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Identification of concanavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting
ANALYTICAL
BIOCHEMISTRY
Identification
123, 143-146
(1982)
of Concanavalin A-Binding Proteins after Sodium Dodecyl Sulfate-Gel Electrophoresis and Protein Blotting RICHARD Friedrich
Miescher-lnstitut, Received
HAWKES
Postbox November
273, CH-4002
Basel,
Switzerland
23, 1981
A method is described for the detection of concanavalin A-binding proteins in complex mixtures. The proteins are separated by gel electrophoresis and then are blotted onto a nitrocellulose sheet where they form an immobilized replica of the original gel. Concanavalin A-binding proteins are identified by incubating the blot in concanavalin A followed by horseradish p-eroxidase. The bound concanavalin A-peroxidase complex is detected using the chromogenic peroxidase substrate 4-chloro-l-naphthol.
to the Con A by the gel matrix (the molecular weight of the Con A tetramer is 110,000, making the reaction slow and complicating the use of high acrylamide concentrations); and third, the problem of removing unbound Con A, which can result in unacceptably high background signals. Recently, techniques have been published for the transfer of proteins of polyacrylamide gels onto sheets of cellulose nitrate where the original banding pattern is faithfully reproduced (7,8). These protein “blots” can then be used to detect specific antibody-binding polypeptides (7) or DNA-binding proteins (8). This communication describes a protocol for the detection of Con A-binding proteins on protein blots. This new procedure avoids the above problems and can readily be adapted for use with any lectin.
Lectins are common reagents in analytical biochemistry for the detection and isolation of glycoprotein-containing structures. The most commonly used lectin is concanavalin A (Con A),’ a protein isolated from jack beans which preferentially binds to a-~mannopyranosyl and a-r>-glucopyrosyl residues (1). This property has been exploited to study the glycoprotein composition of numerous macromolecular structures (2). It is often desirable to identify the Con A-binding proteins in complex mixtures after their separation by SDS-polyacrylamide gel electrophoresis. Several methods have been developed to achieve this (3-6) involving removal of the detergent from the gel by extensive washing, incubation of the gel in Con A, elution of the unbound lectin, and subsequent detection of the retained Con A by autoradiography (6), fluorescence (5), or a secondary enzyme-coupled reaction (3,4). These methods all suffer from similar drawbacks: first, the problem of eluting the SDS without concomitantly eluting the proteins; second, the high diffusion barrier presented
MATERIALS
AND METHODS
The methods used for SDS-gel electrophoresis (9,lO) and protein blotting (7,lO) have previously been described. Rat brain subcellular fractions, either synaptosomal plasma membranes (SPM) or postsynaptic densities (PSD), were isolated, separated by SDS-gel electrophoresis, and transferred to nitrocellulose according to Matus et al. (10).
’ Abbreviations used: Con A, concanavalin A; SDS, sodium dodecyl sulfate; SPM, synaptosomal plasma membranes; PSD, postsynaptic density; TBS, Tris-buffered saline. 143
0003-2697/82/090143-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
144
RICHARD
10-4
lo-5
10-6
10-7
to-8
FIG. 1. A dot assay for Con A binding to thyroglobulin. Various dilutions of thyroglobulin in blocking solution (concentrations in mg/ml) were dotted as OS-~1 dots onto a sheet of cellulose nitrate and allowed to dry to promote binding. These dots were then treated exactly as protein blots to detect Con A binding. Each square is 3 mm wide.
A protocol for amido-black staining has been published (7). The apparent molecular weights (il4,) were obtained according to Matus et al. (10). The procedure for the detection of Con Abinding polypeptides on blots is as follows: (i) Incubate the blot for 10 min in a 10% solution of fetal calf serum in TBS (50 mM Tris-HCl, pH 7.4, 200 mM NaCl) to block the remaining protein-binding sites on the filter. Serum glycoproteins, which might themselves bind Con A, do not contribute significant background staining. (ii) Aspirate away the blocking solution and replace it with freshly prepared Con A (50 pg/ml in blocking solution). Incubate for 30 min. Neither higher Con A concentrations nor longer incubation times significantly increase the sensitivity. The Con A binding is performed at pH 7.4 as a compromise between the requirements for optimal binding (best at pH 5 ( 11)) and the need to maintain a polymeric form of Con A (favored by alkaline pH ( 12)). NaCl is included because it has been shown to reduce nonspecific (hydrophobic) Con A binding (13). In these experiments, the addition of 5 mM CaC& and 5 mM MnCl*, both reported to enhance Con A binding (14), had no effect. (iii) Wash the filter for 15 min in three
HAWKES
changes of TBS and then add 50 pg/ml horseradish peroxidase (Sigma) in blocking solution. Incubate for 30 min. (iv) Wash the filter for 15 min in three changes of TBS. The bound peroxidase is detected by the addition of a freshly prepared solution of 0.06% (w/v) 4-chloro-lnaphthol (Merck) and 0.01% hydrogen peroxide in TBS. The 4-chloro-1-naphthol can be stored cold in the dark as a 0.3% (w/v) stock solution in methanol. Con A-binding polypeptide bands appear blue against the white filter background within l-2 min. The stained blots are washed in distilled water, air-dried, and stored in the dark. Concanavalin A (grade A) was supplied by Calbiochem and thyroglobulin (porcine, type 2) by Sigma. RESULTS
Blotting proteins from polyacrylamide gels onto nitrocellulose paper both frees them of detergent and renders them readily accessible to subsequent reactions. This has been amply demonstrated by studies of antibody binding to blotted polypeptide antigens (7,lO). This suggests that all interactions which do not depend on the preservation of tertiary structure can be studied after blotting. Lectin binding is an obvious candidate. The horseradish peroxidase detection method (4) exploits the two facts that at above pH 6.0 Con A is a tetramer with four equivalent binding sites, and that horseradish peroxidase is itself a Con A-binding glycoprotein. When Con A binds to immobilized glycoproteins problems of steric hindrance will probably ensure that not all four sites are occupied. The free sites can then bind horseradish peroxidase which is readily detected with a range of chromogenic substrates. The interaction of Con A with thyroglobulin ( 11) was used in a dot assay ( 17) to estimate the sensitivity of the method. Serial dilutions of thyroglobulin in blocking solution were applied as 0.5~~1 dots onto the ni-
IDENTIFICATION
OF
CONCANAVALIN
trocellulose paper and these dots were tested for Con A binding. The results in Fig. 1 show that it is possible to detect dilutions as low as 1 pg/ml (i.e., 500 pg of thyroglobulin). When it is recalled that between 10 and 100 pg of protein is typically applied to an SDSgel track, it is evident that even very minor Con A-binding proteins should be readily detected. Because of its simplicity, the dot assay for Con A binding may be valuable in both analytical and preparative biochemistry. An example of the use of blotting to detect Con A-binding proteins is given in Fig. 2. Proteins of rat SPM were separated by SDS-gel electrophoresis and electrophoA
280
6
C
A-BINDING
145
PROTEINS
A
180,
'
110) 100,
1
B
0
-
FIG. 2. The concanavalin A-binding proteins of SPM. The rat brain SPM fraction was separated by electrophoresis through a 7% 15% polyacrylamide gradient gel in the presence of SDS (9,lO). The proteins were then transferred from the gel onto a sheet of cellulose nitrate by lateral electrophoresis (7) and the blot was cut into 2-mm-wide test strips. Four of these test strips are shown in the figure: (A) amido-black stained for protein, (B) stained with Con A-peroxidase as described in the text, (C) stained as described but with Con A omitted, and (D) stained as described under Materials and Methods but with 0.1 M a-o-methylmannoside included in the Con A incubation.
FIG. 3. The concanavalin A-binding proteins of PSD. A PSD blot was prepared exactly as for SPM in Fig. I. Strip A shows the Con A-peroxidase staining pattern; strip B shows the amido-black staining pattern. Apparent molecular weights are given for the principal Con A-binding hands.
retically blotted onto cellulose-nitrate. The blot was then sliced into identical 2-mm test strips. Strip A shows the total protein banding pattern, as revealed by amido-black staining, and strip B shows the Con A staining pattern. It is important to note that the set of bands stained by Con A-peroxidase includes some which are not easily detected by amido-black-stained bands. Strip C was treated the same way as B except that Con A was omitted from the first incubation. No bands are stained, which safeguards against direct interaction of peroxidase with the blot. In strip D, a competing sugar, 0.1 M a-~methylmannoside (l), was added along with Con A in the first incubation. Again no bands are stained, suggesting that the Con A-protein interaction is dependent on its sugar-binding site. The same concentration of a noncompeting sugar, D-ribose or D-galactose, had no effect (not shown). Figure 3 shows the Con A-binding poly-
146
RICHARD
peptides of PSD separated, blotted, and stained as described for the SPM in Fig. 1. Of the numerous polypeptides present, only a small fraction bind Con A, a doublet at M, = 180,000, a broad amorphous band at J4, = 110,000, and several minor bands at M, = 110,000 to 100,000. This pattern is essentially the same as that obtained in other laboratories by measuring the binding of ‘251-Con A to proteins in situ in the gel ( 15,16). The only difference is that the doublet at M, = 180,000 has not been resolved before, and this is probably a property of the gradient-gel system we use rather than the Con A binding. As with the SPM blots, the inclusion of a-D-methylmannoside in the first incubation abolished the staining (not shown). DISCUSSION
Through protein blotting, a single gel can provide dozens of identical test strips, which can be stored indefinitely before use and which can be screened in a variety of different ways. It has been demonstrated here that protein blots can be used to detect Con A-binding proteins in complex mixtures with a sensitivity and resolution comparable to those of the more elaborate techniques currently in use. In addition to the examples shown, the technique has been used to identify Con A-binding proteins in preparations from Friend leukemia virus, calf brain microtubules, C6 glioma-conditioned medium, and the seeds of several cereals (Hawkes, R., unpublished observations). To extend the method to other lectins, or indeed to other protein ligands, requires only that an appropriate detection system be employed. For example, Ricinus and Lens agglutinins bind rat brain acetylcholinesterase ( 18,19) and wheat germ agglutinin binds human liver @-galactosidase (20) and suitable (histochemical) detection systems exist for both these enzymes (21). Alternatively a direct detection method could be employed, using covalent lectin-peroxidase complexes. A de-
HAWKES
tection method using antilectin has also been described (22).
antibodies
ACKNOWLEDGMENTS 1 thank M. Ackermann and G. Pehling for their technical assistance and A. Matus for his advice.
REFERENCES 1. Goldstein, I. J. (1976) in Concanavalin A as a Tool (Bittiger, H., and Schnebli, H. P., eds.), pp. 5765, Wiley-Interscience, New York. 2. Sharon, N., and Lis, H. (1975) Methods Membr Biol 3, 147-200. 3. Avigad, G. (I 978) Anal. Biochem. 86, 443-449. 4. Wood, J. G., and Sarinana, F. 0. 0975) Anal. Biochem. 69, 320-322. 5. Furlan, M., Perret, B. A., and Beck, E. A. (1979) Anal. Biochem. 96, 208-214. 6. Gurd, J. W., and Evans, W. H. (1976) Canad. J. Biochem. 54, 477-480. I. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nat. Acad. Sci. USA 76, 4350-4354. 8. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) Nucleic Acids Res. 8, l20. 9. Laemmli, U. K. (I 970) Nature (London) 227,680685. 10. Matus, A., Pehling, G., Ackermann, M., and Maeder, J. (1980) J. Cell Biol. 87, 346-359. II. Dulaney, J. T. (1979) Anal. Biochem. 99,254-267. 12. McKenzie, G. H., Sawyer, W. H., and Nichol, L. W. (1972) Biochim. Biophys. Acta 263, 283293. J. T. (1979) Mol. Cell. Biochem. 21, 1313. Dulaney, 63. 14. Kalb, A. J., and Levitzki, A. (1968) Biochem. J. 109, 669-672. C. W. (1977) J. Biol. 15. Kelly, P. T., and Cotman, Chem. 252, 786-793. 16. De Silva, N. S., Gurd, J. W., and Schwartz, C. (1979) Brain Res. 165, 283-293. 17. Hawkes, R., Niday, E., and Gordon, J. ( 1982) Anal. Biochem. 119, 142- 147. 18. Uhlenbruck, G., Baldo, B. A., Steinhausen, G., Schwick, H. G., Chatterjee, B. P., Horejsi, V., Krajhanzl, A., and Kocourek, J. (1978) J. Clin. Chem. C/in. Biochem. 16, 19-23. 19. Wenthold, R. J., Mahler, H. R., and Moore, W. J. (1974) J. Neurochem. 22, 945-949. A. G. W., and O’Brien, J. S. ( 1974) Bio20. Norden, them. Biophys. Rex Commun. 56, 193-198. 21. Pearse, A. G. E. (1972) Histochemistry: Theoretical and Applied, 3rd ed., Churchill, Edinburgh. 22. Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Annl. Biochem. 115, 219-224.
BIOCHEMISTRY
Identification
123, 143-146
(1982)
of Concanavalin A-Binding Proteins after Sodium Dodecyl Sulfate-Gel Electrophoresis and Protein Blotting RICHARD Friedrich
Miescher-lnstitut, Received
HAWKES
Postbox November
273, CH-4002
Basel,
Switzerland
23, 1981
A method is described for the detection of concanavalin A-binding proteins in complex mixtures. The proteins are separated by gel electrophoresis and then are blotted onto a nitrocellulose sheet where they form an immobilized replica of the original gel. Concanavalin A-binding proteins are identified by incubating the blot in concanavalin A followed by horseradish p-eroxidase. The bound concanavalin A-peroxidase complex is detected using the chromogenic peroxidase substrate 4-chloro-l-naphthol.
to the Con A by the gel matrix (the molecular weight of the Con A tetramer is 110,000, making the reaction slow and complicating the use of high acrylamide concentrations); and third, the problem of removing unbound Con A, which can result in unacceptably high background signals. Recently, techniques have been published for the transfer of proteins of polyacrylamide gels onto sheets of cellulose nitrate where the original banding pattern is faithfully reproduced (7,8). These protein “blots” can then be used to detect specific antibody-binding polypeptides (7) or DNA-binding proteins (8). This communication describes a protocol for the detection of Con A-binding proteins on protein blots. This new procedure avoids the above problems and can readily be adapted for use with any lectin.
Lectins are common reagents in analytical biochemistry for the detection and isolation of glycoprotein-containing structures. The most commonly used lectin is concanavalin A (Con A),’ a protein isolated from jack beans which preferentially binds to a-~mannopyranosyl and a-r>-glucopyrosyl residues (1). This property has been exploited to study the glycoprotein composition of numerous macromolecular structures (2). It is often desirable to identify the Con A-binding proteins in complex mixtures after their separation by SDS-polyacrylamide gel electrophoresis. Several methods have been developed to achieve this (3-6) involving removal of the detergent from the gel by extensive washing, incubation of the gel in Con A, elution of the unbound lectin, and subsequent detection of the retained Con A by autoradiography (6), fluorescence (5), or a secondary enzyme-coupled reaction (3,4). These methods all suffer from similar drawbacks: first, the problem of eluting the SDS without concomitantly eluting the proteins; second, the high diffusion barrier presented
MATERIALS
AND METHODS
The methods used for SDS-gel electrophoresis (9,lO) and protein blotting (7,lO) have previously been described. Rat brain subcellular fractions, either synaptosomal plasma membranes (SPM) or postsynaptic densities (PSD), were isolated, separated by SDS-gel electrophoresis, and transferred to nitrocellulose according to Matus et al. (10).
’ Abbreviations used: Con A, concanavalin A; SDS, sodium dodecyl sulfate; SPM, synaptosomal plasma membranes; PSD, postsynaptic density; TBS, Tris-buffered saline. 143
0003-2697/82/090143-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
144
RICHARD
10-4
lo-5
10-6
10-7
to-8
FIG. 1. A dot assay for Con A binding to thyroglobulin. Various dilutions of thyroglobulin in blocking solution (concentrations in mg/ml) were dotted as OS-~1 dots onto a sheet of cellulose nitrate and allowed to dry to promote binding. These dots were then treated exactly as protein blots to detect Con A binding. Each square is 3 mm wide.
A protocol for amido-black staining has been published (7). The apparent molecular weights (il4,) were obtained according to Matus et al. (10). The procedure for the detection of Con Abinding polypeptides on blots is as follows: (i) Incubate the blot for 10 min in a 10% solution of fetal calf serum in TBS (50 mM Tris-HCl, pH 7.4, 200 mM NaCl) to block the remaining protein-binding sites on the filter. Serum glycoproteins, which might themselves bind Con A, do not contribute significant background staining. (ii) Aspirate away the blocking solution and replace it with freshly prepared Con A (50 pg/ml in blocking solution). Incubate for 30 min. Neither higher Con A concentrations nor longer incubation times significantly increase the sensitivity. The Con A binding is performed at pH 7.4 as a compromise between the requirements for optimal binding (best at pH 5 ( 11)) and the need to maintain a polymeric form of Con A (favored by alkaline pH ( 12)). NaCl is included because it has been shown to reduce nonspecific (hydrophobic) Con A binding (13). In these experiments, the addition of 5 mM CaC& and 5 mM MnCl*, both reported to enhance Con A binding (14), had no effect. (iii) Wash the filter for 15 min in three
HAWKES
changes of TBS and then add 50 pg/ml horseradish peroxidase (Sigma) in blocking solution. Incubate for 30 min. (iv) Wash the filter for 15 min in three changes of TBS. The bound peroxidase is detected by the addition of a freshly prepared solution of 0.06% (w/v) 4-chloro-lnaphthol (Merck) and 0.01% hydrogen peroxide in TBS. The 4-chloro-1-naphthol can be stored cold in the dark as a 0.3% (w/v) stock solution in methanol. Con A-binding polypeptide bands appear blue against the white filter background within l-2 min. The stained blots are washed in distilled water, air-dried, and stored in the dark. Concanavalin A (grade A) was supplied by Calbiochem and thyroglobulin (porcine, type 2) by Sigma. RESULTS
Blotting proteins from polyacrylamide gels onto nitrocellulose paper both frees them of detergent and renders them readily accessible to subsequent reactions. This has been amply demonstrated by studies of antibody binding to blotted polypeptide antigens (7,lO). This suggests that all interactions which do not depend on the preservation of tertiary structure can be studied after blotting. Lectin binding is an obvious candidate. The horseradish peroxidase detection method (4) exploits the two facts that at above pH 6.0 Con A is a tetramer with four equivalent binding sites, and that horseradish peroxidase is itself a Con A-binding glycoprotein. When Con A binds to immobilized glycoproteins problems of steric hindrance will probably ensure that not all four sites are occupied. The free sites can then bind horseradish peroxidase which is readily detected with a range of chromogenic substrates. The interaction of Con A with thyroglobulin ( 11) was used in a dot assay ( 17) to estimate the sensitivity of the method. Serial dilutions of thyroglobulin in blocking solution were applied as 0.5~~1 dots onto the ni-
IDENTIFICATION
OF
CONCANAVALIN
trocellulose paper and these dots were tested for Con A binding. The results in Fig. 1 show that it is possible to detect dilutions as low as 1 pg/ml (i.e., 500 pg of thyroglobulin). When it is recalled that between 10 and 100 pg of protein is typically applied to an SDSgel track, it is evident that even very minor Con A-binding proteins should be readily detected. Because of its simplicity, the dot assay for Con A binding may be valuable in both analytical and preparative biochemistry. An example of the use of blotting to detect Con A-binding proteins is given in Fig. 2. Proteins of rat SPM were separated by SDS-gel electrophoresis and electrophoA
280
6
C
A-BINDING
145
PROTEINS
A
180,
'
110) 100,
1
B
0
-
FIG. 2. The concanavalin A-binding proteins of SPM. The rat brain SPM fraction was separated by electrophoresis through a 7% 15% polyacrylamide gradient gel in the presence of SDS (9,lO). The proteins were then transferred from the gel onto a sheet of cellulose nitrate by lateral electrophoresis (7) and the blot was cut into 2-mm-wide test strips. Four of these test strips are shown in the figure: (A) amido-black stained for protein, (B) stained with Con A-peroxidase as described in the text, (C) stained as described but with Con A omitted, and (D) stained as described under Materials and Methods but with 0.1 M a-o-methylmannoside included in the Con A incubation.
FIG. 3. The concanavalin A-binding proteins of PSD. A PSD blot was prepared exactly as for SPM in Fig. I. Strip A shows the Con A-peroxidase staining pattern; strip B shows the amido-black staining pattern. Apparent molecular weights are given for the principal Con A-binding hands.
retically blotted onto cellulose-nitrate. The blot was then sliced into identical 2-mm test strips. Strip A shows the total protein banding pattern, as revealed by amido-black staining, and strip B shows the Con A staining pattern. It is important to note that the set of bands stained by Con A-peroxidase includes some which are not easily detected by amido-black-stained bands. Strip C was treated the same way as B except that Con A was omitted from the first incubation. No bands are stained, which safeguards against direct interaction of peroxidase with the blot. In strip D, a competing sugar, 0.1 M a-~methylmannoside (l), was added along with Con A in the first incubation. Again no bands are stained, suggesting that the Con A-protein interaction is dependent on its sugar-binding site. The same concentration of a noncompeting sugar, D-ribose or D-galactose, had no effect (not shown). Figure 3 shows the Con A-binding poly-
146
RICHARD
peptides of PSD separated, blotted, and stained as described for the SPM in Fig. 1. Of the numerous polypeptides present, only a small fraction bind Con A, a doublet at M, = 180,000, a broad amorphous band at J4, = 110,000, and several minor bands at M, = 110,000 to 100,000. This pattern is essentially the same as that obtained in other laboratories by measuring the binding of ‘251-Con A to proteins in situ in the gel ( 15,16). The only difference is that the doublet at M, = 180,000 has not been resolved before, and this is probably a property of the gradient-gel system we use rather than the Con A binding. As with the SPM blots, the inclusion of a-D-methylmannoside in the first incubation abolished the staining (not shown). DISCUSSION
Through protein blotting, a single gel can provide dozens of identical test strips, which can be stored indefinitely before use and which can be screened in a variety of different ways. It has been demonstrated here that protein blots can be used to detect Con A-binding proteins in complex mixtures with a sensitivity and resolution comparable to those of the more elaborate techniques currently in use. In addition to the examples shown, the technique has been used to identify Con A-binding proteins in preparations from Friend leukemia virus, calf brain microtubules, C6 glioma-conditioned medium, and the seeds of several cereals (Hawkes, R., unpublished observations). To extend the method to other lectins, or indeed to other protein ligands, requires only that an appropriate detection system be employed. For example, Ricinus and Lens agglutinins bind rat brain acetylcholinesterase ( 18,19) and wheat germ agglutinin binds human liver @-galactosidase (20) and suitable (histochemical) detection systems exist for both these enzymes (21). Alternatively a direct detection method could be employed, using covalent lectin-peroxidase complexes. A de-
HAWKES
tection method using antilectin has also been described (22).
antibodies
ACKNOWLEDGMENTS 1 thank M. Ackermann and G. Pehling for their technical assistance and A. Matus for his advice.
REFERENCES 1. Goldstein, I. J. (1976) in Concanavalin A as a Tool (Bittiger, H., and Schnebli, H. P., eds.), pp. 5765, Wiley-Interscience, New York. 2. Sharon, N., and Lis, H. (1975) Methods Membr Biol 3, 147-200. 3. Avigad, G. (I 978) Anal. Biochem. 86, 443-449. 4. Wood, J. G., and Sarinana, F. 0. 0975) Anal. Biochem. 69, 320-322. 5. Furlan, M., Perret, B. A., and Beck, E. A. (1979) Anal. Biochem. 96, 208-214. 6. Gurd, J. W., and Evans, W. H. (1976) Canad. J. Biochem. 54, 477-480. I. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nat. Acad. Sci. USA 76, 4350-4354. 8. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) Nucleic Acids Res. 8, l20. 9. Laemmli, U. K. (I 970) Nature (London) 227,680685. 10. Matus, A., Pehling, G., Ackermann, M., and Maeder, J. (1980) J. Cell Biol. 87, 346-359. II. Dulaney, J. T. (1979) Anal. Biochem. 99,254-267. 12. McKenzie, G. H., Sawyer, W. H., and Nichol, L. W. (1972) Biochim. Biophys. Acta 263, 283293. J. T. (1979) Mol. Cell. Biochem. 21, 1313. Dulaney, 63. 14. Kalb, A. J., and Levitzki, A. (1968) Biochem. J. 109, 669-672. C. W. (1977) J. Biol. 15. Kelly, P. T., and Cotman, Chem. 252, 786-793. 16. De Silva, N. S., Gurd, J. W., and Schwartz, C. (1979) Brain Res. 165, 283-293. 17. Hawkes, R., Niday, E., and Gordon, J. ( 1982) Anal. Biochem. 119, 142- 147. 18. Uhlenbruck, G., Baldo, B. A., Steinhausen, G., Schwick, H. G., Chatterjee, B. P., Horejsi, V., Krajhanzl, A., and Kocourek, J. (1978) J. Clin. Chem. C/in. Biochem. 16, 19-23. 19. Wenthold, R. J., Mahler, H. R., and Moore, W. J. (1974) J. Neurochem. 22, 945-949. A. G. W., and O’Brien, J. S. ( 1974) Bio20. Norden, them. Biophys. Rex Commun. 56, 193-198. 21. Pearse, A. G. E. (1972) Histochemistry: Theoretical and Applied, 3rd ed., Churchill, Edinburgh. 22. Glass, W. F., Briggs, R. C., and Hnilica, L. S. (1981) Annl. Biochem. 115, 219-224.