Selective spin labeling of terminal galactose and N-acetylgalactosamine residues on the membrane surface of erythrocytes

Journal of Biochemical and Biophysical Methods, 10 (1984) 111-120

111

Elsevier BBM 00441

Selective spin labeling of terminal galactose and N-acetylgalactosamine residues on the membrane surface of erythrocytes Bennett T. Farmer II and D. Allan Butterfield * Department of Chemistry, Universityof Kentucky, Lexington, KY 40506, U.S.A. (Received 9 May 1984) (Accepted 21 June 1984)

Summary A method to selectively spin label galactose and N-acetylgalactosamine residues of erythrocyte membrane glycoconjugates is described. The method is based on the activation of the C-6 CHeOH group of these two sugars by galactose oxidase followed by reductive amination with 2,2,6,6-tetramethyl-4aminopiperidine-l-oxyl in the presence of a mild reducing agent, NaBH3CN. The extent and distribution of the spin labeling suggest that the major sialoglycoprotein, PAS-1, incorporates the greatest amount of spin label while the glycolipids incorporate less than 10% of the spin label. Key words: spin labeling; ESR; selectivity for galactose residues; cell surface carbohydrates; $1ycophorin A; erythrocyte membrane.

Introduction

Previously we reported the development of a method for selectively spin labeling sialic acid residues on erythrocyte membranes [1]. This method used periodate in a pH 5.0 acetate buffer to oxidize the C7-9 vicinal hydroxyl groups of this sugar to a C7-aldehyde, to which a primary amine spin label (Tempamine) was then coupled by reductive amination employing NaBH3CN. Residual periodate was reduced by the addition of arsenite in order to limit the extent of oxidation and to maximize the selectivity of this labeling procedure. This labeling method offered a means to examine the physical state of sialic acid in a variety of processes which occur at the cell surface [2]. Potential drawbacks to this procedure center around the necessity of employing a low pH buffer in the periodate oxidation step (in order to give a usable signal-to-noise ratio in the ESR spectrum) and the use of arsenite to destroy excess

* To whom correspondence should be addressed. 0165-022X/84/$03.00 © 1984 Elsevier Science Publishers B.V.

112 periodate. Both spectrin and actin have an isoelectric point of approximately 4.5-5.0 and will begin to precipitate at pH 5.0 [3]. The possibility exists, therefore, that this pH-induced precipitation of membrane-bound spectrin and actin might lead to some irreversible alteration in the skeletal network or in its relationship with the bilayer domain and certain transmembrane proteins, e.g., PAS-2 [4] and band 3 [5]. Additionally, arsenite has been shown to render the erythrocyte acetylcholinesterase inactive (D.A.B., unpublished observations). In view of the possible artifacts that might arise due to this labeling procedure, we have developed a method for selectively labeling terminal galactose or N-acetylgalactosamine residues of human erythrocyte oligosaccharide chains that is performed at pH 8.0 and employs no arsenite. A description of the method and partial characterization of the resulting membranes and ESR spectrum are the subjects of this report. Methods Erythrocyte isolation Erythrocytes were obtained from healthy volunteers by venipuncture into heparinized, glass vacutainer tubes and stored on ice for no longer than 1 h prior to use. Intact cells were obtained by washing the blood twice in 5 mM sodium phosphate/150 mM sodium chloride, pH 8.0 buffer (PBS) by centrifugation at 600 × g for 5 min and aspiration of the supernatant and buffy coat. The cells were then washed one more time in PBS containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM L-l-tosylamide-2-phenylethylchloromethylketone (TPCK), and 0.1 mM N-a-p-tosyl-L-lysinechloromethyl ketone (TLCK) at pH 8.0 (Buffer PBS(P)). Ghost membranes were obtained by hypotonic lysis of intact cells, previously treated with galactose oxidase as described below, with 5 mM sodium phosphate buffer, pH 8.0 (5P8). The membranes were washed at 4 ° C in 5P8 by successive centrifugation for 10 min at 27 000 × g in a refrigerated centrifuge until completely free of hemoglobin. Protein content was estimated by the method of Lowry et al. [6]. Galactose oxidase treatment Galactose oxidase type IV was purchased from Sigma. 3 ml of PBS(P) prewarmed to 37 °C were added to give a final concentration of 150 units/ml galactose oxidase. 20 ~1 of a 28 m g / m l PMSF solution in methanol was also added. The galactose oxidase solution was incubated for 30 rain at 37 o C in order to deactivate any serine proteases that may have been present. Aliquots ranging from 0.2 to 1.4 ml were removed, frozen in liquid nitrogen, and stored at - 10 o C until needed. When stored in this fashion, the enzyme retained its activity for at least 2 months. Galactose oxidase, prepared as described above, was warmed to room temperature and added to intact cells at 37°C in PBS(P) to a final concentration of 15 units/ml to give a final hematocrit of 25%. The enzyme was incubated with the erythrocytes for 30 min at 37°C followed by two washes in PBS at 600 × g at 4°C. No significant hemolysis occurred as a result of the galactose oxidase treatment.

113

NaB3H~ reduction Typically, 0.5 ml of activated cells with a hematocrit of 50-70% were incubated with 10/~1 of NaBaH4 (0.05 mCi//tl in 0.01 M NaOH) (New England Nuclear, 341 mCi/mmol) for 30 rain at room temperature. The erythrocytes were subsequently washed once in PBS, lysed by exposure to 5P8, and washed in 5P8 to yield hemoglobin-free ghosts.

Spin labeling 2,2,6,6-Tetramethyl-4-aminopiperidine-l-oxyl (Tempamine) was obtained from Molecular Probes. Sodium cyanoborohydride was obtained from Aldrich. Solutions of Tempamine and sodium cyanoborohydride, 1.88 and 3.13 mM, respectively, were prepared in both monobasic and dibasic 5 mM phosphate solutions which were used to prepare a solution of pH 8.0. Terminal galactose and N-acetylgalactosamine residues of intact erythrocytes were activated by galactose oxidase, and ghosts were isolated as described above. 1 vol. of these ghosts at a protein concentration of 3.0 mg/ml was covalently labelled by Tempamine by reaction with 4 vols. of the spin label solution (described below) for 16-18 h at 4°C following mixing by successive inversions of the Corex tube. Unreacted spin label was removed by 5-6 washings in 5P8. No detectable ESR signal could be demonstrated in the supernatant of the last wash. Since it has been previously demonstrated in our laboratory that the motion of the spin label, when covalently attached to sialic acid, is sensitive to the protein content [1], all spin-labeled samples were adjusted to a constant protein content as indicated.

Extent and distribution of labeling In order to determine the extent of activation of galactose or N-acetylgalactosamine residues on glycolipids, the lipids were extracted from tritium-labeled ghosts according to the procedure of Folch et al. [7]. The chloroform was evaporated and the dried lipids were then re-extracted in the same manner to remove most of the residual protein contamination. The dried lipids were then re-dissolved in 1 vol. of cyclohexanone (MC/B Chemical, 98% min. purity) and centrifuged for 5 rain in a desk-top, analytical centrifuge. An aliquot (typically 20-30/~1) was removed and placed in a shell vial (Fischer Scientific) to which 4 ml of Biofluor scintillation cocktail (New England Nuclear) were added. The clear mixture was allowed to cool to approximately 11 o C before counting. The labeled membranes were extracted in duplicate, and each extraction sample was counted in triplicate. The percentage of Tempamine incorporated onto glycolipids was also determined by comparison of the resulting ESR spectra of ghosts that had been subjected to extensive pronase treatment (1.0 mg/rnl for 24 h at 37 o C) to that of ghosts kept at 4°C in the absence of pronase. In order to complement this procedure, we also extracted lipids from Tempamine-labeled ghosts as described by Folch et al. [7]. Intensity measurements were made as previously described [1]. The distribution of activated carbohydrate residues and of Tempamine among the glycoproteins was quantitated by comparison of radioactivity profiles of NaB3H4 attached to membrane glycoproteins of previously spin-labeled and non-spin-labeled

114 ghosts that had been treated with galactose oxidase and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis ( S D S - P A G E ) following exposure to NaB3H4 . Other methods

The protein-specific spin label, 2,2,6,6-tetramethyl-4-maleimidopiperidine-l-oxyl was covalently attached to erythrocyte ghosts as described previously [8], and was used to monitor the effects of solution pH resulting from the use of Tempamine, on the physical state of membrane proteins [9,10]. Discontinuous S D S - P A G E employing either a 7.5% resolution gel and 3.5% stacking gel or a 10.0% resolution gel and 3.5% stacking gel was performed according to the method of Laemmli [11]. Typically, 100 #g of protein in 40-50 #1 was loaded per gel tube and electrophoresed at 2.5 mA per gel tube. Acrylamide and bisacrylamide were purchased from Bio-Rad and were > 99% pure (electrophoresis grade). All liquid scintillation counting was performed on a Packard Model 3255 Tricarb Liquid Scintillation Counter. ESR spectra were recorded on a Varian X-band E-109 Century Series ESR spectrometer equipped with an E-238 rectangular cavity supporting a Varian E-238 quartz aqueous sample cell. The cavity and waveguide were purged with dry nitrogen gas at a flow rate of 5 standard f t a / h and the modulation amplitude (0.32 G) and the incident microwave power (14 roW) were set to avoid instrumental line-broadening.

Results

The ESR spectrum of Tempamine, bound to terminal galactose and N-acetylgalactosamine residues of glycoconjugates on the human erythrocyte membrane under conditions fully described below, is shown in Fig. 1. Information on the rotational rate of spin-labeled galactose residues can be obtained from this spectrum by the calculation of an apparent rotational correlation time, ~'A, according to methods previously described [1,12]. Typical rotation correlation times observed ranged from 0.6 to 0.8 ns/rad, which is similar to that obtained for Tempamine incorporated onto sialic acid in erythrocyte ghosts (~'A = 0.8 n s / r a d ) [1]. The conditions required to obtain this spectrum are now described. Fig. 2 shows the degree and distribution of terminal galactose activation by galactose oxidase as a function of the enzyme concentration. Increasing the galactose oxidase concentration to greater than 10-15 units/ml did not lead to an appreciable increase in the number of galactose residues activated. We therefore chose to employ galactose oxidase at a final concentration of 15 units/ml. Fig. 3 shows the effect of varying the time of the galactose oxidase incubation on the relative ESR signal intensity per mg protein ( I r / m g P ) as a function of Tempamine concentration. There is no increase in ESR signal intensity for galactose oxidase incubation times greater than 30 rain. Although the signal intensity appears to increase linearly with Tempamine concentration and a high concentration of Tempamine would, therefore, seem to be

115

favored, Heisenberg spin exchange begins to arise at concentrations much above 1.5-2.0 raM. Heisenberg spin exchange is undesirable since it severely hinders attempts to extract dynamic information from simple considerations of lineshapes. We found that the relative ESR signal intensity is nearly insensitive to the concentration of sodium cyanoborohydride employed above 2.5 raM. To ensure a slight excess of sodium cyanoborohydride for the complete reduction of the unstable imine (formed between the amine group of Tempamine and the activated aldehyde of galactose) to the stable secondary amine, we chose to use Tempamine at a final concentration of 1.5 mM and sodium cyanoborohydride at 2.5 mM. Non-specific labeling (i.e., incorporation of Tempamine into ghosts derived from cells not previously activated with galactose oxidase) accounted for less than 10% of the ESR signal intensity. Fig. 4 presents the results of spin label incorporation as a function of the pH of the spin label solution. The extent of the reaction increases considerably above pH 8.0 but is approximately constant from pH 6.5 to 8.0. Although Tempamine incorporation increases greatly above pH 8.5, the results presented in Fig. 5 demonstrate that the physical state of erythrocyte membrane proteins is significantly altered above pH 8.0, as judged by the W / S ratio of a protein specific spin label (MAL-6). The W / S ratio has been shown to be a sensitive monitor of membrane protein conformation [9,10]. We therefore chose to spin label galactose residues at a pH of 8.0. Table 1 presents the distribution of activated galactose residues among the four major glycoproteins (band 3, PAS-1, PAS-2, band 4.5) [13] and the percentage of the active sites on each individual glycoprotein that is labeled by Tempamine as

_J I

lOG

I

Fig. 1. Typical ESR spectrum of Tempamine selectively and covalently bound to terminal galactose or N-acetylgalactosamine resides of membrane glycoconjugates in human erythrocyte ghosts. ESR spectrometer parameters: scan width, 100 G; modulation amplitude, 0.32 G; microwave power incident on the ESR resonant cavity, 14 mW.

116 d e t e r m i n e d b y p r o c e d u r e s d e s c r i b e d in the M e t h o d s section. A p p r o x i m a t e l y 75% o f t h e a c t i v a t e d g a l a c t o s e r e s i d u e s o n g l y c o p r o t e i n s reside o n P A S - 1 a n d b a n d 4.5, b u t m o r e t h a n t w i c e as m u c h s p i n l a b e l is f o u n d o n P A S - 1 c o m p a r e d to b a n d 4.5. P A S - 1 b e a r s t w o t y p e s o f c a r b o h y d r a t e c h a i n s : 15 serine- o r t h e r o n i n e - l i n k e d t e t r a s a c c h a r i d e s a n d o n e a s p a r a g i n e - N - l i n k e d o l i g o s a c c h a r i d e p e r p r o t e i n m o l e c u l e [14]. E m p l o y i n g e n d o - f l - g a l a c t o s i d a s e w h i c h c l e a v e s the p o l y l a c t o s a m i n y l c a r b o h y d r a t e s t r u c t u r e p r e s e n t o n b a n d 3 a n d b a n d 4.5 a n d the s i m i l a r N - l i n k e d s t r u c t u r e o n P A S - 1 , b u t d o e s n o t c l e a v e the t e t r a s a c c h a r i d e s o n the l a t t e r g l y c o p r o t e i n , F u k u d a a n d F u k u d a s h o w e d t h a t t h e vast m a j o r i t y o f a c t i v a t e d g a l a c t o s e r e s i d u e s o n P A S - 1 w e r e n o t o n the t e t r a s a c c h a r i d e s a n d are t h e r e f o r e a s s u m e d to r e s i d e o n the N - l i n k e d o l i g o s a c c h a r i d e [15]. W e t h e r e f o r e p r e s u m e t h a t t h e T e m p a m i n e i n c o r p o r a t e d o n t o

:5,600 3,200 2,800 J

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16

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26

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31

36

41

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o ' , ~ 0.5

1.5

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{Tempomine] m M

Fig. 2. Reduction by NaB3H4 of aldehyde groups formed by reaction of galactose oxidase with terminal galactose residues in human erythrocyte membrane glycoconjugates as a function of galactose oxidase concentration. After the membranes were reacted with the enzyme and NaB3H4 as described in Methods, ghosts were subjected to SDS-PAGE. 2 mm sfices of the gel were dissolved in 200/d of 30% H202, cooled, and counted in 4.0 ml of Biofluor as described. - - , 30 units/ml; - - - - - - , 20 units/mi; . . . . . . , 10 units/ml; . . . . . , 0 units/ml. Fig. 3. Relative intensity (Jr) of Tempamine ESR resonance line (M ! = 0) per mg protein as a function of Tempamine concentration for varying times of incubation with galactose oxidase. The concentration of sodium cyanoborohydride was 6.5 mM. Times of enzyme incubation were: x , 0 rain; O, 30 rain; n, 60 min; z~, 120 nun. 1, = h .AH2pp where h is the peak-to-peak amplitude and AHpp the peak-to-peak width of the first-derivative hyperfine line.

117 TABLE 1 DISTRIBUTION AND REACTIVITY TOWARDS TEMPAMINE OF THE ACTIVATED GALACTOSE AND N-ACETYLGALACTOSAMINE RESIDUES ON ERYTHROCYTE GLYCOPROTEINS Glycoprotein

% Distribution of activated residues among the 4 major glycoproteins

% Activated sites on each individual glycoprotein which are labeled by Tempamine

Band 3 PAS 1 PAS 2 Band 4.5

15+3a 29+5 14 + 5 43+2

10+3 42+3 =0 13+13

a The reported values are the mean + S.E. for two separate measurements.

P A S - 1 f o l l o w i n g g a l a c t o s e o x i d a s e t r e a t m e n t is p r i m a r i l y o n t h e N - l i n k e d o l i g o s a c charide chain. The percentage of Tempamine on glycolipids was determined in two independent fashions: pronase degradation of exposed glycopeptides and lipid extraction. Table 2 p r e s e n t s t h e r e s u l t s . B e t w e e n 1 a n d 10% o f t h e t o t a l E S R s i g n a l i n t e n s i t y is a s c r i b e d to lipid-bound Tempamine, the variance probably being due to contaminating

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120

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7.0

7.5 pH

8.0

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8.5

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Fig. 4. Tempamine spin label incorporation onto activated terminal galactose residues of membrane glycoconjugates in human erythrocyte ghosts as a function of Tempamine solution pH. * The value of the relative intensity I r, of the M l = 0 ESR Nine of Tempamine at pH 8.5 is highly significantly different ( P < 0.005) from that at pH 8.0. The values present are means+ S.E. for two separate experiments. Fig. 5. Relative conformation of membrane proteins in human erythrocyte ghosts exposed to buffers of various pH compared to ghosts exposed to 5P8 as judged by the W / S ratio of the protein specific spin label MAL-6 [9,10]. Each point on the graph represents the mean+S.E, of two different samples. * P < 0.002; ** P < 0.0005 relative to a mean value of 1.0.

118

TABLE 2 Q U A N T I T A T I O N OF T E M P A M I N E A T T A C H E D TO G L Y C O L I P I D G A L A C T O S E Expt.

Lipid extraction

Pronase treatment

1 2 3

6~ 15 10

0 0 3

Mean + S.E.

10 + 2.6

1.0 + 1.0

a The values presented are % Tempamine on glycolipids to total incorporated Tempamine.

protein in the lipid extraction. In contrast, 28% of the NaB3H4 incorporation occurred on glycolipids, suggesting that glycoproteins are preferentially labeled by Tempamine. This suggestion is in agreement with the observations of Grant et al. [16] concerning the crypticity of membrane glycolipids. Reduction kinetics with membrane impermeable ascorbate demonstrated that labeling intact cells with Tempamine is not possible due to the large, intracellular incorporation of Tempamine (data not shown).

Discussion A procedure to selectively spin label galactose and N-acetylgalactosamine residues on erythrocyte membranes by reductive amination and partial characterization of the binding sites of the amine spin label employed has been described. The utility of galactose oxidase in radiolabeling cell surface glycoproteins, especially in erythrocyte membranes, is well documented. Among other workers, Gahmberg [17,18] has used a labeling procedure employing galactose oxidase treatment followed by reduction with sodium borotritide (NaB3H4) to demonstrate the existence of at least 20 glycoproteins in the human erythrocyte membrane, the existence of asymmetry in the distribution of the carbohydrate on these glycoproteins (exposed only to the outside), and the existence of differences in glycoproteins from fetal erythrocytes as compared to those from adults or in the rare blood type E n ( a - ). Sharon and co-workers [19] have employed galactose oxidase to monitor differences in glycolipids as a function of the antigenic composition of erythrocytes. Additionally, Gattegno et al. [20] have shown that the number of terminal galactose residues decreases with the age of the erythrocyte. We have observed a distribution of activated galactose residues similar to these workers and those of Fukuda and Fukuda [15], In adapting galactose oxidase to a spin labeling method, we hope to open new ways of probing motional dynamics at the erythrocyte cell surface as a function of experimental perturbation of the erythrocyte membrane. Most of the activated galactose residues were on band 4.5 and PAS-1, while most of the Tempamine was found on PAS-1 (Table 1). The apparent selectivity for

119 PAS-1 in the labeling process is not understood. It is possible that less steric hinderance near the PAS-1 galactose residues accounts for this selectivity. The enhanced spin labeling of PAS-1 over b a n d 3, b a n d 4.5, and PAS-2 suggests a difference in the microenvironment of activated galactose residues on these glycoproteins. Based on the work of others [15], it is inferred that the majority of the spin label incorporated onto PAS-1 is on galactose residues on the N-linked oligosaccharide chain. This assignment differs from the sites of spin label incorporation onto PAS-1 following periodate treatment, which is most likely predominantly on the tetrasaccharides of PAS-1 [1]. The method for selective spin labeling of terminal galactose residues described in this report yields ESR spectra with an improved signal-to-noise ratio over that resulting from selective spin labeling of sialic acid [1] b y a factor of approximately 2. This slight improvement in signal-to-noise permits a more precise measurement of the peak-to-peak width of the M I = 0 line used in the calculation of cA [1]. The primary reason for this improvement in signal-to-noise is p r o b a b l y the higher concentration of both T e m p a m i n e and sodium c y a n o b o r o h y d r i d e employed in this study in contrast to concentrations of these substances used in the sialic acid-specific procedures [1]. Feix and Butterfield [1] employed the highest concentrations of T e m p a m i n e and sodium c y a n o b o r o h y d r i d e possible that did not result in Heisenberg spin exchange. The lower concentrations of T e m p a m i n e required to induce Heisenberg spin exchange for sialic acid spin labeling are perhaps due to the nature of the carbohydrate chains being labeled. As noted above, there are 15 tetrasaccharide chains per glycophorin A molecule, each with terminal sialic acid residues and most with penultimate galactose residues [14]. Spin labeling of sialic acid residues with higher concentrations of T e m p a m i n e than used by Feix and Butterfield [1] m a y lead to interactions between neighboring chains, depending on their proximity and their motional freedom, and because of the intrinsic proximity of adjacent sialic acid residues on individual chains, excessive spin labeling m a y predispose the system to spin exchange. These characteristics should not exist for the single oligosaccharide chain of glycophorin A bearing the terminal galactose residue.

Simplified description of the method and its applications

Selective spin labeling of galactose residues of erythrocyte membrane glycoconjugates is achieved by enzymatic activation of galactose residues with galactose oxidase, followed by reductive amination with an amine spin label in the presence of NaBH3CN. The procedure described in this report has alleviated the necessity for strong oxidizing agents such as periodate, for subjecting the membranes to a pH 5 buffer where precipitation of spectrin and actin may occur, and for strong reducing agents such as arsenite. Optimal labeling conditions are: 15 units/ml galactose oxidase for 30 rain at 37 o C, in the presence of several protease inhibitors, followed by reaction with a solution of Tempamine and sodium cyanoborohydride at a final concentration of 1.5 and 2.5 mM, respectively, pH = 8.0 for 16-18 h at 4°C. This procedure can be exploited to investigate cell surface phenomena and to probe the interaction of skeletal and bilayer components with transmembrane proteins, and can be used in conjunction with spin labeling experiments involving lSN-labeled Tempamine. All these experiments are currently in progress in our laboratory.

120 Acknowledgements T h i s w o r k was s u p p o r t e d in p a r t b y N I H g r a n t s A G - 0 0 0 8 4 a n d A G - 0 2 7 5 9 a n d a g r a n t f r o m the A l z h e i m e r ' s D i s e a s e a n d R e l a t e d D i s o r d e r s A s s o c i a t i o n , Inc.

References 1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Feix, J.B. and Butterfield, D.A. (1980) FEBS Lett. 115, 185-188 Feix, J.B., Green, L.L. and Butterfield, D.A. (1982) Life Sci. 31, 1001-1009 Elgsaeter, A., Shotton, D.M. and Branton, D. (1976) Biochim. Biophys. Acta 426, 101-122 Mueller, T.J. and Morrison, M. (1981) in Erythrocyte Membranes 2: Recent Clinical and Experimental Advances (Eaton, K.W., Kruckeerg, W.C. and Brewer, G.J., eds.), pp. 95-112, Alan R. Liss, Inc., New York Bennett, V. and Stenbuck, P.J. (1979) J. Biol. Chem. 254, 2533-2541 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Foich-Pi, J., Lees, M. and Stanley, G.H.S. (1957) J. Biol. Chem. 226, 497-509 Butterfield, D.A., Roses, A.D., Appel, S.H. and Chesnut, D.B. (1976) Arch. Biochem. Biophys. 177, 226-234 Butterfield, D.A. and Markesbery, W.R. (1981) Biochem. Int. 3, 517-525 Butterfield, D.A. (1982) Biol. Magn. Resonance 4, 1-78 Laemmli, U.K. (1970) Nature (London) 227, 680-685 Nordio, P.L. (1976) in Spin Labeling: Theory and Applications (Berliner, L.J., ed.), pp. 29-35, Academic Press, New York Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617 Yoshima, H., Furthmayr, H. and Kobata, A. (1980) J. Biol. Chem. 255, 9713-9718 Fukuda, M. and Fukuda, M.N. (1981) J. Supramol. Struct. 17, 313-324 Peters, M.W., Singleton, C., Barber, K.R. and Grant, C.W.M. (1983) Biochim. Biophys. Acta 731, 475-482 Gahmberg, C.G. and Hakomori, S. (1973) J. Biol. Chem. 248, 4311-4317 Gahmberg, C.G. (1976) J. Biol. Chem. 251,510-515 Lis, H., Jaffe, C.L. and Sharon, N. (1982) FEBS Lett. 147, 59-63 Gattegno, L., Perret, G., Fabia, F., Baldier, D. and Cornillot, P. (1981) Carbohydr. Res. 95, 283-290