SHORT COMMUNICATION
Microheterogeneity and phosphate content of myelin basic protein from ‘freeze-blown’ guinea-pig brains (Received 5 February 1976. Accepted 26 April 1976)
for any possible surviving enzymes to act. The pH 3.0 extract was dialyzed overnight against 0.1 M-acetic acid (pH 2.9) and lyophilized. All procedures were carried out at 0 to 5°C. Procedures for gel filtration and polyacrylaet a/., 1972) ionmide gel electrophoresis (DEIBLER & MARTENSON. 1973), exchange chromatography (DEIBLER ct a/.. 1975) and phosamino acid analysis (MARTENSON et al.. 1975) have been dephorus determination (DEIBLER scribed. Polyacrylamide gels containing separated proteins stained with Amido black were scanned densitometrically at 690 nm. The relative areas under the peaks were determined by transferring the curves to thin cardboard and cutting out and weighing the appropriate areas. Myelin basic protein is the only protein present in significant amounts in pH 3.0 extracts which migrates cathodiel al., 1971b). Hence, we cally at pH 10.6 (MARTENSON were able to examine the protein’s microheterogeneity prior to the actual isolation of the pure protein. Electrophoresis of the pH 3.0 extracts at pH 10.6 (Fig. 1) showed that the basic protein consisted of three major forms, components 1, 3 and 5. The minor components (2 and 4) were poorly resolved. N o significant differences in the profiles were found among the five preparations examined. Component 1 plus 2’ accounted for 6 5 . 8 : ~ f 1.13 (s.D.) of the total basic protein; component 3 plus 4, 29.8aL 0.86; and component 5, 4.4% 0.34. The combined pH 3.0 extracts (1.06 g) were subjected to repeated gel filtration through a column (3.2 x 95 cm) of Sephadex G-100 in 0.01 N - H C ~at 5°C to remove most of the high and low molecular weight contaminants; 0.45 g of partially purified basic protein was obtained. The procedure was monitored by gel electrophoresis at both pH 2.4 and 10.6 to insure that no significant amounts of basic protein were discarded. The partially purified basic protein was chromatographed on a column of CM-cellulose according to the procedure of DEIBLER & MARTENSON (1973). The fractions eluted were analyzed for protein and phosphorus (Fig. 2). Electrophoresis of the chromatographic fractions at pH 10.6 (Fig. 3) showed that fractions 1 to 4 consisted of component 5 and material of lower electrophoretic mobility, while fractions 5 to 8 consisted predominantly of components 3 and 4. It can be seen (Fig. 2 and Fig. 3) that the two peaks of phosphorus concentration, fractions 3 and 8, coincided with electrophoretic In view of the limited resolution. each of the two sets components 5 and 3. From the chromatographic profile of components ( I plus 2; 3 plus 4) was treated as a single it was estimated that component 1 plus 2, 3 plus 4, and et a/., 1975) have shown 5 made up, respectively, about 65, 30 and 57; of the total entity. Previous studies (DEIBLER that components 1 and 2 contain no phosphorus. while basic protein eluted. Hence. the component composition components 3 and 4 have essentially the same phosphorus of the protein had not been significantly altered by purification (compare with Fig. 1). contents. 1529 \ r ?7;6-- P THEBASIC protein of CNS myelin exists in multiple electrophoretic and chromatographic forms at alkaline pH (MARTENSON Pt al., 1971~;DEIBLER & MARTENSON, 1973). Five forms (components I, 2, 3, 4 and 5 in decreasing order of basicity) have been detected in the total basic protein; & these have been isolated and characterized (DEIBLER MARTENSON, 1973; DEIBLERet a/.. 1975). The five components differ sequentially by a single charge and differ in their contents of phosphoserine and phosphothreonine and carboxy-terminal arginyl residues (DEIBLERet al., 1975). The complete spectrum of components observed in the total protein results from a combination of differential phosphorylation, loss of one or two of the arginines at the carboxy terminus, and, possibly, deamidation of glutaminyl and asparaginyl residues. Unlike phosphorylation. loss of arginine and deamidation might arise as artifacts either in situ during the short interval between the death of the animal and freezing of the brain and/or during subsequent isolation of the basic protein. To eliminate the first possibility, we have instantaneously removed and frozen the brains from conscious guinea-pigs by the method of ‘freeze-blowing’ (VEECH et a/., 1973).In an attempt to eliminate the second possibility, we have extracted the basic protein by a procedure known et al., 1969) and preto inhibit its proteolysis (MARTENSON vent its conversion from the more basic to the less basic & MARTENSON, 1973). forms (DEIBLER Brains were removed from conscious Hartley strain guinea pigs by the freeze-blowing technique of VEECH et nl. (1973) and stored at -70°C until used. Five collections of tissue were made over a I-year period. Myelin basic protein was extracted at pH 3.0 from the delipidated tissue et al., 1972). The residue as described previously (DEIBLER most critical aspect of the procedure was the treatment of the frozen tissue with chloroform-methanol (2: t, v/v), 19 vol per gram: after homogenization in a Waring Blendor, the homogenate (containing the tissue water) was stirred for a prolonged period of time (24 h) to optimize destruction of any enzymes capable of using the basic protein as substrate. The subsequent treatment of the delipidated tissue with deionized water was carried out for only I to 2 h. rather than 24 h, to minimize the time available
Short communication
1530 2.0 -
-0.6
-E
-0.5
C
0 Q. 9
-0.4
$
1.0-
-0.3
C
0
-f!
-0.2
2 A 4
10 l 1 12
100
0
2
4
Cm f r o m Origin FIG.I . Electrophoresis of appron 150 pg of pH 3.0 extract. The profile is that of one of the five preparations: the range of values (per cent of basic protein as components I plus 2. 3 plus 4 and 5) obtained for the five preparations (one run of each) is shown. Electrophoresis was carried out toward the cathode in 5"; polyacrylamide gels containing 0.01 M-sodium glycinate. pH 10.6. and 8 M-urea for 3 h at 3.75 mA per gel. Fractions 1 to 4 (largely component 5) and fractions 5 to 8 (mixture of components 3 and 4) were combined separately and chromatographed on Sephadex G-100 to remove any polymeric basic protein and contaminants of low molecular weight. The amino acid compositions of the final, purified materials were essentially identical to that of fraction 12 (pure component 1). The purified basic protein of fractions 5 to Y and 1 to 3 contained. respectively, 0.6 and 0.5 g-atom of phosphorus per mol.' This study has shown that myelin basic protein isolated from freeze-blown brain displays a charge microheterogeneity that cannot be accounted for by phosphorylation alone. Had it resulted solely from phosphorylation. the protein would have consisted only of components 1. 3 and 5 with respective phosphorus concentrations of 0. I and 2 g-atom per mol. It could not have contained components 2 and 4. Furthermore. since the total basic protein contains only 0.2 g-atom of phosphorus per mol (MIYAMOTO & KAKIUCHI.1974; DEIBLER rt a!.. 1975),component 1 would have had to comprise at least 80", of the total protein. None of these requirements were found to hold experimentally. This study has also shown that there were no significant differences in the composition of basic protein isolated from freeze-blown as compared to quick-frozen (2 min et a/., post-mortem) brain: in a previous study (DEIBLER 1975) purified basic protein isolated from quick-frozen brain (designated preparation A) consisted of 639; component 1 plus 2, 31:; component 3 plus 4 and 6"; component 5 with respective phosphorus concentrations of 0, 0.5 and 1.0 g-atom per mol. ~
~~~
' The weight of protein was determined
by quantitative amino acid analysis and summation of the residue weights. The molecular weight of the guinea-pig basic protein is 18.300 (DUBLERrt al., 1975).
110
120
130
140
.
150
Tube No.
FIG.2. Fractionation of the basic protein by ion-exchange chromatography. Elution profiles of basic protein and associated phosphorus. The partially purified basic protein (430 mg) was applied to a column (2.5 x 45 cm) of CM-cellulose. Elution was carried out at pH 11 with a linear gradient of NaCI. Fractions of 18.1 ml were collected at a flow rate of 108 ml per h. The eluted basic protein was divided into 12 fractions as shown. exhaustively dialyzed and lyophilized. A portion of each lyophilized fraction was dissolved in 0.01 N-HCI. and the protein was precipitated in 254, trichloroacetic acid, washed with ethanol-ether, dried, weighed and analyzed in duplicate for phosphorus.
Since freeze-blown brains were removed from conscious animals and frozen instantaneously, and since the length of time the tissue remained frozen had no effect on the electrophoretic profile of the basic protein. it is clear that none of the alterations of the protein responsible for the microheterogeneity we observed occurred prior to its isolation. What is unclear at present is whether those alterations not attributable to phosphorylation were present in the protein in the frozen tissue or whether they occurred during the protein's isolation. Deamidation of proteins is known to occur in civo (for summary see ROBINSON.1974), and its rate appears to be related to the rate at which the protein turns over metabolically (ROBINSON et a/.. 1970; & MEHL~R 1973; , ROBINSON, 1974). Thus it MIDELFORT would not be surprising if the native basic protein existed in multiple, partially deamidated forms. Loss of carboxyterminal arginine is a different matter, however. We have & R. E. MARTENSON, unpublished) found (G. E. DEIBLER that nearly complete loss of the two carboxy-terminal arginines can occur if the initial chloroform-methanol treatment is brief ( I min) and if the subsequent treatment of the delipidated tissue with water is prolonged (24 h). In this study these conditions of time were not used, and the reaction was certainly minimized, if not prevented entirely. Still, a critical evaluation of the problem of artifact production during isolation will require extraction of the basic protein from the delipidated tissue under conditions quite different from those currently employed. Ackriow~2edgernents-This work was supported in part by National Multiple Sclerosis Society Grant 828-A-4. We arc
FIG.3. Electrophoresis of the chromatographic fractions shown in Fig. 7. at alkaline pH (see legend to Fig. I for conditions). Components 1 to 5 are identified at the right. Approximately 50 pg of lyophilized samples were applied to each gel.
Short communication
1531
MARTENSON R. E.. DEIBLERG. E. & KIESM. W. (19710) in Immunological Disorders sf the Nerrorts S ~ ~ r r t t ~ . Proceedings of the Association for Resenrch in Nerrous R. E. MARTENSON and Mental Disease (ROWLANDL. P., ed.) Vol. XLIX, Srction on Myelill Chemistry, A. J. KRAMER pp. 76-93. Williams & Wilkins. Baltimore. Laboratory of Cerebral Metabolism. R. E., DEIBLER G. E. & KIES M. W. (1971b) E. DUBLER MAKTENSON Atrtiotial Institute of Mental Health, GLADYS J . Neurocheni. IS, 2417-2426. Bethesda. MD 20014, MARTENSON R. E., KRAMER A. J. & DEIBLERG. E. (1975) U.S. A. Biochemistry, Easton 14, 1067-1073. MIDELFORT C. F. & MEHLER A. H. (1972) Proc. natn. Acad. REFERENCES Sci., U.S.A. 69, 1816-1819. E. & KAKIUCHI S. (1974) J. bid. Chern. 249, DEIHLER G. E. & MARTENSON R. E. (1973) J. biol. Chem. MIYAMOTO 2769-2777. 248, 2392-2396. DEIHLER G. E., MARTENSON R. E. & KIFS M. W.(1972) ROBINSONA. B. (1974) Proc. natn. Acad. Sci., U.S.A. 71. 885-888. Prepur. Biochetn. 2, 139-165. A. B., MCKERROW J. H. & CARYP. (1970) Proc. DEIBLER G. E., MARTENSON R. E., KRAMER A. J., KIESM. ROBINSON natn. Acad. Sci.. U.S.A. 66, 153-757. W. & MIYAMOTO E. (1975) J. bid. Chem. 250, 7931-7938. MARTENSON R. E.. DEIBLER G. E. & KIFS M. W. (1969) VEECHR. L., HARRISR. L.. VELOW D. & VEECHE. H. (1973) J. Neurochem. 20, 183-188. J . biol. Chern. 244. 42614267. indebted to Dr. R. L. VEECH and co-workers for making available their brain-blower and instructing us in its use.
Microheterogeneity and phosphate content of myelin basic protein from ‘freeze-blown’ guinea-pig brains (Received 5 February 1976. Accepted 26 April 1976)
for any possible surviving enzymes to act. The pH 3.0 extract was dialyzed overnight against 0.1 M-acetic acid (pH 2.9) and lyophilized. All procedures were carried out at 0 to 5°C. Procedures for gel filtration and polyacrylaet a/., 1972) ionmide gel electrophoresis (DEIBLER & MARTENSON. 1973), exchange chromatography (DEIBLER ct a/.. 1975) and phosamino acid analysis (MARTENSON et al.. 1975) have been dephorus determination (DEIBLER scribed. Polyacrylamide gels containing separated proteins stained with Amido black were scanned densitometrically at 690 nm. The relative areas under the peaks were determined by transferring the curves to thin cardboard and cutting out and weighing the appropriate areas. Myelin basic protein is the only protein present in significant amounts in pH 3.0 extracts which migrates cathodiel al., 1971b). Hence, we cally at pH 10.6 (MARTENSON were able to examine the protein’s microheterogeneity prior to the actual isolation of the pure protein. Electrophoresis of the pH 3.0 extracts at pH 10.6 (Fig. 1) showed that the basic protein consisted of three major forms, components 1, 3 and 5. The minor components (2 and 4) were poorly resolved. N o significant differences in the profiles were found among the five preparations examined. Component 1 plus 2’ accounted for 6 5 . 8 : ~ f 1.13 (s.D.) of the total basic protein; component 3 plus 4, 29.8aL 0.86; and component 5, 4.4% 0.34. The combined pH 3.0 extracts (1.06 g) were subjected to repeated gel filtration through a column (3.2 x 95 cm) of Sephadex G-100 in 0.01 N - H C ~at 5°C to remove most of the high and low molecular weight contaminants; 0.45 g of partially purified basic protein was obtained. The procedure was monitored by gel electrophoresis at both pH 2.4 and 10.6 to insure that no significant amounts of basic protein were discarded. The partially purified basic protein was chromatographed on a column of CM-cellulose according to the procedure of DEIBLER & MARTENSON (1973). The fractions eluted were analyzed for protein and phosphorus (Fig. 2). Electrophoresis of the chromatographic fractions at pH 10.6 (Fig. 3) showed that fractions 1 to 4 consisted of component 5 and material of lower electrophoretic mobility, while fractions 5 to 8 consisted predominantly of components 3 and 4. It can be seen (Fig. 2 and Fig. 3) that the two peaks of phosphorus concentration, fractions 3 and 8, coincided with electrophoretic In view of the limited resolution. each of the two sets components 5 and 3. From the chromatographic profile of components ( I plus 2; 3 plus 4) was treated as a single it was estimated that component 1 plus 2, 3 plus 4, and et a/., 1975) have shown 5 made up, respectively, about 65, 30 and 57; of the total entity. Previous studies (DEIBLER that components 1 and 2 contain no phosphorus. while basic protein eluted. Hence. the component composition components 3 and 4 have essentially the same phosphorus of the protein had not been significantly altered by purification (compare with Fig. 1). contents. 1529 \ r ?7;6-- P THEBASIC protein of CNS myelin exists in multiple electrophoretic and chromatographic forms at alkaline pH (MARTENSON Pt al., 1971~;DEIBLER & MARTENSON, 1973). Five forms (components I, 2, 3, 4 and 5 in decreasing order of basicity) have been detected in the total basic protein; & these have been isolated and characterized (DEIBLER MARTENSON, 1973; DEIBLERet a/.. 1975). The five components differ sequentially by a single charge and differ in their contents of phosphoserine and phosphothreonine and carboxy-terminal arginyl residues (DEIBLERet al., 1975). The complete spectrum of components observed in the total protein results from a combination of differential phosphorylation, loss of one or two of the arginines at the carboxy terminus, and, possibly, deamidation of glutaminyl and asparaginyl residues. Unlike phosphorylation. loss of arginine and deamidation might arise as artifacts either in situ during the short interval between the death of the animal and freezing of the brain and/or during subsequent isolation of the basic protein. To eliminate the first possibility, we have instantaneously removed and frozen the brains from conscious guinea-pigs by the method of ‘freeze-blowing’ (VEECH et a/., 1973).In an attempt to eliminate the second possibility, we have extracted the basic protein by a procedure known et al., 1969) and preto inhibit its proteolysis (MARTENSON vent its conversion from the more basic to the less basic & MARTENSON, 1973). forms (DEIBLER Brains were removed from conscious Hartley strain guinea pigs by the freeze-blowing technique of VEECH et nl. (1973) and stored at -70°C until used. Five collections of tissue were made over a I-year period. Myelin basic protein was extracted at pH 3.0 from the delipidated tissue et al., 1972). The residue as described previously (DEIBLER most critical aspect of the procedure was the treatment of the frozen tissue with chloroform-methanol (2: t, v/v), 19 vol per gram: after homogenization in a Waring Blendor, the homogenate (containing the tissue water) was stirred for a prolonged period of time (24 h) to optimize destruction of any enzymes capable of using the basic protein as substrate. The subsequent treatment of the delipidated tissue with deionized water was carried out for only I to 2 h. rather than 24 h, to minimize the time available
Short communication
1530 2.0 -
-0.6
-E
-0.5
C
0 Q. 9
-0.4
$
1.0-
-0.3
C
0
-f!
-0.2
2 A 4
10 l 1 12
100
0
2
4
Cm f r o m Origin FIG.I . Electrophoresis of appron 150 pg of pH 3.0 extract. The profile is that of one of the five preparations: the range of values (per cent of basic protein as components I plus 2. 3 plus 4 and 5) obtained for the five preparations (one run of each) is shown. Electrophoresis was carried out toward the cathode in 5"; polyacrylamide gels containing 0.01 M-sodium glycinate. pH 10.6. and 8 M-urea for 3 h at 3.75 mA per gel. Fractions 1 to 4 (largely component 5) and fractions 5 to 8 (mixture of components 3 and 4) were combined separately and chromatographed on Sephadex G-100 to remove any polymeric basic protein and contaminants of low molecular weight. The amino acid compositions of the final, purified materials were essentially identical to that of fraction 12 (pure component 1). The purified basic protein of fractions 5 to Y and 1 to 3 contained. respectively, 0.6 and 0.5 g-atom of phosphorus per mol.' This study has shown that myelin basic protein isolated from freeze-blown brain displays a charge microheterogeneity that cannot be accounted for by phosphorylation alone. Had it resulted solely from phosphorylation. the protein would have consisted only of components 1. 3 and 5 with respective phosphorus concentrations of 0. I and 2 g-atom per mol. It could not have contained components 2 and 4. Furthermore. since the total basic protein contains only 0.2 g-atom of phosphorus per mol (MIYAMOTO & KAKIUCHI.1974; DEIBLER rt a!.. 1975),component 1 would have had to comprise at least 80", of the total protein. None of these requirements were found to hold experimentally. This study has also shown that there were no significant differences in the composition of basic protein isolated from freeze-blown as compared to quick-frozen (2 min et a/., post-mortem) brain: in a previous study (DEIBLER 1975) purified basic protein isolated from quick-frozen brain (designated preparation A) consisted of 639; component 1 plus 2, 31:; component 3 plus 4 and 6"; component 5 with respective phosphorus concentrations of 0, 0.5 and 1.0 g-atom per mol. ~
~~~
' The weight of protein was determined
by quantitative amino acid analysis and summation of the residue weights. The molecular weight of the guinea-pig basic protein is 18.300 (DUBLERrt al., 1975).
110
120
130
140
.
150
Tube No.
FIG.2. Fractionation of the basic protein by ion-exchange chromatography. Elution profiles of basic protein and associated phosphorus. The partially purified basic protein (430 mg) was applied to a column (2.5 x 45 cm) of CM-cellulose. Elution was carried out at pH 11 with a linear gradient of NaCI. Fractions of 18.1 ml were collected at a flow rate of 108 ml per h. The eluted basic protein was divided into 12 fractions as shown. exhaustively dialyzed and lyophilized. A portion of each lyophilized fraction was dissolved in 0.01 N-HCI. and the protein was precipitated in 254, trichloroacetic acid, washed with ethanol-ether, dried, weighed and analyzed in duplicate for phosphorus.
Since freeze-blown brains were removed from conscious animals and frozen instantaneously, and since the length of time the tissue remained frozen had no effect on the electrophoretic profile of the basic protein. it is clear that none of the alterations of the protein responsible for the microheterogeneity we observed occurred prior to its isolation. What is unclear at present is whether those alterations not attributable to phosphorylation were present in the protein in the frozen tissue or whether they occurred during the protein's isolation. Deamidation of proteins is known to occur in civo (for summary see ROBINSON.1974), and its rate appears to be related to the rate at which the protein turns over metabolically (ROBINSON et a/.. 1970; & MEHL~R 1973; , ROBINSON, 1974). Thus it MIDELFORT would not be surprising if the native basic protein existed in multiple, partially deamidated forms. Loss of carboxyterminal arginine is a different matter, however. We have & R. E. MARTENSON, unpublished) found (G. E. DEIBLER that nearly complete loss of the two carboxy-terminal arginines can occur if the initial chloroform-methanol treatment is brief ( I min) and if the subsequent treatment of the delipidated tissue with water is prolonged (24 h). In this study these conditions of time were not used, and the reaction was certainly minimized, if not prevented entirely. Still, a critical evaluation of the problem of artifact production during isolation will require extraction of the basic protein from the delipidated tissue under conditions quite different from those currently employed. Ackriow~2edgernents-This work was supported in part by National Multiple Sclerosis Society Grant 828-A-4. We arc
FIG.3. Electrophoresis of the chromatographic fractions shown in Fig. 7. at alkaline pH (see legend to Fig. I for conditions). Components 1 to 5 are identified at the right. Approximately 50 pg of lyophilized samples were applied to each gel.
Short communication
1531
MARTENSON R. E.. DEIBLERG. E. & KIESM. W. (19710) in Immunological Disorders sf the Nerrorts S ~ ~ r r t t ~ . Proceedings of the Association for Resenrch in Nerrous R. E. MARTENSON and Mental Disease (ROWLANDL. P., ed.) Vol. XLIX, Srction on Myelill Chemistry, A. J. KRAMER pp. 76-93. Williams & Wilkins. Baltimore. Laboratory of Cerebral Metabolism. R. E., DEIBLER G. E. & KIES M. W. (1971b) E. DUBLER MAKTENSON Atrtiotial Institute of Mental Health, GLADYS J . Neurocheni. IS, 2417-2426. Bethesda. MD 20014, MARTENSON R. E., KRAMER A. J. & DEIBLERG. E. (1975) U.S. A. Biochemistry, Easton 14, 1067-1073. MIDELFORT C. F. & MEHLER A. H. (1972) Proc. natn. Acad. REFERENCES Sci., U.S.A. 69, 1816-1819. E. & KAKIUCHI S. (1974) J. bid. Chern. 249, DEIHLER G. E. & MARTENSON R. E. (1973) J. biol. Chem. MIYAMOTO 2769-2777. 248, 2392-2396. DEIHLER G. E., MARTENSON R. E. & KIFS M. W.(1972) ROBINSONA. B. (1974) Proc. natn. Acad. Sci., U.S.A. 71. 885-888. Prepur. Biochetn. 2, 139-165. A. B., MCKERROW J. H. & CARYP. (1970) Proc. DEIBLER G. E., MARTENSON R. E., KRAMER A. J., KIESM. ROBINSON natn. Acad. Sci.. U.S.A. 66, 153-757. W. & MIYAMOTO E. (1975) J. bid. Chem. 250, 7931-7938. MARTENSON R. E.. DEIBLER G. E. & KIFS M. W. (1969) VEECHR. L., HARRISR. L.. VELOW D. & VEECHE. H. (1973) J. Neurochem. 20, 183-188. J . biol. Chern. 244. 42614267. indebted to Dr. R. L. VEECH and co-workers for making available their brain-blower and instructing us in its use.
