant proportion of this is in the plasma membrane; hence, of the observed 8.6 nmol of fucose incorporated into plasma membrane glycoprotein in 23 hr, 4.2 nmol must be turned over. Direct observation of material in the medium characterized 75-80% of this as free fucose and 20-25% as glycopep-

204

PREPARATIONS

[ 18]

of other labeled monosaccharides. In addition, one could utilize any of the variety of microdetermination methods (Table IV, especially GLC) to quantify the monosaccharide purified through the various steps we have described (Fig. 4) from the cell pools of interest in order to quantitate the process, given that sufficient quantities could be obtained. Acknowledgments This research was supported by grants from the National Institutes of Health (CA-13402, CA-06576). Part of the data in this paper is from a thesis (P. D. Y.) submitted in partial fulfillment for the Degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. P. H. A. is an Established Investigator of the American Heart Association (75-174).

Fig. 7 (continued) tide or glycoprotein fucose. Finally, the ratio of measured specific radioactivities of fucose in the medium to that in the GDP-fucose pool shows that 10.2 nmol 23 h r - ' (10 r cells)-' of fucose bound to GDP comes from an endogenous source, probably GDP-mannose [D. W. Foster and V. Ginsburg, Biochim. Biophys. Acta 54,376 (1961)], while 0.9 nmol comes from the medium. The pathway of fucose to CO2 is minor because little net loss of '4(2 label from HeLa is seen [R. L. Kaufman and V. Ginsburg, Exp. Cell Res. 50, 127 (1968)]. Reprinted with permission from P. D. Yurchenco and P. H. Atkinson [Biochemistry 16, 944 (1977)]. Copyright by the American Chemical Society.

[18] Tritium Labeling of Cell-Surface Glycoproteins and Glycolipids Using Galactose Oxidase B y CARL G . G A H M B E R G

Principle. Galactose oxidase (EC 1.1.3.9) oxidizes o-galactosyl and N-acetyl-D-galactosaminyl residues at nonreducing terminals of glycoproteins and glycolipids to carbon-6 aldehydes.' These aldehydes are then reduced back to galactose/N-acetylgalactosamine with tritiated borohydride. 2~ When intact cells are treated with the enzyme, only surfaceexposed glycoconjugates are oxidized and subsequently reduced, because the enzyme is unable to penetrate the cell plasma membrane. 4,5 Because sialic acids often are linked to penultimate galactosyl residues, more efficient labeling is achieved by the simultaneous use of neuraminidase. 1 G. Avigad, D. Amaml, C. Asensio, and B. L. Horecker, J. Biol. Chem. 237, 2736 (1962). 2 A. G. Morell and G. Ashwell, this series Vol. 28 [14]. 3 y. Suzuki and K. Suzuki, J. LipidRes. 13, 687 (1972). 4 C. G. Gahmberg and S.-i. Hakomori, J. Biol. Chem. 248, 4311 (1973). •~ T. L. Steck and G. Dawson, J. Biol. Chem. 249, 2135 (1974).