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Separation of fodrin subunits by affinity chromatography on calmodulin-Sepharose
ANALYTICAL BIOCHEMISTRY 19,364-368
Separation
(1985)
of Fodrin Subunits by Affinity Chromatography on Calmodulin-Sepharose JOHN R. GLENNEY,
JR.* AND KLAUS
WEBERt
*Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138, and tThe Max-Planck-lnstitute/br Biophysical Chemistry, D-3400 Goettingen-Nikolausberg. West Germany Received May 24, 1985 Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Caz+dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 M IU + 1 mM Ca*+ and applied to CaM-Sepharose the fi subunit (235kDa) passed through unretarded whereas the a subunit (240-kDa) bound and could be eluted with ethylene glycol his@-aminoethyl ether)N,N’-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed. Q 1985Academic hss, Inc. KEY WORDS: calmodulin; spectrin; fodrin; electron microscopy; proteins; protein subunits.
Spectrin is the major membrane-skeletal protein of the red blood cell and an analog, termed fodrin or nonerythroid spectrin, is present in most other cells (1). The spectrins are composed of two nonidentical high-molecular-weight subunits which self-associate to form first heterodimers and then tetramers. Although one of these subunits (M, 240,000) seems identical between different spectrins in avian cells (2) the subunits are clearly two separate gene products in mammalian cells (3). One characteristic of spectrin is its ability to interact with calmodulin Ca2+-dependently. Using a gel overlay technique it was shown that this binding activity resides in the common 240,000 iI& subunit of avian spectrins (2,4) and has been detected in many cell types (5). Given the potential importance of the Ca*+-calmodulin regulation of the subcortical microfilament system we have been analyzing this interaction between nonerythroid spectrin and calmodulin in some detail. We were surprised to find that although this interaction is relatively insensitive to changes in pH, an increase in salt concentration actually enhanced 0003-2697185 $3.00 Copyright All i-i&s
8 1985 by Academic Press. Inc. of reproduction in any form re?en’ed.
this interaction. In addition, it was found that the subunits of spectrin dissociate in the presence of molar amounts of chaotropic salts. This allows the rapid and quantitative separation of fodrin subunits based on the calciumdependent affinity chromatography of 240kDa fodrin on a CaM-Sepharose column in the presence of chaotropic salts. When the procedure was used with human erythrocyte spectrin, only the 240-kDa subunit was retained by CaM-Sepharose to any appreciable extent. We have visualized the subunits sep arated in this way either alone or reconstituted into tetramers, by metal shadowing electron microscopy. Whereas the 240-kDa subunit shows little tendency to self associate, the 23% kDa one aggregates on its own in the absence of the 240-kDa subunit. Reconstituted tetramers appear indistinguishable from native spectrin.
364
MATERIALS
AND METHODS
Fodrin was isolated from bovine brain by a modification of previously published proce-
SEPARATION
II
c
OF FODRIN
SUBUNITS
r)
FIG. I. Effect of pH and salt concentration on the interaction between fodrin and calmodulin. Fodrin was immobilized on nitrocellulose membranes and incubated in a solution of ‘251-calmodulin in (I) 10 mM imidazole, I mM CaC&, 2.5% BSA and the indicated amount of KC1 (m), pH 7.0, or in (2) IO mM sodium acetate, IO mM Tris, 100 mM KCI, 1 mM CaClz, and 2.5% BSA at the specified hnal pH (A). After incubation at room temperature I h in a Bio-Rad Bio-dot apparatus the filters were rinsed with the same buffers without calmoduhn, dried, and counted in a gamma counter. All points represent the average of three separate assaysand subtracting nonspecific binding to the filter without fodrin.
Volume
BY AFFINITY
CHROMATOGRAPHY
dures (6). Calmodulin was purified from bovine brain (7) and coupled to activated Sepharose 4B (Pharmacia). Spectrin was isolated from outdated human blood (8). The interaction between fodrin and ‘% calmodulin was assayed by spotting native fodrin onto nitrocellulose membranes using a Bio-Rad Bio-dot apparatus essentially as described previously (3). Variations in the basic assay are described in the legend to Fig. 1. The fodrin subunits were separated as follows. Fodrin was precipitated at pH 5.5 by adjusting to 10 mM sodium acetate and then adjusting the pH with acetic acid. After 30 min at 4 C, the solution was centrifuged at 1O,OOOg for 5 min and the pellet resuspended in a small volume of water. The suspension was adjusted to a final concentration of 2 M KI, 10 mM Tris (pH 7.5) 2 mM DTT, and 1 mM CaCl* from a 2.5X stock solution. The solution was then sonicated briefly, further incubated 1 h at room temperature, and centrifuged at 20,OOOg for 15 min. The clear supernatant (1 mg fod’ Abbreviations used: CaM, calmodulin; DTT, dithiothreitol; EGTA, ethylene glycol bis(@-aminoethyl ether) N,N’-tetraacetic acid; BSA, bovine serum albumin.
lmll
--
FIG. 2. Affinity chromatography of fodrin on calmodulin-Sepharose in the presence of 2 M Kl. Fodrin was dissolved in 10 mM imidazole, 2 M KI, 1 mM CaCIZ, I mM dithiothreitol, pH 7.3, and applied to a column of CaM-Sepharose at room temp. At the point indicated Ca*+ in the buffer was replaced by 5 mM EGTA. Absorbance at 280 nm was monitored continuously and fractions corresponding to the flow-through peak (A) and EGTA-eluted peak (B) were run on SDS-PAGE and compared to unfractionated fodrin (F), inset.
365
366
GLENNEY
AND WEBER
SEPARATION
OF FODRIN
SUBUNITS
rin) was applied to a I-ml column of calmodulin (10 mg calmodulin/ml settled gel) and after the washing with 3-4 column volumes of buffer alone, the 240-kDa subunit was eluted with the same solution but substituting 5 mM EGTA for Ca2+. Protein was monitored by absorbance at 280 nm and peak fractions of the unretarded or bound and eluted protein were dialyzed into 20 IIIM KCI, 5 IIIM Tris, 20 mM 2-mercaptoethanol, 65% glycerol. Yields of the 240- and 235-kDa subunits were generally greater than 80%. In some experiments samples were dialyzed first against 5 mM Tris, 20 mM 2-mercaptoethanol, and 7 M urea overnight, and then against the high-glycerol buffer. For metal shadowing, samples were diluted to 20-50 pg/ml in the glycerol buffer, sprayed onto freshly cleaved mica, dried under vacuum, and rotary shadowed with tantalum/ tungsten at an angle of 3-5” as described previously (6).
RESULTS
To analyze the parameters affecting the interaction between fodrin and calmodulin, we used a sensitive solid-phase assay in which native fodrin was immobilized on nitrocellulose and iodinated calmodulin was then applied. Controls in which fodrin was omitted were always run in parallel and subtracted to determine specific binding. As shown in Fig. 1, binding is relatively insensitive to pH between 5.5 and 8.0, but is greatly enhanced by relatively high salt concentrations. Similar results were observed when NaCl or KI replaced KCl. It can be seen that even molar amounts of various salts do not reduce the binding to fodtin. Control experiments, however, show that when CaZC is replaced by EGTA the amount of calmodulin bound to fodrin decreases to
BY AFFINITY
CHROMATOGRAPHY
367
less than 5% even in the presence of high levels of other salts (not shown). Other studies have shown that chaotropic salts at high levels could dissociate subunits of multi-subunit proteins (9- 12). We were interested, then, in whether the fodrin subunits became dissociated under conditions of high salt where the calmodulin-binding site was still expressed. As shown in Fig. 2, when bovine fodrin in 2 M KI was passed through a calmodulin column in the presence of calcium, the smaller subunit (235-kDa) passed through the column unretarded whereas the larger subunit (240-kDa) was bound to the calmodulin column and could be eluted with EGTA containing buffers (Fig. 2). The larger peak of uv-absorbing material in the unretarded fractions is due to contaminants in the fodrin preparation. When the same procedure was applied to human spectrin only the 240-kDa subunit was partially retained by CaM-Sepharose and could be eluted with EGTA (not shown). The two separated subunits of fodrin behaved quite differently as observed by electron microscopy (Fig. 3). The isolated 240-kDa subunit appeared as short rods approximately 100 nm in length or about half the length of the fodrin tetramer (2,4). They appeared well separated from each other with few aggregates observed. By contrast, the 235-kDa subunit appeared highly aggregated with only few profiles observed which could be the monomer (Fig. 3). To try and avoid this aggregation we dialyzed the 235-kDa subunit first into 7 M urea, followed by dialysis into the buffer used for metal shadowing, but again aggregation was observed. In no instances did we observe the typical fodrin tetrameric unit (0.2 pm length, doublestranded rods) in preparations of either subunit alone. When the isolated 240- or 23.5kDa
FIG. 3. Visualization of fodrin subunits alone or mixed. Fodrin subunits were separated as described in Fig. 2 and dialyzed into 5 mM Tris, 2 mM fl-mercaptoethanol, 20 mM KCI, and 65% glycerol, sprayed onto mica, dried, and metal shadowed. (A) 240-kDa subunit; (B) 235-kDa subunit: (Ct 240- + 23%kDA subunits mixed.
368
GLENNEY
subunits were mixed together and then dialyzed, however, a large number of tetrameric profiles were observed (Fig. 3). DISCUSSION
Dissociation of subunits and reconstitution into functional units is a classical approach for the study of the structure and function of proteins. Previous studies have shown that subunits of tightly associated multi-subunit proteins can be dissociated using chaotropic salts (9- 12). As discussed by Hatefi and Hanstein (1 l), this is probably due to a decrease in water structure, allowing the transfer of apolar groups to an aqueous environment. The mechanism of dissociation by chaotropic ions probably does not involve denaturation since a-helical conformation is usually retained (9-11).
Previously the subunits of spectrin had been separated by treatment with high urea followed by chromatography on hydroxylapatite (1315) or gel filtration in SDS (16). From these methods the subunits are substantially pure as judged by SDS-PAGE and can be at least partially renatured to form the intact molecule. In the present report we describe an alternative method for the separation of nonerythroid spectrin (fodrin) subunits which is rapid and quantitative, and does not involve strongly denaturing conditions. This method relies on the dissociation of fodrin subunits with the chaotropic salt KI and separation of these subunits using an affinity column in which the larger subunit (240-kDa) binds to calmodulin in the presence of Ca2+ and can be eluted with EGTA. The subunits isolated in this way can be reconstituted into the fodrin tetramer, indistinguishable from unfractionated fodrin. When this procedure was applied to human erythroid spectrin, the 240-kDa subunit bound to and was eluted from the CaM-Sepharose,
AND WEBER
although most was unbound. This is probably due to the lower affinity of mammalian spectrin for calmodulin (3). In a preliminary report, however, it was suggested that CaM binds to the lower molecular weight (220-kDa) subunit of mammalian spectrin (17). The reason for the discrepancy with the results presented here is unknown. REFERENCES I. Goodman, S. R., and Shiffer, K. (1983) J. Amer. Physiol. Sot. C121-C141. 2. Glenney, J. R., Jr., Glenney, P., and Weber, K. (1982) Proc. Natl. Acad. Sci. USA 79,4002-4005. 3. Glenney, J. R., Jr., and Glenney, P. (1984) Eur. J. Biochem. 144,529-539. 4. Glenney, J. R., Jr., Glenney. P., Osbom, M., and Weber, K. (1982) Cell 28,843-854. 5. Palfrey, H. C., Scheibler, W., and Gmengard, P. ( 1982) Proc. Natl. Acad. Sci. USA 79, 3780-3184. 6. Glenney, J. R., Jr., Glenney, P., and Weber, K. (1982) J. Biol. Chem. 257,9781-9787. 7. Gopalakrishna, R., and Anderson, W. B. (1982) Biochem. Biophys. Res. Commun. 104,830-836. 8. Brenner, S. L., and Kom, E. D. (1980) J. Biol. Chem. 255,84 I-844. 9. Bums, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 257,8638-8641. 10. Winkler, F. K., and Stanley, K. K. (1983) EMBO J. 2, I393- 1400. I I. Hatefi, Y., and Hanstein, W. G. (1974) in Methods in Enzymology (Fleixher, S., and Packer, L., eds.), Vol. 3 1, Part A, pp. 770-790, Academic Press,New York. 12. Kikuchi, T., Yumoto, N., Sasaki, T., and Murachi, T. (1984) Arch. B&hem. Biophys. 234, 639-645. 13. Calvert, R., Bennett, P., and Gratzer, W. (1980) Eur. J. Biochem. 107, 355-361. 14. Yoshino, H., and Marchesi, V. T. (1984) J. Biol. Chem. 259,4496-4500. 15. Davis, J., and Bennett, V. (1983) J. Biol. Chem. 258, 7757-7766. 16. Speicher, D. W., Morrow, J. S., Knowles, W. J., and Marchesi, V. T. (1980) Proc. Nat/ Acad. Sci. USA 77,5673-5671. 17. Sears, D. E., Morrow, J. S., and Marchesi, V. T. (1982) J. Cell Biol. 95, 25 la.
Separation
(1985)
of Fodrin Subunits by Affinity Chromatography on Calmodulin-Sepharose JOHN R. GLENNEY,
JR.* AND KLAUS
WEBERt
*Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138, and tThe Max-Planck-lnstitute/br Biophysical Chemistry, D-3400 Goettingen-Nikolausberg. West Germany Received May 24, 1985 Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Caz+dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 M IU + 1 mM Ca*+ and applied to CaM-Sepharose the fi subunit (235kDa) passed through unretarded whereas the a subunit (240-kDa) bound and could be eluted with ethylene glycol his@-aminoethyl ether)N,N’-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed. Q 1985Academic hss, Inc. KEY WORDS: calmodulin; spectrin; fodrin; electron microscopy; proteins; protein subunits.
Spectrin is the major membrane-skeletal protein of the red blood cell and an analog, termed fodrin or nonerythroid spectrin, is present in most other cells (1). The spectrins are composed of two nonidentical high-molecular-weight subunits which self-associate to form first heterodimers and then tetramers. Although one of these subunits (M, 240,000) seems identical between different spectrins in avian cells (2) the subunits are clearly two separate gene products in mammalian cells (3). One characteristic of spectrin is its ability to interact with calmodulin Ca2+-dependently. Using a gel overlay technique it was shown that this binding activity resides in the common 240,000 iI& subunit of avian spectrins (2,4) and has been detected in many cell types (5). Given the potential importance of the Ca*+-calmodulin regulation of the subcortical microfilament system we have been analyzing this interaction between nonerythroid spectrin and calmodulin in some detail. We were surprised to find that although this interaction is relatively insensitive to changes in pH, an increase in salt concentration actually enhanced 0003-2697185 $3.00 Copyright All i-i&s
8 1985 by Academic Press. Inc. of reproduction in any form re?en’ed.
this interaction. In addition, it was found that the subunits of spectrin dissociate in the presence of molar amounts of chaotropic salts. This allows the rapid and quantitative separation of fodrin subunits based on the calciumdependent affinity chromatography of 240kDa fodrin on a CaM-Sepharose column in the presence of chaotropic salts. When the procedure was used with human erythrocyte spectrin, only the 240-kDa subunit was retained by CaM-Sepharose to any appreciable extent. We have visualized the subunits sep arated in this way either alone or reconstituted into tetramers, by metal shadowing electron microscopy. Whereas the 240-kDa subunit shows little tendency to self associate, the 23% kDa one aggregates on its own in the absence of the 240-kDa subunit. Reconstituted tetramers appear indistinguishable from native spectrin.
364
MATERIALS
AND METHODS
Fodrin was isolated from bovine brain by a modification of previously published proce-
SEPARATION
II
c
OF FODRIN
SUBUNITS
r)
FIG. I. Effect of pH and salt concentration on the interaction between fodrin and calmodulin. Fodrin was immobilized on nitrocellulose membranes and incubated in a solution of ‘251-calmodulin in (I) 10 mM imidazole, I mM CaC&, 2.5% BSA and the indicated amount of KC1 (m), pH 7.0, or in (2) IO mM sodium acetate, IO mM Tris, 100 mM KCI, 1 mM CaClz, and 2.5% BSA at the specified hnal pH (A). After incubation at room temperature I h in a Bio-Rad Bio-dot apparatus the filters were rinsed with the same buffers without calmoduhn, dried, and counted in a gamma counter. All points represent the average of three separate assaysand subtracting nonspecific binding to the filter without fodrin.
Volume
BY AFFINITY
CHROMATOGRAPHY
dures (6). Calmodulin was purified from bovine brain (7) and coupled to activated Sepharose 4B (Pharmacia). Spectrin was isolated from outdated human blood (8). The interaction between fodrin and ‘% calmodulin was assayed by spotting native fodrin onto nitrocellulose membranes using a Bio-Rad Bio-dot apparatus essentially as described previously (3). Variations in the basic assay are described in the legend to Fig. 1. The fodrin subunits were separated as follows. Fodrin was precipitated at pH 5.5 by adjusting to 10 mM sodium acetate and then adjusting the pH with acetic acid. After 30 min at 4 C, the solution was centrifuged at 1O,OOOg for 5 min and the pellet resuspended in a small volume of water. The suspension was adjusted to a final concentration of 2 M KI, 10 mM Tris (pH 7.5) 2 mM DTT, and 1 mM CaCl* from a 2.5X stock solution. The solution was then sonicated briefly, further incubated 1 h at room temperature, and centrifuged at 20,OOOg for 15 min. The clear supernatant (1 mg fod’ Abbreviations used: CaM, calmodulin; DTT, dithiothreitol; EGTA, ethylene glycol bis(@-aminoethyl ether) N,N’-tetraacetic acid; BSA, bovine serum albumin.
lmll
--
FIG. 2. Affinity chromatography of fodrin on calmodulin-Sepharose in the presence of 2 M Kl. Fodrin was dissolved in 10 mM imidazole, 2 M KI, 1 mM CaCIZ, I mM dithiothreitol, pH 7.3, and applied to a column of CaM-Sepharose at room temp. At the point indicated Ca*+ in the buffer was replaced by 5 mM EGTA. Absorbance at 280 nm was monitored continuously and fractions corresponding to the flow-through peak (A) and EGTA-eluted peak (B) were run on SDS-PAGE and compared to unfractionated fodrin (F), inset.
365
366
GLENNEY
AND WEBER
SEPARATION
OF FODRIN
SUBUNITS
rin) was applied to a I-ml column of calmodulin (10 mg calmodulin/ml settled gel) and after the washing with 3-4 column volumes of buffer alone, the 240-kDa subunit was eluted with the same solution but substituting 5 mM EGTA for Ca2+. Protein was monitored by absorbance at 280 nm and peak fractions of the unretarded or bound and eluted protein were dialyzed into 20 IIIM KCI, 5 IIIM Tris, 20 mM 2-mercaptoethanol, 65% glycerol. Yields of the 240- and 235-kDa subunits were generally greater than 80%. In some experiments samples were dialyzed first against 5 mM Tris, 20 mM 2-mercaptoethanol, and 7 M urea overnight, and then against the high-glycerol buffer. For metal shadowing, samples were diluted to 20-50 pg/ml in the glycerol buffer, sprayed onto freshly cleaved mica, dried under vacuum, and rotary shadowed with tantalum/ tungsten at an angle of 3-5” as described previously (6).
RESULTS
To analyze the parameters affecting the interaction between fodrin and calmodulin, we used a sensitive solid-phase assay in which native fodrin was immobilized on nitrocellulose and iodinated calmodulin was then applied. Controls in which fodrin was omitted were always run in parallel and subtracted to determine specific binding. As shown in Fig. 1, binding is relatively insensitive to pH between 5.5 and 8.0, but is greatly enhanced by relatively high salt concentrations. Similar results were observed when NaCl or KI replaced KCl. It can be seen that even molar amounts of various salts do not reduce the binding to fodtin. Control experiments, however, show that when CaZC is replaced by EGTA the amount of calmodulin bound to fodrin decreases to
BY AFFINITY
CHROMATOGRAPHY
367
less than 5% even in the presence of high levels of other salts (not shown). Other studies have shown that chaotropic salts at high levels could dissociate subunits of multi-subunit proteins (9- 12). We were interested, then, in whether the fodrin subunits became dissociated under conditions of high salt where the calmodulin-binding site was still expressed. As shown in Fig. 2, when bovine fodrin in 2 M KI was passed through a calmodulin column in the presence of calcium, the smaller subunit (235-kDa) passed through the column unretarded whereas the larger subunit (240-kDa) was bound to the calmodulin column and could be eluted with EGTA containing buffers (Fig. 2). The larger peak of uv-absorbing material in the unretarded fractions is due to contaminants in the fodrin preparation. When the same procedure was applied to human spectrin only the 240-kDa subunit was partially retained by CaM-Sepharose and could be eluted with EGTA (not shown). The two separated subunits of fodrin behaved quite differently as observed by electron microscopy (Fig. 3). The isolated 240-kDa subunit appeared as short rods approximately 100 nm in length or about half the length of the fodrin tetramer (2,4). They appeared well separated from each other with few aggregates observed. By contrast, the 235-kDa subunit appeared highly aggregated with only few profiles observed which could be the monomer (Fig. 3). To try and avoid this aggregation we dialyzed the 235-kDa subunit first into 7 M urea, followed by dialysis into the buffer used for metal shadowing, but again aggregation was observed. In no instances did we observe the typical fodrin tetrameric unit (0.2 pm length, doublestranded rods) in preparations of either subunit alone. When the isolated 240- or 23.5kDa
FIG. 3. Visualization of fodrin subunits alone or mixed. Fodrin subunits were separated as described in Fig. 2 and dialyzed into 5 mM Tris, 2 mM fl-mercaptoethanol, 20 mM KCI, and 65% glycerol, sprayed onto mica, dried, and metal shadowed. (A) 240-kDa subunit; (B) 235-kDa subunit: (Ct 240- + 23%kDA subunits mixed.
368
GLENNEY
subunits were mixed together and then dialyzed, however, a large number of tetrameric profiles were observed (Fig. 3). DISCUSSION
Dissociation of subunits and reconstitution into functional units is a classical approach for the study of the structure and function of proteins. Previous studies have shown that subunits of tightly associated multi-subunit proteins can be dissociated using chaotropic salts (9- 12). As discussed by Hatefi and Hanstein (1 l), this is probably due to a decrease in water structure, allowing the transfer of apolar groups to an aqueous environment. The mechanism of dissociation by chaotropic ions probably does not involve denaturation since a-helical conformation is usually retained (9-11).
Previously the subunits of spectrin had been separated by treatment with high urea followed by chromatography on hydroxylapatite (1315) or gel filtration in SDS (16). From these methods the subunits are substantially pure as judged by SDS-PAGE and can be at least partially renatured to form the intact molecule. In the present report we describe an alternative method for the separation of nonerythroid spectrin (fodrin) subunits which is rapid and quantitative, and does not involve strongly denaturing conditions. This method relies on the dissociation of fodrin subunits with the chaotropic salt KI and separation of these subunits using an affinity column in which the larger subunit (240-kDa) binds to calmodulin in the presence of Ca2+ and can be eluted with EGTA. The subunits isolated in this way can be reconstituted into the fodrin tetramer, indistinguishable from unfractionated fodrin. When this procedure was applied to human erythroid spectrin, the 240-kDa subunit bound to and was eluted from the CaM-Sepharose,
AND WEBER
although most was unbound. This is probably due to the lower affinity of mammalian spectrin for calmodulin (3). In a preliminary report, however, it was suggested that CaM binds to the lower molecular weight (220-kDa) subunit of mammalian spectrin (17). The reason for the discrepancy with the results presented here is unknown. REFERENCES I. Goodman, S. R., and Shiffer, K. (1983) J. Amer. Physiol. Sot. C121-C141. 2. Glenney, J. R., Jr., Glenney, P., and Weber, K. (1982) Proc. Natl. Acad. Sci. USA 79,4002-4005. 3. Glenney, J. R., Jr., and Glenney, P. (1984) Eur. J. Biochem. 144,529-539. 4. Glenney, J. R., Jr., Glenney. P., Osbom, M., and Weber, K. (1982) Cell 28,843-854. 5. Palfrey, H. C., Scheibler, W., and Gmengard, P. ( 1982) Proc. Natl. Acad. Sci. USA 79, 3780-3184. 6. Glenney, J. R., Jr., Glenney, P., and Weber, K. (1982) J. Biol. Chem. 257,9781-9787. 7. Gopalakrishna, R., and Anderson, W. B. (1982) Biochem. Biophys. Res. Commun. 104,830-836. 8. Brenner, S. L., and Kom, E. D. (1980) J. Biol. Chem. 255,84 I-844. 9. Bums, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 257,8638-8641. 10. Winkler, F. K., and Stanley, K. K. (1983) EMBO J. 2, I393- 1400. I I. Hatefi, Y., and Hanstein, W. G. (1974) in Methods in Enzymology (Fleixher, S., and Packer, L., eds.), Vol. 3 1, Part A, pp. 770-790, Academic Press,New York. 12. Kikuchi, T., Yumoto, N., Sasaki, T., and Murachi, T. (1984) Arch. B&hem. Biophys. 234, 639-645. 13. Calvert, R., Bennett, P., and Gratzer, W. (1980) Eur. J. Biochem. 107, 355-361. 14. Yoshino, H., and Marchesi, V. T. (1984) J. Biol. Chem. 259,4496-4500. 15. Davis, J., and Bennett, V. (1983) J. Biol. Chem. 258, 7757-7766. 16. Speicher, D. W., Morrow, J. S., Knowles, W. J., and Marchesi, V. T. (1980) Proc. Nat/ Acad. Sci. USA 77,5673-5671. 17. Sears, D. E., Morrow, J. S., and Marchesi, V. T. (1982) J. Cell Biol. 95, 25 la.