The phosphorylation of the major proteins of the human erythrocyte membrane

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 195, No. 2, July, pp. 300-314, 1979

The Phosphorylation of the Major Proteins the Human Erythrocyte Membrane1

of

LLOYD WAXMAN The Biological

Laboratories,

Harvard

University,

16 Divinity

Avenue, Cambridge, Massachusetts

02198

Received October 11, 1978; revised “February 20, 1979 Both the major sialoglycoprotein (PAS-l) and the component designated by Fairbanks 10,2606-2617) asBand 3 are shown to be bonafide phosphoproteins by virtue of the presence of covalently bound serine and threonine phosphate residues. In agreement with the findings of others, PAS-l does not seem to be phosphorylated when ghosts are incubated with [y-32P]ATP, but the phosphorylation is significant (about 0.15 molimol) when the cells are incubated in the presence of a2Pi. Band 3 is phosphorylated to the extent of 0.90 moYmo1, and these sites are apparently distributed in several places along the polypeptide chain. Spectrin is also a phosphoprotein containing approximately four molecules of phosphate per 450,000 daltons of protein. The phosphorylation of these three polypeptides is not stimulated by the presence of CAMP. et al. (G. Fairbanks, T. L. Steck, and D. F. H. Wallach, 1971, Biochemistry

Currently there is great interest in the problem of how membrane proteins are designed to function as carriers and channels, receptors and recognition sites, as well as to enable the cell to move and maintain its shape. An important question, then, is how cells regulate these processes in response to external stimuli or cytoplasmic events. It has become an increasingly popular concept that protein phosphorylation may play a key role in modulating a variety of cellular processes. In fact, numerous cases have been reported regarding the presence of protein kinases in virtually all subcellular organelles (for a review, see Rubin and Rosen cl)), although there are only a few instances in which a direct correlation has been established between a phosphorylation event and a physiological change. Indeed, with the exception of the elegant studies on the regulation of mammalian glycogen metabolism (Z-4), the regulation ’ This work was supported by grants from the National Institutes of Health (HL 08893) and from the National Science Foundation (BMS 75-09919)to Guido Guidotti. 2 Fellow of the Leukemia Society of America, 1974-76. Current address: Department of Biological Chemistry, Schoolof Medicine, University of California, Davis, California 95616. 0003-9861/79/080300-15$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

of biological events via phosphorylation remains unclear. To date considerable effort has gone into characterizing CAMP-dependent and independent forms of protein kinases in the erythrocyte membrane with regard to the effect of salts on their activity, their ability to utilize both membrane components and exogenously added proteins as phosphate acceptors, and their solubilization by detergents and salts (5-9). Little is known, however, about the actual levels of phosphorylation in any membrane protein, where the sites of modification are in the protein architecture, or whether this actually has any effect on their function or on the behavior of the cell. In this paper, experiments are presented which address the first two questions with regard to several of the major polypeptides of the erythrocyte membrane, e.g., the major sialoglycoprotein (PAS-l), the anion carrier (Band 3), and spectrin. MATERIALS

AND METHODS

All chemicals were reagent grade. Lis3 was produced from the free acid (Aldrich) and recrystallized 3 Abbreviations used: SDS, sodium dodecyl sulfate; Lis, lithium diiodosalicylate. 300

MAJOR

ERYTHROCYTE

MEMBRANE

as described by Marchesi and Andrews (10). SDS, phosphoserine and phosphothreonine, and phenylmethane sulfonyl flouride were from Sigma. Acrylamide and methylene bisacrylamide were from Eastman and used without further purification. Trypsin treated with L-1-tosylamido-Z-phenylethyl chloromethyl ketone and chymotrypsin were from Worthington. Carrier-free “*P, was obtained from New England Nuclear and incubated with erythrocyte suspensions as described below, or converted to [y-32P]ATP by the method of Glynn and Chappell (11). SDS-polyacrylamide gels were run in tubes using the buffer system of Davies and Stark (12), and on slabs with the system of Laemmli (13). The latter were dried on Whatman 3MM chromatography paper after staining and destaining before autoradiography. CNBr fragments were run as described by Swank and Munkres (14). Proteins were stained with Coomassie brilliant blue (Sigma) as described by Weber et al. (15), and glycoproteins were located with Schiff’s reagent by the procedure of Glossman and Neville (16). The catalytic subunit of the cyclic AMP-dependent protein kinase was purified from beef skeletal muscle by the method of Beavo et al. (17), and the cyclic GMP-dependent protein kinase was purified from bovine cardiac muscle using the method described by Gill ef al. (18) for bovine lung. The cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle (19) was the gift of J. McPherson (University of California, Davis). Paper chromatography and electrophoresis were carried out on Whatman 3MM paper which had been previously washed by decending irrigation with 10% acetic acid. Cellulose thin-layer plates (Eastman) were used in analytical experiments, and in both cases, electrophoresis was performed on a Savant watercooled flat plate. The phosphoamino acids were separated by electrophoresis on paper at pH 1.9 (2 kV, 2.5 h). Amino acids and peptides were detected by means of a cadmium-ninhydrin stain or by autoradiography by exposure to Kodak X-ray film in light-tight film holders. Radioactive slab gels were also processed in this way.

Cyanogen Bromide

Cleavage

PAS-l and spectrin were cleaved with CNBr in 70% formic acid with a 200-fold weight excess of reagent over protein for 10 h at room temperature. The reaction mixture was diluted with 10 vol of water and lyophilized. Band 3 and its 55K dalton chymotryptic derivative formed turbid suspensions in 70% formic acid which failed to clarify even after addition of reagent. Moreover, when the mixture was lyophilized and the peptides were dissolved in SDS for gel electrophoresis, a large percentage of the protein remained on top of the gel.

PROTEIN

PHOSPHORYLATION

301

Instead, the ethanol-precipitated Band 3 material (see below) could be dissolved directly in 70% trifluoracetic acid to give a clear solution. At the end of the reaction (the same conditions as for PAS-l), the CNBr was blown off with a stream of N,, and the digest could be applied directly to a gel filtration column. Alternatively, a few crystals of SDS were added, and the sample was diluted with water and lyophilized. This material redissolved completely in 1% SDS, and left no residue on top of a gel after electrophoresis. This procedure has recently been used with great success to study the orientation of the Band 3 polypeptide in the erythrocyte membrane (20), and 90-95% cleavage is obtained based on the disappearance of methionine after amino acid analysis.

Labeling

of

Proteins

with ?‘Pi

In cells. Freshly drawn cells (from up to 50 ml of blood) were incubated in a buffer consisting of 10 mM sodium phosphate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM MgS04, 1 mM Call,, 1 mgiml glucose, 50 pgiml streptomycin, and 10 units/ml penicillin G. Then, 5-10 mCi carrier-free 32P1 were added to a 50% suspension, and the cells were incubated at room temperature with intermittent shaking every few hours. They were then washed several times in 0.9% saline to remove large amounts of radioactivity and then lysed. Ghosts. The method of Guthrow et al. (5) was used to label membrane proteins with [y-“*P]ATP (lo-12 Ci/mmol). Following phosphorylation, the ghosts were washed in 20 mM sodium phosphate, pH 6.5, and dissolved in SDS or extracted with Lis as described below to remove spectrin and other loosely bound membrane proteins.

Purification

Procedures

Preparation of erythrocytes. Erythrocytes were obtained from the Boston Chapter of the American Red Cross, and when fresh cells were required, they were drawn into 0.9% saline containing several thousand units of heparin (Sigma). In either case the cells were washed in 0.9% saline and resuspended in an appropriate buffer or else lysed in 5 mM sodium phosphate, pH 8.0, and this process was repeated until the ghosts were pink. Putijication of PAS-I. The major sialoglycoprotein was obtained by the chloroform-methanol extraction procedure of Hamaguchi and Cleve (21). This was followed by chromatography on phosphocellulose (Whatman Pll) (lo), and then by gradient elution from DEAE-cellulose (Whatman DE-52) (22). Although the latter steps separate a few impurities, they failed to resolve other minor sialoglycoprotein components. Pu@ation of‘ Band 3. This polypeptide was purified by the method described by Drickamer (23).

302

LLOYD WAXMAN

After pooling and lyophilizing the appropriate column fractions, the resulting white powder was dissolved in a small volume of water and 9 vol cold 60% ethanol were added which precipitated Band 3 material but left the sialoglycoproteins and SDS in solution. After settling on ice for 30 min, the precipitate was collected by centrifugation, and could be used for phosphate determinations or cyanogen bromide cleavage. In addition, for many experiments, intact Band 3 was ‘purified from 6% SDS polyacrylamide gels to which Lis-extracted material had been applied. The appropriate regions were localized by staining a parallel gel or by autoradiography, and eluted from the gel by shaking the slices overnight at 37°C in a large volume of water. The 55K-dalton chymotryptic derivative of Band 3 was prepared as described by Steck et al. (24) except that the cells had been previously incubated in the presence of 32Pi, as described above. Finally, the trypsinization of ghosts at 0°C was carried out as described by Steck et al. (24) except that in these experiments the membrane proteins had also been previously labeled with 32PSpectrin. To obtain natiie spectrin, extraction of ghosts with 0.1 mM EDTA was performed as described by Fairbanks et al. (25). The extract was then precipitated by the addition of 0.1 vol 1 M sodium acetate, pH 5.1, collected by centrifugation at 5000 rpm for 10 min in a Sorvall SS-34 rotor, and redissolved in a small volume of 1 M Tris-Cl, pH 8.5. This material was then further purified by gel filtration on Sepharose 4B in 10 mM Tris-Cl (pH 8.0), 0.1 mM EDTA, 0.1 M NaCl, or by DEAE ion exchange chromatography in 10 mM Tris-Cl (pH 8.0). Large-scale purification was carried out somewhat differently. Since the EDTA procedure removes at best two-thirds of the spectrin from the membrane, the protein quantitatively extracted using 25 mM Lis, essentially as described by Steck and Yu (26). Following high-speed centrifugation, the supernatant was dialyzed against water at 4°C overnight to remove most of the Lis, and the extract was submitted to isoelectric precipitation as described above. The precipitate was collected, redissolved in 1 M Tris-Cl (pH 8.5), made 3% in SDS, and boiled for 10 min. After incubation at room temperature for 30 min in 1% 2-mercaptoethanol, the solution was applied to a column of Sepharose 4B in 0.1% SDS, 40 mM Trisacetate, pH 7.4. The spectrin, which eluted near the void volume, was pure except for a small amount of band 2.1 (27). Appropriate fractions were then lyophilized, and the power was redissolved in 4-5 ml water. Addition of 10 vol ice-cold ethanol precipitated the spectrin and removed the SDS. The pellet obtained after centrifugation was washed twice more in ethanol, and dissolved in 70% formic acid before cleavage with CNBr. Because the pellet was insoluble

in few other solvents, it was not useful for proteolysis experiments. Consequently, following solubilization in 70% formic acid, the spectrin was dialyzed against 6 M urea at 4°C to remove the formic acid, and then against water to precipitate the protein as a fluffy mass which was readily digested by all proteases which were tested.

Analytical

Methods

Protein concentrations were estimated by a modification of the method of Lowry et al. (28) using bovine serum albumin as the standard. These determinations were often done in the presence of 0.2% SDS to solubilize membrane proteins. Sialic acid was assayed by the resorcinol method as modified by Jourdian et al. (29) or by the thiobarbituric acid method of Warren (30). Lipid was extracted from protein by the procedure of Goldstein and Hasty (31). Amino acid analysis were carried out on a Beckman Model 12OC, and amino sugars were detected on this instrument as previously described (32). The dansyl reaction and the detection of dansyl end groups were performed as described by Weiner et al. (33). To assay labeled membrane proteins or fragments which had been separated on SDS-polyacrylamide gels, the stained gels were scanned on a Zeiss PM &II spectrophotometer with a linear transport accessory. The gels were then cut into 2-mm slices, which were placed in individual glass vials containing 0.2 ml 30% H202, and incubated at 110°C for 2 h to dissolve the gel. Then 2 ml of Aquasol (New England Nuclear) were added to each after the residue had been dissolved in 0.2 ml 50% acetic acid, and the samples were counted in a Beckman liquid scintillation spectrometer. Total inorganic phosphate was determined by the method of Ames (34). Since the ashing procedure was impractical for large amounts of protein, samples were first hydrolyzed in polypropylene tubes in 1 N NaOH for 1 h at 110°Cto liberate base-labile phosphate. This mixture was then measured directly for free phosphate, and a small aliquot was taken for quantitative protein determination by amino acid analysis. Generally 0.5-1.5 mg were used in each assay. Protein-bound phosphate was also measured by determining the specific radioactivity of membrane polypeptides obtained from cells incubated with 32Pi for 50-60 h. At these times, the specific radioactivity of the proteins reaches a constant level, and the y-phosphoryl group of exchangeable ATP has equilibrated with the pool of inorganic phosphate (35). Thus, the specific radioactivity of free phosphate should be a direct measure of the specific radioactivity of that pool of cellular ATP which is exchangeable. Membrane polypeptides were fractionated by SDS-gel electrophoresis, the appropriate slices of the gel were hydrolyzed in 6 N HCl for 24 h, and the amino acids

MAJOR ERYTHROCYTE

MEMBRANE

303

PROTEIN PHOSPHORYLATION

glycoproteins levels off within several hours, while incorporation into spectrin continues for at least 2 days. As first noted by Palmer and Verpoorte (361, and then by Guthrow et al. (5), only the smaller of the spectrin polypeptides is phosphorylated. The amount of covalently bound phosphate was quantitated in two ways as described under Materials and RESULTS Methods, and the results are presented in Spectrin Phosphorylation Table I. Highly purified spectrin contains Although a large number of polypeptides 3.5-4 mol total phosphate, although only are apparently phosphorylated in the 2 mol are exchangeable. Moreover, both erythrocyte membrane, it would be quite serine and threonine residues are modified difficult to try to correlate any of these with (Fig. 2). It was of interest, then, to a specific membrane function. For several of determine the distribution of phosphorylated sites after limited tryptic digestion of the the major components, however, tentative native material. Unfortunately, these sites roles have been ascribed, and in this are rapidly removed and digested by paper an examination of their phosphorylatrypsin even when the protein itself has tion has been undertaken. been only partially degraded (Fig. 3). This Figure 1 shows that addition of phosphate suggests that the sites may be at one or both to a suspension of red cells results in a ends of the spectrin polypeptide. time-dependent incorporation of [3zP]phosWhen spectrin purified by column chrophate into three major polypeptides of the erythrocyte membrane: spectrin, Band 3, matography in SDS to separate it from and PAS-l. The incorporation into the two other proteins and low molecular weight phosphorylated compounds such as lipids and nucleotides was subjected to trypsin released were quantitated by amino acid analysis while a portion of the hydrolysate was assayed for radioactivity. Concomitantly, the inorganie phosphate in the red cells was isolated by ion-exchange chromatography by the method of Bartlett (361, and its specific radioactivity was determined. In these experiments the concentration of phosphate was 10 mM and the specific activity ranged from 40 to 180cpminmol.

I

I

TABLE I QUANTITATIONOF PHOSPHATE CONTENTOF SPECTRIN

0

20

30 40 TIMEthours)

50

Moles of phosphate per 450,000 g Number of spectrin determinations

60

FIG. 1. Time course of incorporation of radioactivity into erythrocyte membrane proteins. Erythrocytes were incubated with 32Pi and glucose as an oxidizable substrate as described under Materials and Methods. Cells were withdrawn at the times indicated, ghosts were made, and the proteins were separated on SDS-polyacrylamide gels. In the cases of Band 3 and PAS-I, the Lis pellet and the crude CHCl,-CH,OH extracts were used, respectively. The appropriate bands were excised and hydrolyzed in 6 N HCl. Protein was then quantitated by amino acid analysis, and aliquots of the hydrolyzate were counted. The specific activity in this experiment was 84 cpminmol. Molecular weights of 450,000, 95,000, and 31,000 were assumed for the spectrin dimer, Band 3, and PAS-l, respectively.

Procedure 1” (determined as cold phosphate)

3.6 f 0.2

6

Procedure 2” (determined from specific activity of labeled spectrin)

2.1 2 0.3

6

Procedure lb

3.65 + 0.2

2

Procedure 2

2.05 + 0.2

n See Materials and Methods for description of each procedure. b Both procedures were applied to the same sample to test the consistency of these determinations.

LLOYD WAXMAN

‘i

fractions which eluted from the peptide column and partially hydrolyzed, phosphothreonine was found primarily in the first radioactivity-containing peak while phosphoserine was found in all three peaks (Fig. 3). Preliminary experiments with CNBrcleaved spectrin show that the majority of phosphate is in two fragments of molecular weight 18,500 and 14,000 which are resolved on SDS-polyacrylamide gels (Fig. 3). PAS-l

Phosphorglation

The possibility that PAS-l might be a phosphoprotein was suggested by the observation that the labeling patterns on SDS-polyacrylamide gels of ghosts from cells incubated with 32Pi and ghosts incubated SW-P with [Y-~~P]ATP were not identical. When these two preparations were first extracted with 25 mM Lis to remove peripheral proteins (see Materials and Methods) and then electrophoresed on SDS-polyacrylThr-P amide gels, two major peaks of radioactivity were observed in the case of ghosts derived from cells incubated with i 32Pi, but only one in ghosts incubated with [Y-~*P]ATP (Fig. 4). Thus, in contrast to the FIG. 2. Autoradiogram of the hydrolysis products of findings of Shapiro and Marchesi (3’7),PAS-l 32P-labeled spectrin. 3ZP-labeled spectrin was hydrolyzed in 2 N HCl for 4 (left) or 8 (right) h, and the could not be significantly phosphorylated in products were separated by electrophoresis at pH ghosts by the methods employed in these experiments. 1.9, 2 kV, 2.5 h. Phosphoamino acids were identified Purified PAS-l is depicted in Fig. 5 which by the inclusion of standards which were localized by staining with ninhydrin. shows that the phosphate label travels with the protein as well as the sialic acid. The amino acid composition of PAS-l was treatment and the digest was fractionated in agreement with the published values (38). by gel filtration, several radioactive peaks In order to identify the phosphoamino were obtained (Fig. 3). This pattern was acids present in PAS-l, samples which had highly reproducible and was the same been purified from erythrocytes previously whether the spectrin was phosphorylated in incubated with 32Pi and glucose (see cells or in ghosts. In addition, spectrin Materials and Methods) were hydrolyzed, prepared as described in this paper is and the radioactive products separated by readily solubilized by trypsin because paper electrophoresis (Fig. 5). Clearly, this negligible amounts of protein were found to protein contains both phosphoserine and elute in the void volume of the peptide phosphothreonine; the extraneous radiocolumn (Fig. 3). It is not clear at this time active spots are probably due to lipid if the three fragments are unique. Further contaminants because they are not seen if degradation does not occur if each is the protein is passed over a gel filtration redigested with trypsin, and other proteo- column in SDS. lytic enzymes are being used to explore this Quantitation of the actual number of question. When aliquots were taken from moles of phosphate per mole of protein is

MAJOR

ERYTHROCYTE

MEMBRANE

PROTEIN

PHOSPHORYLATION

305

FRACTION E

B

4000

,:: zk2t

3000

1

LYY

-I

F

I='3

2003 1000 0K 0 mm from top of gel

20

40 60 mm from lop of gel

01

Frc. 3. The fragmentation of 32P-labeled spectrin. Left. Time course of digestion of native spectrin by trypsin. “‘P-labeled spectrin extracted from red cells incubated with 32P1was purified by DEAEcellulose chromatography and incubated with trypsin (150:1, w/w) at 0°C in 10 mM Tris-Cl, pH 8.0. The reaction was stopped at 1 min by boiling in 2% SDS. Samples (20 kg) were electrophoresed on 6% SDS-polyacrylamide gels which were stained, scanned, sliced, and counted. A: 0 min. B: 1 min. C: Distribution of radioactivity at 0 and 1 min. D: Gel filtration of a tryptic digest of “*P-labeled spectrin. Y’P-labeled spectrin (50 mg) was purified by column chromatography in SDS and digested with trypsin (50:1, w/w) for 16 h in 1% NH,HCO, with an equal addition of trypsin at 8 h. The reaction was terminated by the addition of phenylmethane sulfonyl fluoride and applied directly to a P-10 column (2.2 x 125 cm) in 1% NH,HCO,. The flow rate was 9-10 ml/h, and 3.5-ml fractions were collected. A total of 50 ~1 was taken to assay for radioactivity. E: Autoradiogram of the hydrolysis products of :‘*P-labeled tryptic fragments of spectrin separated by gel filtration; 500-~1 aliquots of various column fractions were hydrolyzed for 8 h, and the products were separated as in Fig. 2. The anode is at the top. F: Separation of “2P-labeled spectrin CNBr cleavage products. “2P-labeled spectrin was treated with CNBr in ‘70% HCOOH (16 h, lOO:l, w/w), and the products were separated on urea-SDS-104 polyacrylamide gels (14). The gels were stained, sliced, and counted.

complicated by the fact that, as a membrane protein, PAS-l binds large amounts of lipid very tightly, and initially very high values were obtained. A.fter delipidation by the procedure of Goldstein and Hasty (31),

however, the major glycoprotein was found to have 0.2 mol phosphate/m01 protein (Table II). Alternatively, PAS-l was purified by SDS-polyacrylamide gel electrophoresis.

306

LLOYD WAXMAN

FIG. 4. Comparison of glycoprotein phosphorylation in cells and ghosts. Cells were incubated with 32P,,and ghosts were phosphorylated with [yz2P]ATP as described under Materials and Methods. Ghosts were then prepared from the S2Pi-labeled cells, and both preparations were extracted with 25 mM Lis. The 10% SDS-polyacrylamide gels were run on Lis pellets from cells incubated with 32PI(A) and ghosts labeled with [Y-~*P]ATP (B).

The crude extract from the procedure of Hamaguchi and Cleve (21) was run on 10% gels which were then stained with Schiff’s reagent to locate PAS-l, and the appropriate region was cut out and acid hydrolyzed. The results (Table II) show that PAS-l still contains about 0.15 mol phosphate/m01 protein. In order to locate the actual sites of phosphorylation on PAS-l, advantage was taken of the fact that the sequence of this protein has already been published (39). After fragmentation with CNBr (Fig. 6) it was clear that the radioactivity travelled with a fragment of molecular weight 6-7000 which suggested that this had to be the C-terminal peptide (40). Delipidated PAS-l was then treated with trypsin (10 mg protein, 0.25 mg trypsin at 37°C for 18 h in 1% NH,HCO,). As has been noted

in several reports, a cloudy precipitate develops which can be spun out and which represents the hydrophobic core of this protein (41, 42). When this procedure was carried out on undelipidated material, the precipitate was found to be highly radioactive but did not contain phosphoamino acids. The soluble portion of the sample was placed on a column of Sephadex G-75 (Fig. S), and no counts were found to be associated with the large glycopeptides. The major peak of radioactivity was pooled and lyophilized, and chromatographed on paper (butanollpyridinelacetic acid/water, 15:10:3:12). The peptide was located by autoradiography, and its composition and N-terminus (lysine) agreed with that of one of the fragments isolated by Tomita and Marchesi (38). Further digestion with chymotrypsin (25 pglO.25 mg peptide for 6 h) (Fig. 6) gave a smaller fragment which had most of the radioactivity and whose composition corresponds to that of fragment CH 5 (29). The amino acid compositions of these peptides is shown in Table III. Thus, the phosphorylation sites on PAS-l are located in a small segment near the C-terminal which is completely exposed to the cytoplasmic surface. This result agrees with the data of Shapiro and Marchesi (37), although the actual amount of phosphate in the PAS-l molecule reported here, while low, is significantly greater than that found by these investigators (0.01 mol/mol). This discrepancy may be due to the fact that neither the ATP nor PAS-l had attained constant specific activity in their experiments (37). Band 3 Phosphorylation

When Band 3 material was isolated from cells which had been incubated with 32Pi or from ghosts phosphorylated with [y-32P]ATP (23) and then submitted to partial acid hydrolysis, electrophoresis of the products gave the same distribution of radioactivity (Fig. 7). This glycoprotein therefore contains primarily phosphoserine and relatively little phosphothreonine. Quantitation of the amount covalently bound phosphate in Band 3 also required careful delipidation (31). Following this

MAJOR

ERYTHROCYTE

MEMBRANE

PROTEIN

307

PHOSPHORYLATION

Ser-P Thr-P

mm

from

top of gel

FIG. 5. Purified PAS-l contains covalently bound phosphate. Left: Gel scan of the PAS-l component. PAS-l was purified from V,-labeled cells as described under Materials and and electrophoresed on 6% SDS-polyacrylamide gels which were stained for carbohydrate periodate-Schiff method (18), sliced, and counted. Right: Autoradiogram of the hydrolysis of 32Pi-labeled PAS-l. Purified glycoprotein was acid hydrolyzed in 2 N HCl for 8 h, and the were separated as described in Fig. 2.

treatment, nearly 1 mol phosphate/m01 protein was obtained (Table II). Band 3 was also isolated after electrophoresis on 10% SDS-polyacrylamide gels of Lis-extracted ghosts obtained from cells incubated with the gels. After staining with Coomassie blue, the Band 3 region was excised and hydrolyzed, and an aliquot was counted to quantitate radioactivity while total protein was determined from amino acid TABLE

II

QUANTITATION OF COVALENTLY BOUND PHOSPHATE IN BAND 3 AND PAS-1

Band 3 PAS-l

Cold phosphate bound” (mol/mol)

Radioactive phosphate bound” (molimol)

0.90 + 0.05' 0.20 + 0.1

0.35 + 0.1 0.15 2 0.05

a Determined by method of Ames (34) after lipid extraction (31) and alkaline hydrolysis. See Materials and Methods. * Determined from specific activity of proteins isolated from cells incubated with 32Pi. See Results. c Each value is the average of six determinations.

purified Methods by the products products

analysis. The results of this experiment (Table II) show that Band 3 contains about 0.35 mol phosphate/m01 protein, again suggesting that some of the covalently bound phosphate does not equilibrate with the pool of phosphate in the cell. Initially, Band 3 was fragmented with CNBr, and the distribution of the peptides on SDS-polyacrylamide gels is shown in Fig. 7. From the amino acid composition of Band 3 (39), there are probably nine methionine residueslmol, and it is quite possible that a number of smaller fragments eluted from the gel either during electrophoresis or staining and destaining. The fragmentation pattern did not change with longer periods of digestion and indicates that Band 3 probably contains more than one site of phosphorylation. Band 3 was then cleaved by proteolysis with either papain or trypsin as described by Steck et al. (24). The most revealing experiment was to carefully trypsinize depleted ghosts isolated from cells that had been previously incubated with “Pi. This procedure has been shown to release a 41K-dalton water-soluble peptide from the cytoplasmic surface while leaving a

308

LLOYD WAXMAN

dJ,JJm 0

20

40

20

60

80 0 20 mm from top of gel

40

60 80 FRACTION

I

40

60

100

120

50r

1

1

0

80

20

40

60 FRACTION

80

b-+--f0 100

120

FIG. 6. Localization of the phosphorylation sites in PAS-l. SDS-polyacrylamide gel electrophoresis (10% acrylamide, 1% methylene bisacrylamide) was carried out on intact (A) and CNBr-cleaved (B) V-labeled PAS-l in the buffer system of Swank and Munkres (14). Gels were sliced and counted as described under Materials and Methods. The large peak of radioactivity at the bottom of the gels is due to labeled lipid. C: Separation of water-soluble tryptic peptides from PAS-l. Peptides were resolved on a column of G-75 in 1% NH,HCO, (1.5 X 90 cm).3-ml fractions were collected at a flow rate of lo-12 ml/h, and analyzed for radioactivity by scintillation counting and sialic acid by the resorcinol method (29); 50-~1 aliquots were taken for both assays. Fractions 71-80 were pooled for further analysis. D: Separation of the chymotryptic digest of a radioactive tryptic fragment from PAS-l. The digest was resolved on a column of G-50 (1.5 x 90 cm) in 1% NH,HCO,. The flow rate was 12 ml/h, and 1.5-ml fractions were collected; 50 ~1was taken to assay for radioactivity. Fragment I was rich in proline and corresponds to CH 4; fragment II was high in glutamic acid and corresponds to CH 5 (39).

52K-dalton fragment in the membrane (24). Further work with the 52K-dalton piece was hindered by the fact that it runs in a region with several other proteins on SDSpolyacrylamide gels. However, it can be seen that this fragment does not seem to be phosphorylated (Fig. 7). When the water-soluble fragments were

examined for radioactivity, the 22K-dalton piece, which wasshown by Steck et al. (24) to be derived from the 41K fragment by prolonged trypsinization, did contain label, while the other product of this cleavage, a 16K-dalton fragment did not. Thus, the major sites of phosphorylation of Band 3 are situated on a 22K-dalton fragment

MAJOR

ERYTHROCYTE

TABLE AMINO

ACID

MEMBRANE

III

COMPOSITION OF PHOSPHOPEPTIDES ISOLATED FROM PAS-l”

Peptide Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine NH,-Terminal u The compositions drolysates.

Tryptic

Chymotryptic

2.2 0.1 0.0 5.2 2.2 5.8 4.3 6.1 0.3

0.3 0.1 0.2 2.0 1.1 2.6 4.2 1.3 0.1

1.0

0.1

3.5 0.0 1.0 2.0 0.1 0.1

1.2 0.0 1.0 0.2 0.0 0.1

Lysine

Serine

are the average of two 24-h hy-

which has been shown to be at the end of the 95K polypeptide which is also in contact with the cytoplasm (24). Recent work by Jenkins and Tanner (43) has called into question the position of this fragment in the Band 3 polypeptide. Nonetheless, the 22K-dalton piece does contain the major phosphorylated sites of this protein. Finally, it can be seen that in chymotrypsin-treated phosphorylated cells (Fig. 7), the majority of radioactivity now travels with the 55K-dalton component which is known to span the membrane bilayer (24,30). The other product of this cleavage, a 38K-dalton peptide is very labile (24), but superposition of the Coomassie-stained gel and the autoradiograph show that it is not phosphorylated. Since it is thought not to extend to the cytoplasmic surface, it would be expected that this fragment not be phosphorylated. A phosphorylated polypeptide can be seen which seems to arise as the result of treating cells with trypsin

PROTEIN

PHOSPHORYLATION

309

and which migrates just slightly more slowly than the 38K-dalton fragment. This is probably a cleavage product of PAS-l because it is the only other major phosphoprotein in NaOH-extracted ghosts. Variability

of Protein

Phosphorylation

To determine whether any agents which alter red cell deformability have an effect on the phosphorylation of membrane proteins, two kinds of experiments were performed. In the first, protein kinase assays were carried out in the presence of different concentrations of these substances and [Y-~~P]ATP; the membrane proteins themselves served as phosphate acceptors. Samples of the various membrane preparations were examined by SDS-gel electrophoresis and compared to controls after slicing and counting the gels. No change in incorporation of radioactivity into protein was found with epinephrine, isoproterenol, prostaglandin E, (lo-“-lo-” M), or with phalloidon (1O-y-1O-6 M), a mushroom metabolite known to accelerate the polymerization of actin (44). Thus, these compounds do not act directly on a membrane protein to render it more available to phosphorylation. CAMP (1 PM), on the other hand, was found to elevate the levels of phosphorylation only slightly in spectrin, but nearly lo-fold in a component of 48,000 daltons, in agreement with earlier reports (5, 45) (Table IV). In a second experiment, aliquots of cells which had been incubated with “‘Pi for 4-5 h were transferred to centrifuge tubes containing substances known to change erythrocyte deformability, and incubated for lo-15 min at 37°C. These are the conditions employed by Allen and Rasmussen in their deformability assay (46), except that the proteins are now prelabeled with 32P. After the incubation period, the cells were lysed in ice-cold 5 mM sodium phosphate (pH 8), and ghosts were prepared. Over the same concentration range as used in the first experiment, no reproducible changes were seen in the state of phosphorylation of any major membrane protein. This result suggests that new sites are not phosphorylated nor are occupied ones

310

LLOYD

WAXMAN

Ser-P

Thr-P

B

20

40 60 mmfrom top of gel

.20

.40 MOBILITY

D

I 0

.60

80

FIG. 7. Localization of the phosphorylation sites in Band 3. A: Partial acid hydrolysis of Band 3 purified from cells which had been incubated with Vi for 48 h was carried out in 2 N HCl for 8 h, and the products were separated as described in Fig. 2. The anode is at the top of the autoradiogram. C: SDS-polyacrylamide gel electrophoresis of the CNBr fragments of Band 3. CNBr fragments were prepared from Band 3 material purified on Sepharose 4B in Triton X-100 (23), and run on 10% acrylamide gels in the system of Swank and Munkres (14). The gel was scanned, sliced, and counted as described under Materials and Methods. The approximate molecular weights of the fragments are: A-20,000; B (doublet)-15,100 and 13,500; C (doublet)-11,000 and 10,000; D (doublet)-6700 and 5900. B.: SDS-polyacrylamide slab gel of the proteolytic cleavage products of Band 3. Ghosts from 32Pi-labeled cells were subjected to various proteolytic cleavages described by Steck et al. (24) and then electrophoresed on 10% acrylamide gel slabs. The gels were then stained and autoradiographed. The upper photograph is the autoradiograph of the lower, Coomassiestained gel slab. From left to right: (1) depleted ghosts (extracted with 150 mM NaCl [O”C, 30 min] followed by 0.1 InM EDTA [37“C, 15 min]); (2) 0.1 N NaOH-extracted ghosts from trypsin-treated cells; (3) 0.1 N NaOH-extracted ghosts from chymotrysin-treated cells first treated with trypsin (55K-dalton derivative of Band 3; (4) molecular weight standards; (5) depleted ghosts treated with trypsin, followed by extraction with 0.1 N NaOH; this treatment leaves the membrane-bound 52K-dalton fragment of Band 3; (6) the supernatant after depleted ghosts were treated with trypsin and then spun out. In this experiment only a small amount of the 41K-dalton fragment was produced, but a large amount of the 22K-dalton peptide was produced. D: Molecular weight calibration curve for the SDS-polyacrylamide gel ofthe CNBr fragments of Band 3, shown at the right. The standards used were: 1 -aspartate transcarbamylase, R chain; 2-cytochrome c, dimer; 3-aspartate transcarbamylase, C chain; 4-cytochrome c; 5 through ‘I-cytochrome c, CNBr fragments, l-80, l-65, 66-104.

MAJOR

ERYTHROCYTE

MEMBRANE TABLE

PROTEIN

311

PHOSPHORYLATION

IV

PROTEIN KINASE SPECIFICITY IN THE PHOSPHORYLATION OF ERYTHROCYTE MEMBRANE PROTEINS” Counts per minute incorporated Conditions A. Control Control Control Control Control

+ + + +

1 ~.LMCAMP 1 PM CAMP - 5 pgiml I* 1 PM CAMP + 50 pgiml I” 50 Fgiml I”

B. Control Control Control Control cGMP

+ + + +

C subunit” (5 @g/ml) 50 ItIM Mg(CH,COO)z 50 mM Mg(CH,COO), + 4 PM cG kinaseh (10 pg/ml)

Spectrin

Band 3

48K

9,250 10,400 11,400 9,600 12,000

12,800 12,600 12,900 11,600 12,250

650 5,250 2,700 250 400

2,400 4,400 1,250

1,150 2,750 1,300

150 7,900 150

1,150

500

150

” Phosphorylation of membranes was carried out in 100 pl(80 pg protein) exactly as described by Guthrow et al. (5). The reaction was stopped by the addition of 25 ~1 10% SDS, and the entire sample was electrophoresed on 6% SDS-polyacrylamide gels. The gels were sliced and counted, and the counts per minute in each protein were estimated from its area on a plot of counts per minute vs gel slice. In (A) phosphorylation was allowed to proceed for 5 min; in (B) the reaction was stopped after 1 min. The specific activity of the ATP was 100 cpmfpmol. Note that in (B), MgZ+ apparently inhibits endogenous kinase activity. b I is the heat stable protein kinase inhibitor from rabbit skeletal muscle (19), C is the catalytic subunit of the CAMP-dependent protein kinase (17), and cG kinase is the cGMP-dependent protein kinase from bovine cardiac muscle (18).

dephosphorylated when cells are treated with these compounds, but it does not eliminate the possibility that their mode of action may be through changing the rate of turnover of phosphate in one or more proteins. Specificity

of Protein

Phosphorylation

Although the human erythrocyte appears to lack adenylate cyclase activity (l), the phosphorylation of several membrane components has been shown to be enhanced by the addition of CAMP (5, 45). That this effect is most clearly due to a CAMPdependent protein kinase is shown in Table IV. By the addition of the inhibitor which is specific for this kinase, and therefore useful as a diagnostic tool, it can be seen that phosphorylation of the 48K-dalton component, which is CAMP-dependent, is completely eliminated, while phosphorylation of the other major phosphoproteins is unchanged. Moreover, when free catalytic subunit of the CAMP-dependent protein kinase from skeletal muscle is incubated

with ghosts and [Y-~~P]ATP, only the 48K-dalton component is significantly phosphorylated. The cGMP-dependent protein kinase, which seems to have a specificity similar to that of the CAMP-dependent enzyme, did not phosphorylate any of the membrane proteins even under conditions which are optimal for this enzyme (18). DISCUSSION

The results presented above show that spectrin, Band 3, and PAS-l polypeptides are all phosphorylated at several sites. I have shown that the major sialoglycoprotein of the human erythrocyte, PAS-l, is phosphorylated when cells are incubated in the presence of 32Pi. In agreement with several reports, however, incubation of ghosts with [Y-~*P]ATP produces no detectable phosphorylation of this component. This suggests that the sites of phosphorylation of this component. This suggests that the sites of phosphorylation are nearly completely occupied and turn over very slowly, or that the kinase specific for this protein has been

312

LLOYD WAXMAN

lost during cell lysis. The very low levels (less than 0.2 mol/mol) of phosphate found in PAS-l may indicate that this form of protein modification has neither physiological nor functional significance. On the other hand, one cannot rule out the possibility that a subpopulation of PAS-l actually carries most of the phosphate and that this could have an important role in cell function. It is interesting in this regard that people whose erythrocytes are missing this protein are otherwise normal (47, 48). Band 3 is of greater interest because it is a constituent of the anion carrier (38,49), and may include the transport system for water as well (50). Moreover, an increasing body of evidence suggests that it is a homogeneous protein, and that it is at least a dimer in the membrane (23, 24, 27). Although previous reports have shown that the phosphorylation of Band 3 is not CAMP-stimulated (9, 45), its modification may still play an important functional or structural role. Steck’s laboratory (51, 52) has shown that both glyceraldehyde 3-phosphate dehydrogenase and aldolase bind to Band 3, and from the data presented here, this probably occurs near the phosphorylated region of the polypeptide. However, the same component is phosphorylated in the erthrocytes of a number of animals, including the rat, rabbit, cow, dog, and chicken (L. Waxman, unpublished observations). Not unexpectedly, the distribution of radioactivity among the CNBr fragments when the protein has been phosphorylated in cells or in ghosts is nearly the same as it is in the human red cell. Spectrin is also phosphorylated and it contains approximately 4 mol of phosphate/m01 of protein, although 2 mol must turn over very slowly or not at all; these results are in substantial agreement with a recent report by Wolfe and Lux (53). On the other hand, Wyatt et al. (54) have found that a single peptide contains most of the label which was incorporated into spectrin after incubating red cells for 20 h in the presence of 32Pi. Disagreement with the data presented in this paper may be explained by arguing that only the most rapidly exchanging site is significantly labeled at 20 h, or that the very low

levels of radiolabel used by Wyatt et al. (54) would make it difficult to detect any but the most heavily labeled site. The biological significance of spectrin’s phosphorylation is not completely clear, but recently Birchmeier and Singer (55) have shown that changes in the shape of erythrocyte ghosts are associated with the phosphorylation of a serine residue and argue that this might provide a mechanism for the control of red cell shape by ATP (56). More interestingly, Pinder et al. (57) have found that spectrin can induce the polymerization of muscle actin, and that this effect depends on the phosphorylation of spectrin by a CAMP-independent kinase derived from red cells (58). It is striking that the evidence presented here indicates that several of the major erythrocyte membrane proteins are multiply phosphorylated. This may be interpreted as heterogeneity in the proteins themselves. On the other hand, as Cohen has postulated (59), multi-site phosphorylation might offer a higher form of control than that provided by single-site phosphorylation. In any event, it is clear that there is more than one protein kinase associated with the membrane (‘7,8,60,61), at least one of which is CAMP-dependent. It is possible, therefore, that different sites are recognized by different kinases. Although the significance of the phosphorylation of these proteins is not obvious, it is not likely to be a spurious event. There is evidence that the phosphorylation is specific. Incubation of ghosts with [Y-~*~]ATP, CAMP, and the Walsh inhibitor (14) eliminated the phosphorylation of the 48,000-dalton component, but had little effect on the phosphorylation of spectrin or Band 3. The function of this polypeptide is not known, although Rubin (8) has clearly shown that it is not the regulatory subunit of a CAMP-dependent protein kinase, nor is it an integral membrane protein because it is extracted by 25 mM Lis along with spectrin (L. Waxman, unpublished observations). Consequently, the 48K-dalton protein was readily phosphorylated by the catalytic subunit of the CAMP-dependent kinase from bovine skele-

MAJOR

ERYTHROCYTE

MEMBRANE

tal muscle, while spectrin and Band 3 were poor substrates for this enzyme. Another purified protein kinase, the cGMP-dependent enzyme from bovine heart, did not phosphorylate any of these substrates (Table IV). These results indicate that none of the three proteins in this study contains sites which are recognized by the CAMPdependent protein kinase, although from the sequence of PAS-l (39) and an understanding of the substrate specificity of this kinase (62), several serine residues could be potential sites of phosphorylation. The fragmentation procedures described in this paper for the major erythrocyte phosphoproteins have two important applications. The first of these is to aid in the purification of protein specific kinases from the erythrocyte membranes. Thus, although there has been at least one group which has described the partial purification of several of these enzymes (58, 63), it will be necessary to show that the sites which are phosphorylated in viva are the same as those modified using purified components, and this is now possible. The second application concerns the molecular basis for the observation that the levels of phosphorylation of spectrin in ghosts incubated with [ y-32P]ATP are elevated 30-40% in mothers of patients with Duchenne muscular dystrophy (64). Two possibilities are that new sites are phosphorylated in vivo or that the steady-state levels of phosphate in spectrin are significantly less than 4 mol/mol in the Duchenne red cells and thus many more sites are available to accept phosphate when ghosts are incubated with [y-32P]ATP than in ghosts from normal patients; both alternatives can now be examined. REFERENCES 1. RUBIN, C. S., AND ROSEN, 0. M. (1975) Annu. Rev. Biochem. 44, 831-887. 2. FISCHER, E. H., HEILMEYER, L. M. G., AND HASCHKO, R. H. (1971) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 4, pp. 211-251, Academic Press, New York. 3. WALSH, D. A., AND KREBS, E. G. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. 8, pp. 555-581, Academic Press, New York.

PROTEIN

PHOSPHORYLATION

313

4. LARNER, J., AND VILLAR-PALASI, C. (1971) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 3, pp. 195-236, Academic Press, New York. 5. GUTHROW, C. E., JR., ALLEN, J. E., AND RASMUSSEN, H. (1972) J. Biol. Chern. 247, 8145-8153. 6. ROSES, A. D., AND APPEL, S. H. (1973) J. Biol. Chem. 248, 1408-1411. 7. RUBIN, C. S., ERLICHMAN, J., AND ROSEN, 0. M. (1972) J. Biol. Chem. 247, 6135-6139. 8. RUBIN, C. S. (1975) J. Biol. Chm. 250,9044-9052. 9. FAIRBANKS, G., AND AVRUCH, J. (1974) Biochemistry 13, 5514-5521. 10. MARCHESI, V. T., AND ANDREWS, E. P. (1971) Science 174, 1247-1248. 11. GLYNN, I. M., AND CHAPPELL, J. B. (1964) Biochem. J. 90, 147-149. 12. DAVIES, G. E., AND STARK, G. R. (1970) PTOC. Nat. Acad. Sci. USA 66, 651-656. 13. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 14. SWANK, R. T., ANDMUNKRES, K. D. (1971)Anal. Biochem. 39, 462-477. 15. WEBER, K., PRINGLE, J. R., AND OSBORN, M. (1972) in Methods in Enzymology (Hirs, C. H., and Timasheff, S. N., eds.), Vol. 26, Pt. C, pp. 3-27, Academic Press, New York. 16. GLOSSMAN, H., AND NEVILLE, D. M., JR. (1971) J. Biol. Chem. 246, 6339-6346. 17. BEAVO, J. A., BECHTEL, P. J., AND KREBS, E. G. (1974) in Methods in Enzymology (O’Malley, B., and Hardman, J. G., eds.), Vol. 36, pp. 299-308, Academic Press, New York. 18. GILL, G. N., HOLDY, K. E., WALTON, G. M., AND KANSTEIN, C. B. (1976) Proc. Nat. Acad. Sci. USA 73, 3918-3922. 19. ASHBY, C. D., and WALSH, D. A. (1974) in Methods in Enzymology (O’Malley, B., and Hardman, J. G., eds.), Vol. 36, pp. 350-358, Academic Press, New York. 20. DRICKAMER, L. K. (1977) J. Biol. Chem. 252, 6909-6917. 21. HAMAGUCHI, H., AND CLEVE, H. (1972)Biochem. Biophys. Res. Commun. 44, 390-395. 22. CAREY, J. C., WANG, C. S., AND ALAUPOVIC, P. (1976) Fed. Eur. Biochem. Sot. Lett. 65, 159-162. 23. DRICKAMER, L. K. (1976) J. Biol. Chem. 251, 5115-5123. 24. STECK, T. L., RAMOS, B., AND STRAPAZON, E. (1976) Biochemistry 15, 1154-1161. 25. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochemistry 10, 2606-2617. 26. STECK, T. L., AND Yu, J. (1973) J. Supramol. St?xct. 1, 220-232. 27. STECK, T. L. (1972) J. Mol. Biol. 66, 295-305.

314

LLOYD

28. BAILEY, J. L. (1967) Techniques in Protein Chemistry, 2nd ed., p. 293, Elsevier, New York. 29. JOURDIAN, G. W., DEAN, L., AND ROSEMAN, S. (1971) J. Biol. Chem. 246, 430-435. 30. WARREN, L. (1959)J. Biol. C&m. 234,1971-1975. 31. GOLDSTEIN, J. L., AND HASTY, M. A. (1973) J. Biol. Chem. 248, 6300-6307. 32. WAXMAN, L. (1975)5. Biol. Chem. 250,3796-3806. 33. WEINER, A. M., PLATT, J., AND WEBER, K. (1972) J. Biol. Chem. 247, 3242-3251. 34. AMES, B. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. 115-118, Academic Press, New York. 35. KABAT, D. (1972) J. Biol. Chem. 247, 5338-5344. 36. BARTLETT, G. R. (1959) J. Biol. C&m. 242, 459-465. 37. SHAPIRO, D. L., AND MARCHESI, V. T. (1977) J. Biol. Chem. 252, 508-517. 38. TOMITA, M., AND MARCHESI, V. T. (1975) PT-OC. Nat. Aead. Sci. USA 72, 2964-2968. 39. Ho, M. K., AND GUIDOTTI, G. (1975) J. Biol. Chem. 250, 675-683. 40. SEGREST, J. P., KAHANE, I., JACKSON, R. L., AND MARCHESI, V. T. (1973) Arch. Biochem. Biophys. 155, 16’7-183. 41. WINZLER, R. J. (1969) in Red Cell Membranes: Structure and Function (Jamieson, G. A., and Greenwalt, T. S., eds.), pp. 157-171, J. B. Lippincott, Philadelphia. 42. JACKSON, R. L., SEGREST, J. P., KAHANE, I., AND MARCHESI, V. T. (1973) Biochemistry 12, 3131-3138. 43. JENKINS, R. E., AND TANNER, M. J. A. (1977) Biochem. J. 161, 131-138. 44. WIELAND, T., AND GOVINDAN, V. M. (1974) FEBS Lett. 46, 361-363. 45. RUBIN, C. S., AND ROSEN, 0. M. (1973) Biochem. Biophys. Res. Commun. 50, 421-429. 46. ALLEN, J. E., AND RASMUSSEN, J. (1971) Science 174, 512-514.

WAXMAN 47. DARNBOROUGH, J., DUNSFORD, J., AND WALLACE, J. A. (1969) VOX Sang. 17, 241-255. 48. FURUHJELM, U., MYLLYL~, G., NEVANLINNA, H. R., NORDLING, S., PIRKOLA, A., GAVIN, J., GOOCH, A., SANGER, R., AND TIPPETT, P. (1969) VOX Sang. 17, 256-278. 49. CABANTCHIK, Z. I., AND ROTHSTEIN, A. (1976) J. Membrane Biol. 15, 207-226. 50. BROWN, P. A., FEINSTEIN, M. B., AND SHAAFI, R. I. (1975) Nature (London) 254, 523-525. 51. Yu, J., AND STECK, T. L. (1976) J. Biol. Chem. 250, 9176-9184. 52. STRAPAZON, E., AND STECK, T. L. (1976) Biochemistry 15, 1421-1424. 53. WOLFE, L. C., AND Lux, S. E. (1978) J. Biol. Chem. 253, 3336-3342. 54. WYATT, J. L., GREENQUIST, A. C., AND SHOHET, S. B. (1977) Biochem. Biophys. Res. Commun. 79, 1279- 1285. 55. BIRCHMEIER, W., AND SINGER, S. J. (1977) J. Cell Biol. 73, 647-659: 56. MAKATO, N., NAKAO, T., AND YAMAZOE, S. (1960) Nature (London) 187, 945-946. 57. PINDER, J. C., BRAY, D., AND GRATZER, W. B. (1977) Nature (London) 270, 752-754. 58. HOSEY, M. M., AND TAO, M. (1977) Biochemistry 16, 4578-4583. 59. COHEN, P. (1976) Trends Biochem. Sci. 1, 38-40. 60. AVRUCH, J., AND FAIRBANKS, G. (1974) Biochemistry 13, 5507-5514. 61. FAIRBANKS, G., AND AVRUCH, J. (1974) Biochemistry 13, 5514-5521. 62. KEMP, B. E., BYLUND, D. B., HUANG, T.-S., AND KREBS, E. G. (1975) Proc. Nat. Acad. Sci. USA 72, 3448-3452. 63. HOSEY, M. M., AND TAO, M. (1977) Biochim. Biophys. Acta 482, 348-357. 64. ROSES, A. D., ROSES, M. J., MILLER, S. E., HULL, K. L., JR., AND APPEL, S. H. (1976) N. Engl. J. Med. 294, 193-198.