Purification and Characterization of a Membrane-bound Protein Kinase from Human Erythrocytes*

Transcription

1 THE JOURNAL OF BOLOGCA. CHEMSTRY Vol. 2, No. 6, ssue of March 2, pp Prrnted n U.S.A. Purification and Characterization of a Membrane-bound Protein Kinase from Human Erythrocytes* (Received for publication, October 19,1979) Mariano Tao, Richard Conway, and Safinaz Cheta From the Department of BioZogicaE Chemistry, University of llinois ut the MedicaE Center, Chicago, LEinois A protein kinase which catalyzes the phosphorylation of spectrin and other membrane and non-membrane proteins has been purified to homogeneity from human erythrocyte membranes. The purification procedure was relatively simple and took advantage of the association-dissociation phenomena of the enzyme. After salt extraction of the enzyme from spectrin-depleted membranes, the extract was passed through a Sephadex G-100 column equilibrated with 0.1 M NaC1. The enzyme was eluted at the void volume together with a number of high molecular weight proteins including spectrin. t appears that in low salt, the kinase has a tendency to undergo self-aggregation and also complex formation with spectrin. A second chromatography on Sephadex G-100 in the presence of high salt (0. M NaCl) caused the enzyme to dissociate and elute at a molecular weight of approximately 34,000. This second column separated the enzyme from other high molecular weight membrane proteins. The purified enzyme preparation exhibited a single protein-staining component when electrophoresed in sodium dodecyl sulfate-polyacrylamide gel. Based on sucrose density gradient centrifugation, sodium dodecy1 sulfate-polyacrylamide gel electrophoresis and gel filtration, an apparent molecular weight of approximately 32,000 to 34,000 was estimated for the enzyme. The kinase catalyzed the phosphorylation of band 2 of spectrin, band 3, casein, and phosvitin using ATP as the phosphoryl donor. n addition, incubation of the kinase with [Y-~~P]ATP and Mg+ led to the labeling of the enzyme, presumably through an autophosphorylation reaction. The cell membrane contains a number of enzyme systems which are essential for membrane function. n recent years, considerable interest has been focused on the membrane autophosphorylation system (1, 2). The phosphorylation-dephosphorylation of membrane proteins conceivably could provide a mechanism for alteration or regulation of membrane structure and membrane metabolic activity. nformation concerning membrane phosphorylation has been largely obtained from the study of the human erythrocyte membrane system although similar studies in other cell membranes have also been quite extensive (1, 2). Based on a number of analyses, it appears that human erythrocyte mem- * This work was supported in part by grants from the National nstitutes of Health (AM 2304) and from the American Heart Association (79-701), with funds provided in part by the Chicago Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. branes contain at least two phosphorylating systems: one is catalyzed by a cyclic AMP-dependent protein kinase whereas the other by a cyclic AMP-independent enzyme (3-6). The principal substrates of the cyclic AMP-dependent protein kinase are the minor membrane polypeptides, bands 2.1, 4.1, 4., and 4.8 (). On the other hand, the two major membrane polypeptides, spectrin and band 3, are phosphorylated by the cyclic-amp-independent protein kinase. The significance of membrane phosphorylation, although not completely understood, seems apparent since these same polypeptides are shown to be labeled in vivo when the intact erythrocytes are incubated with [ P]orthophosphate (7). Recently, evidence has been presented to indicate that spectrin phosphorylation may be responsible for the MgATP-dependent shape change of ghosts from crenated spheres to smooth biconcave discs (8, 9). n an effort to gain further insight into the membrane phosphorylation systems and to extend our knowledge of membrane phenomena in general, we have focused our attentions on the cyclic AMP-independent protein kinase and its membrane-bound substrates. Our initial report shows that the cyclic AMP-independent protein kinase can be readily solubilized from the membranes by extraction with high salt (10). The solubilized enzyme catalyzes the phosphorylation of casein but not histones. Although casein is not a natural substrate of the enzyme, it, however, provides a valuable tool for the assay of the enzyme. n this study, we have devised a novel method for the purification of the enzyme. Using this method, a homogeneous enzyme preparation is obtained. Hence, we have now identified a minor erythrocyte membrane protein as a protein phosphotransferase. EXPERMENTAL PROCEDURES Materials Outdated human erythrocytes were obtained from the University of llinois Blood Bank or from the Chicago Blood Services. [y- P]- ATP was purchased from either New England Nuclear or CN. Casein was supplied by Schwarz/Mann and other reagents for enzyme assay were obtained from Sigma Chemical Co. Membrane Preparation The outdated (within 10 days) human erythrocytes were washed with 0.16 M NaCl by centrifugation (2,000 rpm for 10 min) in a JA-10 rotor using a Beckman 2-21 preparative centrifuge. The washing and centrifugation was repeated at least three times; each time, the buffy coat was removed by aspiration. Erythrocyte membranes were prepared essentially according to the procedure of Steck and Kant (11). Briefly, the red cells were lyzed in mm sodium phosphate buffer, ph 8.0, and the membranes sedimented by centrifugation at 10,OOO rpm for 30 min in a JA-10 rotor. The membranes were washed repeatedly with the phosphate buffer until free of hemoglobin as judged by the disappearance of reddish color. f not used immediately, the membranes were stored frozen at -80 C. 263

2 264 Erythrocyte Membrane Protein Kinase Preparation of Spectrin-depleted Ghosts and Spectrin Spectrin was extracted from the human erythrocyte membranes according to the procedure of Marchesi (12). The membranes were extracted twice with 0.1 mm EDTA (ph 8.0) and the spectrin-depleted membranes were stored ftozen at -80 C. Spectrin was precipitated from the combined extracts with ammonium sulfate (0% saturation), dissolved in a ph 7. buffer (Buffer A) containing 0.1 M NaCl, 10 mm sodium phosphate, and rn EDTA, and dialyzed overnight against this buffer. Spectrin was further purified by gel filtration in Sepharose(CL)-4B column (2.6 X 80 cm) according to the procedure of Ralston (13). The major spectrin peak fractions eluted from the column were pooled and concentrated by ammonium sulfate precipitation, dissolved in and dialyzed against Buffer A containing 3 M urea. The spectrin solution was reapplied to a Sepharose(CL)-4B column except that the elution was carried out in the presence of 3 M urea. We found that this latter step was necessary to rid the spectrin of low molecular weight contaminants including actin. The final spectrin preparation was dissolved in Buffer A and kept frozen at -80 C until use. Protein Kinase Assays with 0.1 mm EDTA, ph 8.0, not only released spectrin but also some of the kinase activities. However, the majority of the enzyme activities appeared to remain in the spectrindepleted membranes. To solubilize these enzyme activities from the membranes, solidnaclwas added to the ghost suspension to a final concentration of 0. M and kept at 0" for 30 min. The suspension was centrifuged at 20,OOO rpm for 30 min in a Beckman JA-21 rotor and the membrane residues were extracted once more with?h volume of 0. M NaC1. The two extracts were combined, placed in a dialysis bag, and concentrated at 4 C by covering the bag with flakes of polyethylene glycol (average M, - 20,000). The concentrated salt extract was clarified by centrifugation and applied to a Sephadex G-100 column (2.6 X 8 cm) which had been equilibrated with 20 Tris-HC1, ph 7., buffer containing 1 mm dithiothreitol and 0.1 M NaCl (low salt buffer). The column was eluted with the low salt buffer. Fractions of 4. ml were collected and assayed for kinase activity using Method A. Fig. L4 shows that the major kinase activity peak together with the major protein absorption peak elute as high molecular weight components at the void volume. The active frac- Method A-This method used casein as the phosphoryl acceptor. The assay procedure was essentially the same as that described by Hosey and Tao (10). The reaction mixture (0.2 ml) contained: 0 mm Tris-HC, ph 7.; mm MgCL; 10 mm KC; 2 mg/ml of casein; 0.2 mm [y3'p]atp (20 to 60 cpm/pmol); and enzyme protein. The tions under this peak were pooled and concentrated in a Diaflo incubation was carried out at 37 C for 20 min, unless indicated ultrafiltration unit equipped with a PM-30 membrane. otherwise. The reaction was quenched with 2 ml of 10% (w/v) trichloroacetic acid. The protein precipitated was collected on a Whatman Second Sephadex G-100 Gel Filtration-To the concen- GF/C glass filter disc, washed five times with 2-ml portions of 10% trated enzyme solution, NaCl was added to a final concentratrichloroacetic acid, and counted in ml of Budget-Solve (Research tion of 0. M. The solution was clarified by centrifugation and Products nternational Corp.). Protein concentrations were deter- applied to a Sephadex G-100 column (2.6 X 90 cm) which had mined by the Lowry method (14) or by the dye-binding method (1) been equilibrated with 20 mm Tris-HC1, ph 7., containing 1 using bovine serum albumin as standard. mm dithiothreitol and 0. M NaCl (high salt buffer). Method B-The incubation mixture contained: 0 mm Tris-HC1, ph 8.; 10 mmmgc12; 0.2 mm [y-32p]atp; 1 mg/ml of spectrin; and The elution profile of the enzyme in the high salt buffer is enzyme protein. After terminating the reaction with 10% trichloro- shown in Fig. 1B. n the presence of high salt, the major acetic acid, 0.2 mg of bovine serum albumin was added to co-precipi- kinase activity peak was eluted as a low molecular weight tate the spectrin. Other experimental conditions were as described in component and clearly resolved from the high molecular Method A. weight protein contaminants. The small amount of kinase Method C-Spectrin phosphorylation was conducted as in Method activity eluted at the void volume appeared to represent B except that the volume of the reaction mixture was scaled down to incomplete dissociation. Additional kinase activity was re- 0 pl and the spectrin concentration used was 0.4 mg/ml. The incubation was carried out at 37'C for 20 min and the reaction terminated leased into the low molecular weight component when this by the addition of % volume of a sodium dodecyl sulfate-containing void volume fraction was reapplied to the high salt column electrophoresis sample buffer (30 mm Tris-HC1, ph 8.0; 3 m~ EDTA (data not shown). The active kinase fractions were pooled, 1 % sucrose; 6% sodium dodecyl sulfate; 0.2 mg/ml of bromphenol concentrated by Diaflo ultrafiltration, dialyzed against the low blue; and 20 mg/ml of dithiothreitol) followed by heating in a boiling salt buffer, and stored in liquid nitrogen. water bath for 2 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted in a Hoefer SE 00 slab gel unit at a Comments on Purification current of 6 ma/slab according to the procedure described earlier (). The gels were stained with Coomassie Brilliant Blue R-20 and The use of EDTA-extracted membranes rather than undried on Whatman No. 3" filter papers. The radioactive phosphate incorporated was analyzed by either 1) cutting the spectrin band from treated ghosts as the starting enzyme source allowed us to the dried gel and counting in a liquid scintillation spectrometer or 2) r 1 scanning the radioautogram prepared from the dried gel with a Zeineh Soft-Laser densitometer. This assay method was used since it was more sensitive and required less substrates. t also provided a measurement of 32P incorporation into spectrin alone and not into other proteins, such as the kinase, which may be present in the reaction mixture. The same procedure was also used for the assay of the autophosphorylation of F".<710* NUYBER F".CT!O* *Y*.l" the kinase in the absence of an exogenous phosphoryl acceptor. 1 Preparation of Band 3 Band 3 was prepared according to the procedure described by Yu et ae. (16). The hemoglobin-free ghosts were washed twice with 10 mnn Tris-HC1, ph 8.0, and extracted with volumes of 0.% Triton X- 100 in 6 mm sodium borate, ph 8.0, at 0 C for 30 min. The Triton residues were separated from the Triton extract by centrifugation and FG. 1. Chromatography of crude membrane kinase preparations on Sephadex G-100 under aggregating and disaggregating conditions. A, low salt column. About 6 ml(24 mg of protein) of the concentrated NaCl extract was applied to the column (2.6 X 8 cm). The column was equilibrated and eluted with the low salt buffer. Fractions, each containing 4. ml, were collected and 0-p1 aliquots of alternate fractions were assayed for kinase activity using Method A. washed once with 10 volumes of the Triton/borate buffer. Band 3 The void volume of the column was 130 ml and the specific activity was enriched in the fust extract; the second extract was discarded. of [y-"patp used was 37 cpm/pmol. B, high salt column. About 6.2 ml(16 mg of protein) of the concentrated low salt column void volume RESULTS kinase fraction of which the NaCl concentration had been adjusted to Purification of Membrane Kinase 0. M was applied to the column (2.6 X 90 cm). The column was equilibrated and eluted with the high salt buffer. Other experimental Solubilization-Extraction of the erythrocyte membranes conditions were as described under A and in the text.

3 Low 26 Erythrocyte Membrane Protein Kinase purify both spectrin and kinase from a single ghost prepara- activity. Theconcentratedenzymepreparation could be tion. stored in liquid nitrogen or at -80 C for several weeks with Fig. 2 shows the sodiumdodecyl sulfate-polyacrylamide gel only a slight degree of inactivation. electrophoretic profiles of proteinfractionsobtained from The molecularweight of the kinase has been estimated various stages of purification. T h e gel shows that the salt using various methods. On Sephadex G-100 column, the enextract(lane 3) is enriched in minormembraneprotein zyme exhibited a molecular weight of about 34,000. A slightly components. A small amount of spectrin was also extracted lower value, about 32,000, was obtained in sodium dodecyl by the high salt. Passage of the salt extract through the low sulfate-polyacrylamide gel electrophoresis andin sucrose densalt column removed someof the low molecular weight com- sity gradient centrifugation (data not shown). Studies using ponents migratingin the band4. and 29,000 to 43,000 regions Sephadex G-100 gel filtration and sucrose density gradient kinase centrifugation were conducted in the presenceof 0. M NaCl. (Lane ). The void volumefractioncontainingthe activity washeavily contaminated with other membrane com-the molecular weight standards used were: phosphorylase h ponents (Lane 4). The kinase preparation obtained from the (94,000); bovine serum albumin (67,000); ovalbumin (43,000); high salt column appeared to be homogeneous and exhibited carbonic anhydrase (29,000); and cytochrome c (12,000). Withcasein asthephosphorylacceptor,the kinase exa single protein-staining band in the gel (Lane 7). The overall purification procedure for a typical preparation hibited a broad ph activity profile. Optimum activity was is summarized in Table. There was a considerable loss of observed a t ph values between 6 and 8.. The apparent K,, enzyme activity during concentration of the salt extract and for casein was estimated to be about 1 mg/ml and that for in the low salt Sephadex G-100 column due perhaps to inac- ATP, 11 VM. tivation. This resulted in a lower degree of purification in Step Substrate Specificity 3 than in Step 2. As expected, the highest degreeof purificathe phosphoryl acceptor specificity of the human erythrotion was obtained from the high salt column. The overall purification was about 190-fold with a yield of approximately cyte membrane kinase was examined using both exogenous 4%. T h e kinase appears to represent a minor component of and endogenous substrates.as shown in Table 11, the kinase exhibited a high degree of preference toward casein and to a the erythrocyte membraneproteins. lesser degree toward phosvitin and spectrin. Histones, protageneral Properties mines, and bovine serum albumin were poor substrates. The T h e kinase wasrelatively unstablein dilute protein solution. phosphorylation of spectrin was further examined using sof not concentrated immediately, the enzyme in the various dium dodecyl sulfate-polyacrylamide gel electrophoresis and fractions obtained from the high salt column rapidly lost its radioautography. Fig. 3 shows that phosphorylation occurs primarily in band 2 of spectrin. The possibility that the purified kinase may also catalyze the phosphorylation of other membrane proteins was examined using intact ghosts, Triton-extracted ghosts (cytoskeleton), and solubilized band 3. Fig. 4 shows that as a result of endogenous kinase activity, bands2 and 3 are heavily labeled with.12pwhen intact ghosts are incubated with [y-""p]atp (Lane 1). The addition of purified kinase did not appreciably increase the labeling of these two components but appeared -94K 3to increase the phosphorylation of several minor membrane *,24.'"67K polypeptides (Lane 2). n the Triton-extracted ghosts, exogenous kinase enhanced the phosphorylationof bands 2,3, and K several minor protein components migrating in the region TU -29K - 12K FG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein fractions obtained from various stages of purification. Electrophoresis was carried out on %gel and 28 pg of protein was applied to each well except for Lane 7 which received only 9 pg. (, 8)intact erythrocyte membranes; (2) EDTA-extracted membranes; (3) NaCl extract; (4) low salt column void volume fraction; () low salt column retarded fraction; (6) high salt column void volume fraction; (7) high salt column retarded fraction. TABLE 1 Substrate specificity of membrane kinase Each incubation mixture contained 10 pg of enzyme protein. The assay was conducted as under "Method A", except the incubation was for 10 min. After termination of the reaction with trichloroacetic acid, 0.2 mg of bovine serum albumin was added as co-precipitating agent. Substrate "P incorporated Casein Phosvitin Spectrin Histones Protamines Bovine serum albumin prnol TABLE Purification of human erythrocyte membraneprotein kinase Step 1. Spectrin-depleted membranes 2. NaCl extract 3. salt fraction (concentrated) 4. High salt fraction (concentrated) ' Volume rnl Total protein mg Total activity units" Specific activity units/mp Purification -fold 1.o One unit of activity is defined as that amount of enzyme which catalyzes the incorporation of 1 nmol of '"P into casein/min Yield c

4 266 Erythrocyte Membrane Protein Kinase t s GTP, UTP, and CTP had no effect. The membrane kinase can partially utilize GTP as the phosphoryl donor (17). n contrast to the cyclic AMP-dependent protein kinase, the purified membrane kinase was not activated but rather slightly inhibited (about 10%) by 1 PM cyclic AMP or cyclic GMP. Effect of Sulfhydryl Reagents The enzyme activity was significantly higher in the presence of 1 mm dithiothreitol. The increase varied from 1 to 0% depending on the enzyme preparation. The addition of 1 mm mercuric acetate completely inhibited the enzyme activity. The inhibition could be partially reversed by 1 mm dithiothreitol. Based on these observations, dithiothreitol (1 mm) 1 Mr -2 MK - lk - -3 MK-.-.. UK - nk - U W -MU CB RA FG. 3. Phosphorylation of band 2 of spectrin. The phosphorylation of purified spectrin was carried out as described under Method C in the presence of 3 pg of membrane kinase (MK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on 48 gel and the radioautogram (RA) was exposed for days. The specific activity of [y- PATP was 260 cpm/pmol. CB, Coomassie blue stain corresponding to molecular weights of 3,000 to,000 (Lane 4). t is of interest to note that the Triton-extracted membranes contain appreciable kinase activity which seems to phosphorylate spectrin to a greater extent than band 3 (Lane 3). Whether the kinase is associated preferentially with spectrin remains to be determined. The Triton extract, which comprised mainly of bands 3, 4.2 (M,- 72,000), and a small amount of spectrin, contained very little if any protein kinase activity (Lane ). Fig. 4 (Lane 6) shows that the solubilized band 3 is a substrate of the purified kinase. Effects of Divalent Cations and Nucleotides on Casein Phosphorylation The divalent cation requirement of the kinase activity was FG. 4. Phosphorylation of human erythrocyte membrane determined using casein as substrate. Table 11 shows that the proteins by the purified membrane kinase. The phosphorylation kinase is most active in the presence of 1 to mm Mg. A reactions were carried out essentially as described under Method C slight activity was observed when Mg was replaced by Mn, in the presence and absence of purified membrane kinase (MK, 3 pg). Ca )+, or Zn +, but not by Cu2+ or Fe. The incubation was for 10 min and the specific activity of [y-, P]ATP was 00 cpm/prnol. Top figure, Coomassie blue-stained gel; bottom The possibility that the casein kinase activity may be affected by various nucleotides was investigated. At 0.2 mm (same concentration as the phosphoryl donor, [y- P]ATP), ADP inhibited the kinase activity about 4% whereas AMP, WK- 61 K - U K -? K- 12K MK figure, radioautogram (3-day exposure). (2) whole ghosts (2 pg); (2) whole ghosts (2 pg) + membrane kinase; (3) Triton-extracted ghosts (30 pg); (4) Triton-extracted ghosts (30 pg) + membrane kinase; () Triton extract (20 pg); (6) Triton extract (20 pg) + membrane kinase.

5 was routinely added to the enzyme preparation during purification. Spectrin Phosphorylation The phosphorylation of spectrin by the purified membrane Erythrocyte Membrane Protein Kinase TABLE 11 Effect of divalent cations on kinase activity The kinase activity was assayed using Method A except that the cation was varied and the incubation was for 10 min. Each incubation mixture contained 10 ue of enzvme orotein. Cation Concentration.'lP incorporated mm pmol Mi Ca' Mn' Fez+ 1 0 CU" ?+,n 1.9 FG. 6. ph profile of autophosphorylation of membrane kinase. The autophosphorylation of membrane kinase (3 pg) was conducted according to "Method C" but using different buffer systems. The phosphorylated samples were electrophoresed on % gel. The specific activity of [y-'('p]atp was 400 cpm/pmol. (M), sodium phosphate; (0-- -O), Tris-HC; (A-A), glycine-naoh. CB RA FG.. Autophosphorylation of membrane kinase. Autophosphorylation was carried out as described under "Method C." The reaction mixture contained 4. pg of membrane kinase and the incubation was for 1 min. The specific activity of [y-"2p]atp was 300 cpm/pmol. CB, Coomassie blue stain; RA, radioautogram. kinase was investigated in greater detail. n contrast to casein phosphorylation, the reaction exhibited a ph optimum at about 8. to 9.0 and a maximum rate at about 10 mm Mg'+. The apparent K,,, for spectrin was estimated to be about 0.4 mg/ml (assayed by Method B). As shown earlier in Fig. 3, only band 2 of spectrin was phosphorylated. Autophosphorylation of Membrane Kinase As shown earlier in Fig. 3 and also in Fig. 4, a radioactive band co-migrating with the membrane protein kinase was observed when phosphorylation of endogenous substrates was camed out in the presence of purified kinase. The possibility that this band may represent phosphorylated kinase was investigated. The membrane kinase was incubated with [y- :"P]ATP and Mg'+; the incubation mixture was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by radioautography. The result shown in Fig. indicates that the kinase can catalyze self-phosphorylation. The selfphosphorylation reaction was time-dependent (data not shown) and exhibited a ph optimum between 8. and 9.0 (Fig. 6). The effect of ph was very similar to that for spectrin phosphorylation. DSCUSSON This is the first report of the purification to homogeneity of a membrane-bound cyclic AMP-independent protein kinase. The procedure used is relatively simple and should be applicable to the isolation of similar enzymes from other membrane preparations. The purified enzyme exhibits a molecular weight of about 32,000 to 34,000 and catalyzes the phosphorylation of the two major erythrocyte membrane proteins, spectrin and band 3. These two proteins were previously shown not to be phosphorylated by the cyclic AMP-dependent protein kinases (, 18). The purified human erythrocyte membrane protein kinase has properties somewhat similar to those of rabbit erythrocyte membrane protein kinase 1 (10, 17). Both these enzymes are extracted with high salt and are found to be unaffected by the regulatory subunit of the cyclic AMP-dependent protein kinase (10). The two enzymes appear to have the same phosphoryl acceptor specificities although the activity of the hu-

6 268 Erythrocyte Membrane Protein Kinase + spectrin mo + spectrin 0 O 20 " 30 1% FRACTON NUMBER % FG. 7. Effects of salt and spectrin on the sedimentation profile of membrane kinase in sucrose density gradient. The sucrose gradients were prepared in either low salt (A, B) or high salt (C, D) buffer. Each gradient contained about 6 pg of membrane kinase and where indicated, mg of spectrin. The centrifugation was carried out in a SW 0.1 rotor at 4,000 rpm for h. Aliquots of 100 pl were assayed for kinase activity using Method A except that incubation was for 30 min. That the fastsedimentingkinasecomponent in Gradient B contained bound spectrin was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of [y3'p]atp was 1 cpm/pmol. man erythrocyte membrane kinase toward phosvitin is considerably less than that of kinase 11. Also, the human kinase has a lower K, for ATP (11 PM) than the kinase 1 (6 PM). The high molecular weight form of human membrane kinase observed in low salt condition may be due to aggregation of the kinase either with itself or with contaminating proteins (such as spectrin) or both. Preliminary studies using sucrose density gradient centrifugation in the presence of high and low salt indicate that the enzyme is "sticky" and can interact either with itself or with spectrin. Fig. 7 shows that in low salt, the kinase sediments as a dimer. However, if spectrin is present, the kinase appears to co-sediment with spectrin. Thus, this property of the kinase may explain its elution profile in the low salt column. As shown in Fig. 2, the salt extract applied to the low salt column contained spectrin among other protein contaminants. t is of interest that the membrane kinase can catalyze selfphosphorylation. The significance of this reaction is not known. t remains to be determined whether the phosphoenzyme represents an intermediate of the phosphotransferase reaction. Autophosphorylation has also been observed in other protein kinase preparations. For example, the cycle AMPdependent protein kinases can catalyze self-phosphorylation of the regulatory (19) and of the catalytic (20) subunit. n a recent report, Hathaway and Traugh (21) showed that a casein kinase isolated from rabbit reticulocytes could similarly catalyze self-phosphorylation. The significance of erythrocyte membrane protein phosphorylation is not completely understood. There is indirect evidence to indicate that spectrin phosphorylation-dephosphorylation may play an important role in determining the shape and deformability of the red cells (8, 9). However, Sheetz et al. (22) believe that a membrane ATPase rather than a spectrin kinase is responsible for controlling the rate of red cell shape transformation. The availability of the purified membrane kinase preparations should permit us to further evaluate the role of protein phosphorylation in these membrane processes and also to determine the effect of phospho- rylation on the activities of other membrane proteins, such as the anion transporter, band 3. Acknowledgments-We thank Susan Estes for her excellent technical assistance and Mr. W. Davis from the Chicago Blood Services for kindly supplying us with red blood cells. REFERENCES 1. Hosey, M. M., and Tao, M. (1977) Curr. Top. Membranes Transp. 9, WeLler, M. (1979) Protein Phosphorylation, Pion Ltd., London 3. Avruch, J., and Fairbanks, G. (1974) Biochemistry 13, Fairbanks, G., and Avruch, J. (1974) Biochemistry 13, Hosey, M. M., and Tao, M. (1976) Biochemistry 1, Fairbanks, G., Avruch, J., Dino, J. E., and Patel, V. P. (1978) J. Supramol. Struct Plut, D. A,, Hosey, M. M., and Tao, M. (1978) Eur. J. Biochem. 82, Birchmeier, W., and Singer, S. J. (1977) J. Cell Biol. 73, Sheetz, M. P., and Singer, S. J. (1977) J. Cell Biol. 73, Hosey, M. M., and Tao, M. (1977) Biochim. Biophys. Acta 482, Steck, T. L., and Kant, J. A. (1974) Methods Enzymol. 31A, Marchesi, V. T. (1974) Methods Enzymol. 32B, Ralston, G. B. (1976) Biochim. Biophys. Acta 4, Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (191) J. Biol. Chem. 193, Bradford, M. M. (1976) Anal. Biochem. 72, Yu, J., Fischman, D. A., and Steck, T. L., Jr. (1973) J. Supramot. Struct. 1, Hosey, M. M., and Tao, M. (1977) Biochemistry 16, Hosey, M. M., and Tao, M. (1977) J. Biol. Chem. 22, Rangel-Aldao, R., and Rosen, 0. M. (1976) J. BLoZ. Chem. 21, Chiu, Y. S., and Tao, M. (1978) J. Biol. Chem. 23, Hathaway, G. M., and Traugh, J. A. (1979) J. Biol. Chem. 24, Sheetz, M. P., Sawyer, D., and Jackowski, S. (1978) in The Red Cell (Brewer, G. J., ed) pp , Alan R. Liss, nc., New York