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Evidence for the presence of N-Acetyl-α-l -aspartyl-l -glutamate in human brain
ARCHIVES
OF
BIOCHEMISTRY
Evidence
AND
BIOPHYSICS
for the Presence
22-28 (1967)
119,
of N-Acetyi-cx-L-Aspartyl-L-Glutamate
in Human ERIK
J. OLSON, LITTLETON
Department
Brain’
H. WADE,
AND
JOSEPH V. AUDITORE
of Pharmacology, Mehurry Medical College, and Department of Pathology, Nashville Memorial Hospital, Nashville, Tennessee 37208 Received
June 10, 1966
A peptide composed of equimolar quantities of glutamic and aspartic acids was isolated from a protein-free extract of human brain by a combination of ion-exchange and paper chromatography. Hydraeinolysis studies established that glutamic acid was C-terminal and that the amino group of the aspartyl residue was substituted with an acetyl group. To determine which aspartyl carboxyl group was linked to glutamic acid, N-acetyl-aand N-acetyl-P-aspartyl glutamate were synthesized. When the partial acid hydrolyzates of the synthesized acetylated peptides were chromatographed on the amino acid analyzer, two distinct elution patterns were observed. The elution pattern of the brain dipeptide resembled the alpha rather than the beta derivative. When acetylated amino acids prepared from brain dipeptide were incubated with hog L-specific kidney acylase, aspartic and glutamic acids were liberated.
brain of horse and rabbit neurotaxis by Curatolo (3) and Curatolo et al. (4). The complete structural elucidation of the peptide remained to be achieved. The principle structures considered were Nacetyl-a-L-aspartyl-L-glutamate and N-acetyl-fl-L-aspartyl-L-glutamate as shown below.
In 1964 Auditore and Hendrickson (1) reported the isolation of an acetylated peptide composed of equimolar quantities of aspartic and glutamic acids from human brain. Later investigations established that glutamic acid is the C-terminal amino acid and that the terminal amino group is 0 0 H~c-iLL-~-~-c-oH HCH COH 0
0
0
0
0
Hg-C-:-:-~-C-!&,, HCH
CEH
HCH
HCH
0
HCH
COH
COH
0
0
Alpha
Beta
This report presents evidence which indicates that the acetylated peptide from human brain is N-acetyl-a-L-aspartyl-Lglutamate.
substituted with an acetate moiety (2). The compound was therefore tentatively identified as N-acetyl-aspartyl glutamate. A similar acylated peptide was found in the 1 This investigation was supported by U. S. Public Health research grant B-3520, National Institute of Neurological Diseases and Blindness; and grant GB-1721 from the National Science Foundation.
MATERIALS
AND
METHODS
Preparation of tissue extract. Human brains were obtained as soon after death as possible, and placed at -80% for 24 hours. Each brain was cut 22
N-ACETYL-(Y-L-ASPARTYL-L-GLUTAMATE into small segments with a cleaver andmacerated in 2 volumes of 20% trichloroacetic acid in a highspeed, one gallon size Waring Blendor for 3 minutes at, 15,500 rpm, and for 1 hour at 2500 rpm at 4”. The homogenate was centrifuged in a glass container at 2500 rpm for 30 minutes; the supernatant solution was decanted and the sediment was ext,racted wit,h 1 volume of water. The aqueous layer was recovered by centrifugation and added to the first supernatant solution; the sediment was discarded. The pooled aqueous extract was treated with liter portions of diethyl ether until the pH of the extract was 3.4. The volume of the extract was reduced to 50 ml under vacuum at 30” in a rotary flash evaporat,or. with AG60-X4 procedures Chromatographic (Dowex-50) column. Twenty five ml of the concentrated extract was applied to a column of resin (3.3 X 100 cm) in the hydrogen ion form. The extract was washed into the resin bed with a small volume of water and the column was developed with 700 ml of water. The flow rate was set at 40 ml per hour and 20-ml fractions were collected. The fractions were dried under vacuum and the sediment was dissolved in 0.2 ml of water. A small aliquot of each fraction was spot,ted on Whatman No. 1 chromatography paper (23 X 57 cm) and developed in n-butanol:ethanol:acetic acid:water (8:2:1:3; vol) employing the descending technique at 22-26” for 19-24 hours; the solvent front ran an average distance of 35-45 cm. Each chromatogram was tested for acylated amino acids and peptides by the chlorination-starch-iodide (CSI) reaction described by Rydon and Smith (5). This reaction involves placing the chromatogram in an atmosphere of chlorine for 10 minutes, ventilating for 30 minutes, and spraying with 1% starch in 1% KI. The reactive substances appear as blue-black spots on a faint blue background. Amino acids were detected on the chromatogram by the ninhydrin reaction. The chromatogram was sprayed with 0.25y0 ninhydrin in water-saturated butanol and heated at 8&100” for 10 minutes for development. Preparation of paper eluate of Dowex-CO jraclions. Fractions (Nos. 2130) that contained the bulk of the CSI reactive substances were pooled and chromatographed on several sheets of Whatman No. 1 paper, 23 X 57 cm, with a streaking technique. After development in n-butanol: ethanol:acetic acid:water (8:2:1:3; vol) small strips were cut from each side of a chromatogram and stained. These strips served as indicators to map the location of the desired reactive substance. The CSI band between an Rp value of 0.50 and 0.75 was selected, excised, placed in a large beaker filled with water, and mechanically agitated for several hours. The aqueous fluid was collected
IN
HUMAN
BRAIN
23
and taken to dryness in a rotating flash evaporator at 30”, and the residue was dissolved in a desired volume of water. Chromatographic procedures with AGl-X4 resin. The eluate was applied to a 1.75 X 100 cm AGl-X4 resin column in the chloride form and developed with a discontinuous gradient of glacial acetic acid. Three hundred ml of 1,2,3,6, and8 N glacial acetic acid were passed successively over the column. The flow rate was 40 ml per hour and 16ml fractions were collected. Fractions were taken to dryness and the residue was dissolved in a small amount (0.2 ml) of water. An aliquot of each fraction was chromatographed in n-butanol:ethanol: acetic acid:water (8:2:1:3; vol) using Whatman No. 1 paper. Peptides and other reactive compounds were detected on chromatograms by the chlorination-starch-iodide reaction. This isolation procedure clearly separates the acetylated peptide from N-acetyl-L-aspartate and N-acetyl-r-glutamate (2). Synfhesis of N-acetyl-o-L-aspartyl-L-glutamate. N-Carbobenzoxy-a-benzyl-L-aspartate was the starting compound for the synthesis of N-acetylp-L-aspartyl-L-glutamate. The aspartic acid derivative was synthesized from N-carbobenzoxyL-aspartate according to the methods described by Fisher and Whetstone (6). The melting point of the compound was 83”; the reported melting point is 85” (6). We obtained this compound in 30yo yield. Dibenzyl-L-glutamate toluenesulfonate was prepared by heating equimolar quantities of Lglutamic and para-toluene sulfonic acid to a temperature of 120” for 15 minutes in benzyl alcohol (7). It was recrystallized from ethanol:ether (1: 1; vol) to a melting point of 142”. The yield was 80% of the theoretical yield. N-Carbobenzoxy-aand dibenzyl-L-glutamate benzyl-L-aspartate toluenesulfonate were condensed in the presence of dicyclohexyl-diimide dissolved in dichloromethane to form N-carbobenzoxy-tribenzyl-D-Laspartyl-L-glut,amate (8). N-carbobenzoxy-tribenzyl-a-L-aspartyl-L-glutamate, recrystallized from ethanol, had a melting point of 111” (8). The yield was about 40% of the theoretical yield. Blocking groups were removed with hydrogen and palladium to make B-L-aspartyl-L-glutamate. This compound was identified by electrophoresis and degradation experiments as described in the results section. The dipeptide was acetylated by exposure to a 50y0 aqueous solution of acetic anhydride for 1 hour at 50”. The resulting product gave negative ninhydrin and hydroxamic acid tests (9). This product gave aspartic and glutamic acid on acid hydrolysis and glutamic acid on hydrazinolysis. Synthesis of N-acetyl-a-L-aspartyl glutamate. The starting compound for the above peptide,
24
OLSON,
WADE,
@-benxyl-L-aspartate, was synthesized (10) and after recrystallizing the compound from water to its reported melting point of 21%220”, the yield was 40% of theoretical. P-Benzyl-L-aspart,ate was converted to its carbobenzoxy derivative by the method devised by Benoiton (10). The carbobenzoxy derivative was recrystallized from water to the observed melting point, of 107-108”. NCarbobenzoxy-fl-benzyl-L-aspartate was condensed with dibenzyl-glutamate para-toluenesulfonate in the presence of dicyclohexyldiimide in dichloromethane to form N-carbobenzoxy-tribenzyl-cu-L-aspartyl-L-glutamate. After recrystallization from ethanol it. melted at 100” (8). The yield from this reaction was about 40% of the theoretical yield. The blocking groups were removed with hydrogen and palladium to form or-L-aspartyl-L-glutamate. The identification of this dipeptide was based on electrophoretic mobility and degradation experiments (see Results section). The compound was acetylated at room temperature by dropwise addition of acetic anhydride to an aqueous solution of the dipeptide with constant stirring until no ninhydrin color was observed. Care was taken to avoid formation of an acetic anhydride phase. The entire reaction required 5 hours and involved the addition of 15 ml of acetic anhydride to approximately 200 mg of cu-L-aspartyl-L-glutamate in 35 ml of water. When chromatographed in butanol:ethanol:acetic acid: water, the acetylated preparation gave two chlorine, starch iodide-positive components with RF values of 0.4 and 0.6. Both substances yielded aspartic and glutamic acids after acid hydrolysis but only the RF 0.6 component yielded glutamic acid and acetyl hydrazide after hydrazinolysis. Therefore, the Rp 0.6 component wa8 considered to be N-acetyl-a-L-aspartyl-L-glutamate. Electrophoretic methods. Electrophoresis was performed with a horizontal pressure cooling plate electrophoresis assembly obtained from the E-C Apparatus Corp. One-dimensional paper electrophoresis was performed on Whatman No. 1 paper at a set voltage. Electrophoresis was conducted at approximately 15” using a voltage gradient of 38 V/cm. Hydrazinolysis of the dipeptide. Samples of the synthesized and naturally occurring peptides were subjected to hydrazinolysis by a modification of the method of Niu and Fraenkel-Conrat (11). An aliquot of the dipeptide was placed in a tube and dried in vacua. The dried substance was dissolved in hydrazine and sealed under nitrogen. The sealed tube was held at 100” for 4 hours, and the excess hydrazine was then removed under vacuum. The dried hydrazinolyzate was dissolved in a small amount of water and subjected to electrophoretic and chromatographic analysis.
AND
AUDITORE
Acid hydrolysis of peptides. The synthesized and naturally occurring acetylated peptides were partially hydrolyzed by exposure to 0.1 N HCl under nitrogen for 2 hours at 100”. The naturally occurring dipeptide was also hydrolyzed under nitrogen in 0.05 N HCl at 100” for 15, 30, and 70 minutes. Exposure to 6 N HCI at 100” for 18 hours completely hydrolyzed the naturally occurring dipeptide. Chromatographic analysis on the amino acid analyzer. After hydrolysis the solutions of the peptides were concentrated to dryness and taken up in pH 2.2 citrate. An amino acid mixture composed of 0.025 pmole of each of the amino acids found in proteins was added to the reconstituted hydrolyzates. The resulting solution was subjected to amino acid chromatography according to the method of Moore et al. (12). Determination of amino acid con$gurations. The complete acid hydrolyzate of the naturally occurring dipeptide was acetylated by exposure to 50% acetic anhydride at 50” for 1.5 hours. After this solution was concentrated to dryness, the acetylated amino acids were subjected to the action of 2 mg of hog kidney acylase in 0.6 ml of 0.05 M phosphate buffer (pH 7.4) at 37” for 1 hour (13). The digest was dialyzed and the dialyzate was concentrated to dryness and subjected to amino acid chromatography. Hog kidney acylase was obtained from Mann Biochemicals. Note added in proof. A sample of human brain N-acetyl-aspartyl-glutamate was hydrolyzed in 6 N HCI at 100” for 18 hours and then dried in vucuo. To separate the mixture of glutamic and aspartic acids the hydrolyzate was applied to a 100 X 1.75 cm AGl-X4 acetate column that was then eluted with 300 ml of 1.0 M ammonium acetate, acetic acid pH 4 buffer, 300 ml of 0.1 N acetic acid, and 300 ml of 1 N acetic acid at a flow rate of 25 ml per hour. Glutamic appeared slightly before aspartic acid, both being isolated in separate 5-ml fractions of the N acetic acid eluate. Both amino acids were then re-crystallized from water and melting points were taken on a “Mel Temp Apparatus.” The observed melting point for commercial Dand L-glutamic acid was 203”, and our sample of glutamic acid melted at 201”. When our sample of glutamic acid was mixed with L-glutamic acid it melted at 203”, and it melted at 195” when mixed with n-glutamic acid. Therefore we consider that all of the glutamic acid from brain N-acetyl-aaspartyl-glutamate is of the L-configuration. Aspartic acid decomposed without melting. RESULTS
Our previous work indicated that the ninhydrin-negative, starch chloride-posi-
N-ACETYL-WL-ASPARTYL-L-GLUTAMATE
IN
HUMAN
BRAIS
“5
tive peptide from human brain was Nacet,yl aspartyl glutamat’e. Structural analysis of the brain dipept’ide revealed that the aspartyl moiet(y could be attached to glutarnic acid through an alpha or beta linkage. The following chemical data indicated that the naturally occurring dipeptide assumes the alpha structure. Synthesis of the unacet’ylated dipeptides was confirmed by electrophoresis, chromatog-
I
Time
30
(Minutes)
I 100
FIG. 1. A portion of the elution pattern observed when synthetic a-L-aspartyl-L-glutamate is co-chromatographed with a known mixture of amino acids.
t
I
( Mmutes)
FIG. 2. A portion of the elution pattern observed when synthetic p-L-aspartyl-L-glutamate is co-chromatographed with a known mixture of amino acids.
(Minutes)
studies. The and degradation electrophoretic mobilit’ies of the unacetylated peptides to the negative pole were, in reference to a mobility of 1.0 for aspartic acid, 1.6 for the alpha and 0.7 for the beta. These values correspond closely to the values (1.4 for alpha and 0.7 for beta) reported by Bryant et al. (8). Exhaustive acid hydrolysis which approximately doubled their ninhydrin color produced glutamic and aspartic acids from both peptides. Hydrazinolysis of the synbhetic dipeptides, on the other hand, yielded only glutamic acid. Application of ion-exchange chromatography as described by Moore et al. (12) clearly differentiated the alpha and beta unacetylated dipeptides. Both peptides were eluted as single peaks; the beta emerged from the column early just prior to aspartic acid, and the alpha appeared much
rwhy,
Time
Time
I 100 FIG. 3. A portion of the elution pattern observed when a partial acid hydrolyzate of synthetic IV-acetyl-cu-L-aspartyl-L-glutamate is cochromatographed with a known mixture of amino acids.
30
26
OLSON, WADE,
AND AUDITORE
later positioning itself between proline and glycine (Figs. 1 and 2). The synthesized acetylated dipeptides and brain acetylated dipeptides were partially converted by acid hydrolysis to the unacetylated derivatives for analysis by ion-exchange chromatography. The acetylated peptides were hydrolyzed in 0.1 N HCl at 100” for 2 hours. Two distinct characteristic elution patterns were observed. Three prominent peaks were clearly evident in each of the three chromatograms (Figs. 3-5). The peaks corresponding to aspartic and glutamic acids were common to all of the hydrolyzates, but the third peak corresponded to the unacetylated dipeptide. It was apparent that the partial acid hydroly-
Time
P I !-I ii ;1
30
(Minutes)
FIG. 5. A portion of the elution pattern observed when a partial acid hydrolyzate of the acetylated brain peptide is cochromatographed with a known mixture of amino acids.
Time
( Minutes)
FIG. 4. A portion of the elution pattern observed when a partial acid hydrolyzate of synthetic N-acetyl-P-n-aspartyl-n-glutamate is cochromatographed with a known mixture of amino acids.
zate elution profile of the brain peptide was identical to the profile obtained with the alpha dipeptide hydrolyzate. That the brain dipeptide possessed the alpha linkage was further substantiated by the fact that cochromatography of the partial acid hydrolyzates of synthetic N-acetyl-ar-n-aspartyl-nglutamate and acetylated brain dipeptide gave the same chromatographic elution pattern. The possibility of the peptide being a tetra or higher homologue was discounted on the basis of its negative biuret test. Furthermore, the yields of glutamic acid obtained after hydrazinolysis, which liberates only C-terminal amino acids, were the same for N-acetyl-or-L-aspartyl-L-glutamate and the dipeptide when the glutamic acid obtained was expressed as the percentage of glutamic acid obtained after total acid
N-ACETYL-LU-L-ASPARTYL-L-GLUTAMATE TABLE I YIELDS OF GLUTAMIC ACID OBTAINED AFTER HYDRAZINOLYSIS AS COMP.4RED WITH YIELDS OF GLUTAMIC OBT.\INED .~FTEH EXHAUSTIVE ACID HYDROLYSIS Compound
N-Acetyl-a-L-aspartyI+glutamate Unknown
Per cent
50 44
hydrolysis (Table 1). Our yields of 50 % for the C-terminal glutamate were in keeping with previously observed yields of Cterminal amino acids from hydrazinolyzates (11). Moreover, the brain dipeptide yielded the same fundamental elution pattern (Fig. 5) when chromatographed after hydrolysis in 0.05 N HCl for 15, 30, and 70 minutes at 100”. Despite a tenfold increase in aspartic acid and a threefold increase in glutamic acid, additional peaks would have been in evidence if the brain peptide was a tetra or a higher polymer peptide. To determine the configuration of the amino acids of the brain dipeptide, the amino acid mixture obtained after total acid hydrolysis was acetylated and digested with hog kidney acylase, which hydrolyzes Nacetylated-D-amino acids. When a concentrated dialyzate of the digest was subjected to amino acid chromatography, a complex elution pattern was obtained probably because of trans peptidase reactions. However, the presence of aspartic and glutamic acids indicated that both amino acids of the acetylated brain dipeptide were of the L-configuration. It was also found that glutamic acid was not liberated when a mixture of N-acetyl-L-aspartic and Nacetyl-D-glutamic acid was subjected to the hog kidney acylase preparation. DISCUSSION Recently we reported the isolation of an acetylated peptide which yielded equimolar quantities of aspartic and glutamic acid on exhaustive acid hydrolysis (2). The absence of ammonia in the hydrolyzate ruled out the possibility of an amide derivative. Hywhich liberates C-terminal drazinolysis, amino acids, liberated glutamic acid and acetyl hydrazide. On the basis of t,his
IX IIUMAN BRAIN
27
evidence and the acetylated peptides negative biuret test, the brain peptide was considered to be either N-acetyl-ol-L-aspartyl-D-glutamate or N-acetyl+L-aspartylD-glutamate. Synthesized unacetylated alpha and beta peptides were easily differentiated by chromatography on polystyrenesulfonate resin with pH 3.28 citrate buffer as the eluting solution. The more rapid chromatographic mobility of the @-L-aspartyl-Dglutamate may be due to the fact that one of its free carboxyl groups is adjacent to the positively charged amino group. The amino group would enhance the ionization of the adjacent carboxyl group and make the molecule more negatively charged at) pH 3.2s. When partial acid hydrolyzat.es of the synthesized acetylated alpha peptide and brain dipeptide were subjected to amino acid chromatography the chromatogrnphic profiles were identical. Co-chromatography of the partial acid hydrolyzates of the synt’hesized acetylated alpha and the brain peptide also yielded the same pattern. However, the possibility that) the acet*ylated brain pept#ide was a larger pept,ide still existed. Therefore, a series of partial acid hydrolyzates of the brain dipeptide was subjected to amino acid chromatography. The elution pattern shown in Fig. 3 persisted regardless of the hydrolysis time, indicating that no other ninhydrin-positive peptides were released. 1/loreover, the ratio of glutamic acid obtained by hydrazinolysis to glutamic acid obtained by exhaustive acid hydrolysis was the same for N-acetyla+aspartyl-D-glutamate and the acetylated brain peptide. Therefore, w’c believe that the acetylated brain peptide is the dipeptide, N-acetyl-a+aspartyl-D-glutamate. Since human brain was used as a source of this peptide one might speculate that this compound is not a normal constituent of brain tissue but merely arises from postmortem catabolism, drug administration, or from a deteriorating physiological condition. This appears unlikely for several reasons. First, the peptide has been found in numerous human brains. Second, we have isolated a similar peptide from t.hc brains of
28
OLSON,
WADE,
healthy rats sacrificed by decapitation. The rat brains used in t’hese experiments were frozen in dry ice in less than one minute after the death of the animal. Furthermore, an apparent,ly similar acet’ylated aspartyl glutamate peptide has been isolated from horse brain and rabbit neurotaxis (3). It is noteworthy that Westall appears to have isolated a ninhydrin-positive peptide composed of aspartic and glutamic acid from human urine (14). Such a compound could arise from the deacylation of the brain dipeptide. A preliminary report of this work was given at the Federation meeting (15). REFERENCES 1. AUDITORE, J. V., AND Intern. J. Pharmacol. 3, 2. AUDITORE, J. V., OLSON, L. H., Arch. Biochem. (1966).
HENDRICKSON, Ii., 1 (1964). E. J., AND W.~DE, Biophys. 114, 452
AND
AUDITORE
3. CURATOLO, A., Abstr. IV Int. Cong. Biochem. N. Y., V. E. 98, (1964). 4. CURATOLO, A., D’ARC~NGELO, P., LINO, A., AND BKANC~TI, A., J. Neurochem. 12, 339 (1965). RYDON, II. N., .~ND SMITH, P. W. G., Nature 169, 922 (1952). FISCHER, It. F., J\~~ WHETSTONE, R. R., J. Am. Chem. Sot. 76, 750 (1955). IZ~MIY~, N., .UXD MAKISUMI, S., Nippon Kagaku Zasshi 76, 662 (1957). BRYANT, P. M., MOORE, R. H., PIMLOTT, P. J., BND YOUNG, G. T., J. Chem. Sot. 3858 (1959). 9. STADTMAN, E. R., .%NDBARKER, H. A., J. Biol. Chem. 184, 769 (1950). 10. BENOITON, L., Can. J. Chem. 40,570 (1962). H., J. 11. Nru, C. I., AND FRAENKEL-CONRAT, Am. Chem. Sot. 77, 5882 (1955). 12. MOORE, S., SPACKMSN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185, (1958). 13. FODOR, P. J., PRICE, V. E., AND GREENSTEIN, J. P., J. Biol. Chem. 178, 503 (1949). 14. WESTALL, R. G., Biochem. J. 60, 247 (1955). 15. OLSON, E. J., AND AUDITORE, J. V., Federation hoc. 26, 560 (1966).
OF
BIOCHEMISTRY
Evidence
AND
BIOPHYSICS
for the Presence
22-28 (1967)
119,
of N-Acetyi-cx-L-Aspartyl-L-Glutamate
in Human ERIK
J. OLSON, LITTLETON
Department
Brain’
H. WADE,
AND
JOSEPH V. AUDITORE
of Pharmacology, Mehurry Medical College, and Department of Pathology, Nashville Memorial Hospital, Nashville, Tennessee 37208 Received
June 10, 1966
A peptide composed of equimolar quantities of glutamic and aspartic acids was isolated from a protein-free extract of human brain by a combination of ion-exchange and paper chromatography. Hydraeinolysis studies established that glutamic acid was C-terminal and that the amino group of the aspartyl residue was substituted with an acetyl group. To determine which aspartyl carboxyl group was linked to glutamic acid, N-acetyl-aand N-acetyl-P-aspartyl glutamate were synthesized. When the partial acid hydrolyzates of the synthesized acetylated peptides were chromatographed on the amino acid analyzer, two distinct elution patterns were observed. The elution pattern of the brain dipeptide resembled the alpha rather than the beta derivative. When acetylated amino acids prepared from brain dipeptide were incubated with hog L-specific kidney acylase, aspartic and glutamic acids were liberated.
brain of horse and rabbit neurotaxis by Curatolo (3) and Curatolo et al. (4). The complete structural elucidation of the peptide remained to be achieved. The principle structures considered were Nacetyl-a-L-aspartyl-L-glutamate and N-acetyl-fl-L-aspartyl-L-glutamate as shown below.
In 1964 Auditore and Hendrickson (1) reported the isolation of an acetylated peptide composed of equimolar quantities of aspartic and glutamic acids from human brain. Later investigations established that glutamic acid is the C-terminal amino acid and that the terminal amino group is 0 0 H~c-iLL-~-~-c-oH HCH COH 0
0
0
0
0
Hg-C-:-:-~-C-!&,, HCH
CEH
HCH
HCH
0
HCH
COH
COH
0
0
Alpha
Beta
This report presents evidence which indicates that the acetylated peptide from human brain is N-acetyl-a-L-aspartyl-Lglutamate.
substituted with an acetate moiety (2). The compound was therefore tentatively identified as N-acetyl-aspartyl glutamate. A similar acylated peptide was found in the 1 This investigation was supported by U. S. Public Health research grant B-3520, National Institute of Neurological Diseases and Blindness; and grant GB-1721 from the National Science Foundation.
MATERIALS
AND
METHODS
Preparation of tissue extract. Human brains were obtained as soon after death as possible, and placed at -80% for 24 hours. Each brain was cut 22
N-ACETYL-(Y-L-ASPARTYL-L-GLUTAMATE into small segments with a cleaver andmacerated in 2 volumes of 20% trichloroacetic acid in a highspeed, one gallon size Waring Blendor for 3 minutes at, 15,500 rpm, and for 1 hour at 2500 rpm at 4”. The homogenate was centrifuged in a glass container at 2500 rpm for 30 minutes; the supernatant solution was decanted and the sediment was ext,racted wit,h 1 volume of water. The aqueous layer was recovered by centrifugation and added to the first supernatant solution; the sediment was discarded. The pooled aqueous extract was treated with liter portions of diethyl ether until the pH of the extract was 3.4. The volume of the extract was reduced to 50 ml under vacuum at 30” in a rotary flash evaporat,or. with AG60-X4 procedures Chromatographic (Dowex-50) column. Twenty five ml of the concentrated extract was applied to a column of resin (3.3 X 100 cm) in the hydrogen ion form. The extract was washed into the resin bed with a small volume of water and the column was developed with 700 ml of water. The flow rate was set at 40 ml per hour and 20-ml fractions were collected. The fractions were dried under vacuum and the sediment was dissolved in 0.2 ml of water. A small aliquot of each fraction was spot,ted on Whatman No. 1 chromatography paper (23 X 57 cm) and developed in n-butanol:ethanol:acetic acid:water (8:2:1:3; vol) employing the descending technique at 22-26” for 19-24 hours; the solvent front ran an average distance of 35-45 cm. Each chromatogram was tested for acylated amino acids and peptides by the chlorination-starch-iodide (CSI) reaction described by Rydon and Smith (5). This reaction involves placing the chromatogram in an atmosphere of chlorine for 10 minutes, ventilating for 30 minutes, and spraying with 1% starch in 1% KI. The reactive substances appear as blue-black spots on a faint blue background. Amino acids were detected on the chromatogram by the ninhydrin reaction. The chromatogram was sprayed with 0.25y0 ninhydrin in water-saturated butanol and heated at 8&100” for 10 minutes for development. Preparation of paper eluate of Dowex-CO jraclions. Fractions (Nos. 2130) that contained the bulk of the CSI reactive substances were pooled and chromatographed on several sheets of Whatman No. 1 paper, 23 X 57 cm, with a streaking technique. After development in n-butanol: ethanol:acetic acid:water (8:2:1:3; vol) small strips were cut from each side of a chromatogram and stained. These strips served as indicators to map the location of the desired reactive substance. The CSI band between an Rp value of 0.50 and 0.75 was selected, excised, placed in a large beaker filled with water, and mechanically agitated for several hours. The aqueous fluid was collected
IN
HUMAN
BRAIN
23
and taken to dryness in a rotating flash evaporator at 30”, and the residue was dissolved in a desired volume of water. Chromatographic procedures with AGl-X4 resin. The eluate was applied to a 1.75 X 100 cm AGl-X4 resin column in the chloride form and developed with a discontinuous gradient of glacial acetic acid. Three hundred ml of 1,2,3,6, and8 N glacial acetic acid were passed successively over the column. The flow rate was 40 ml per hour and 16ml fractions were collected. Fractions were taken to dryness and the residue was dissolved in a small amount (0.2 ml) of water. An aliquot of each fraction was chromatographed in n-butanol:ethanol: acetic acid:water (8:2:1:3; vol) using Whatman No. 1 paper. Peptides and other reactive compounds were detected on chromatograms by the chlorination-starch-iodide reaction. This isolation procedure clearly separates the acetylated peptide from N-acetyl-L-aspartate and N-acetyl-r-glutamate (2). Synfhesis of N-acetyl-o-L-aspartyl-L-glutamate. N-Carbobenzoxy-a-benzyl-L-aspartate was the starting compound for the synthesis of N-acetylp-L-aspartyl-L-glutamate. The aspartic acid derivative was synthesized from N-carbobenzoxyL-aspartate according to the methods described by Fisher and Whetstone (6). The melting point of the compound was 83”; the reported melting point is 85” (6). We obtained this compound in 30yo yield. Dibenzyl-L-glutamate toluenesulfonate was prepared by heating equimolar quantities of Lglutamic and para-toluene sulfonic acid to a temperature of 120” for 15 minutes in benzyl alcohol (7). It was recrystallized from ethanol:ether (1: 1; vol) to a melting point of 142”. The yield was 80% of the theoretical yield. N-Carbobenzoxy-aand dibenzyl-L-glutamate benzyl-L-aspartate toluenesulfonate were condensed in the presence of dicyclohexyl-diimide dissolved in dichloromethane to form N-carbobenzoxy-tribenzyl-D-Laspartyl-L-glut,amate (8). N-carbobenzoxy-tribenzyl-a-L-aspartyl-L-glutamate, recrystallized from ethanol, had a melting point of 111” (8). The yield was about 40% of the theoretical yield. Blocking groups were removed with hydrogen and palladium to make B-L-aspartyl-L-glutamate. This compound was identified by electrophoresis and degradation experiments as described in the results section. The dipeptide was acetylated by exposure to a 50y0 aqueous solution of acetic anhydride for 1 hour at 50”. The resulting product gave negative ninhydrin and hydroxamic acid tests (9). This product gave aspartic and glutamic acid on acid hydrolysis and glutamic acid on hydrazinolysis. Synthesis of N-acetyl-a-L-aspartyl glutamate. The starting compound for the above peptide,
24
OLSON,
WADE,
@-benxyl-L-aspartate, was synthesized (10) and after recrystallizing the compound from water to its reported melting point of 21%220”, the yield was 40% of theoretical. P-Benzyl-L-aspart,ate was converted to its carbobenzoxy derivative by the method devised by Benoiton (10). The carbobenzoxy derivative was recrystallized from water to the observed melting point, of 107-108”. NCarbobenzoxy-fl-benzyl-L-aspartate was condensed with dibenzyl-glutamate para-toluenesulfonate in the presence of dicyclohexyldiimide in dichloromethane to form N-carbobenzoxy-tribenzyl-cu-L-aspartyl-L-glutamate. After recrystallization from ethanol it. melted at 100” (8). The yield from this reaction was about 40% of the theoretical yield. The blocking groups were removed with hydrogen and palladium to form or-L-aspartyl-L-glutamate. The identification of this dipeptide was based on electrophoretic mobility and degradation experiments (see Results section). The compound was acetylated at room temperature by dropwise addition of acetic anhydride to an aqueous solution of the dipeptide with constant stirring until no ninhydrin color was observed. Care was taken to avoid formation of an acetic anhydride phase. The entire reaction required 5 hours and involved the addition of 15 ml of acetic anhydride to approximately 200 mg of cu-L-aspartyl-L-glutamate in 35 ml of water. When chromatographed in butanol:ethanol:acetic acid: water, the acetylated preparation gave two chlorine, starch iodide-positive components with RF values of 0.4 and 0.6. Both substances yielded aspartic and glutamic acids after acid hydrolysis but only the RF 0.6 component yielded glutamic acid and acetyl hydrazide after hydrazinolysis. Therefore, the Rp 0.6 component wa8 considered to be N-acetyl-a-L-aspartyl-L-glutamate. Electrophoretic methods. Electrophoresis was performed with a horizontal pressure cooling plate electrophoresis assembly obtained from the E-C Apparatus Corp. One-dimensional paper electrophoresis was performed on Whatman No. 1 paper at a set voltage. Electrophoresis was conducted at approximately 15” using a voltage gradient of 38 V/cm. Hydrazinolysis of the dipeptide. Samples of the synthesized and naturally occurring peptides were subjected to hydrazinolysis by a modification of the method of Niu and Fraenkel-Conrat (11). An aliquot of the dipeptide was placed in a tube and dried in vacua. The dried substance was dissolved in hydrazine and sealed under nitrogen. The sealed tube was held at 100” for 4 hours, and the excess hydrazine was then removed under vacuum. The dried hydrazinolyzate was dissolved in a small amount of water and subjected to electrophoretic and chromatographic analysis.
AND
AUDITORE
Acid hydrolysis of peptides. The synthesized and naturally occurring acetylated peptides were partially hydrolyzed by exposure to 0.1 N HCl under nitrogen for 2 hours at 100”. The naturally occurring dipeptide was also hydrolyzed under nitrogen in 0.05 N HCl at 100” for 15, 30, and 70 minutes. Exposure to 6 N HCI at 100” for 18 hours completely hydrolyzed the naturally occurring dipeptide. Chromatographic analysis on the amino acid analyzer. After hydrolysis the solutions of the peptides were concentrated to dryness and taken up in pH 2.2 citrate. An amino acid mixture composed of 0.025 pmole of each of the amino acids found in proteins was added to the reconstituted hydrolyzates. The resulting solution was subjected to amino acid chromatography according to the method of Moore et al. (12). Determination of amino acid con$gurations. The complete acid hydrolyzate of the naturally occurring dipeptide was acetylated by exposure to 50% acetic anhydride at 50” for 1.5 hours. After this solution was concentrated to dryness, the acetylated amino acids were subjected to the action of 2 mg of hog kidney acylase in 0.6 ml of 0.05 M phosphate buffer (pH 7.4) at 37” for 1 hour (13). The digest was dialyzed and the dialyzate was concentrated to dryness and subjected to amino acid chromatography. Hog kidney acylase was obtained from Mann Biochemicals. Note added in proof. A sample of human brain N-acetyl-aspartyl-glutamate was hydrolyzed in 6 N HCI at 100” for 18 hours and then dried in vucuo. To separate the mixture of glutamic and aspartic acids the hydrolyzate was applied to a 100 X 1.75 cm AGl-X4 acetate column that was then eluted with 300 ml of 1.0 M ammonium acetate, acetic acid pH 4 buffer, 300 ml of 0.1 N acetic acid, and 300 ml of 1 N acetic acid at a flow rate of 25 ml per hour. Glutamic appeared slightly before aspartic acid, both being isolated in separate 5-ml fractions of the N acetic acid eluate. Both amino acids were then re-crystallized from water and melting points were taken on a “Mel Temp Apparatus.” The observed melting point for commercial Dand L-glutamic acid was 203”, and our sample of glutamic acid melted at 201”. When our sample of glutamic acid was mixed with L-glutamic acid it melted at 203”, and it melted at 195” when mixed with n-glutamic acid. Therefore we consider that all of the glutamic acid from brain N-acetyl-aaspartyl-glutamate is of the L-configuration. Aspartic acid decomposed without melting. RESULTS
Our previous work indicated that the ninhydrin-negative, starch chloride-posi-
N-ACETYL-WL-ASPARTYL-L-GLUTAMATE
IN
HUMAN
BRAIS
“5
tive peptide from human brain was Nacet,yl aspartyl glutamat’e. Structural analysis of the brain dipept’ide revealed that the aspartyl moiet(y could be attached to glutarnic acid through an alpha or beta linkage. The following chemical data indicated that the naturally occurring dipeptide assumes the alpha structure. Synthesis of the unacet’ylated dipeptides was confirmed by electrophoresis, chromatog-
I
Time
30
(Minutes)
I 100
FIG. 1. A portion of the elution pattern observed when synthetic a-L-aspartyl-L-glutamate is co-chromatographed with a known mixture of amino acids.
t
I
( Mmutes)
FIG. 2. A portion of the elution pattern observed when synthetic p-L-aspartyl-L-glutamate is co-chromatographed with a known mixture of amino acids.
(Minutes)
studies. The and degradation electrophoretic mobilit’ies of the unacetylated peptides to the negative pole were, in reference to a mobility of 1.0 for aspartic acid, 1.6 for the alpha and 0.7 for the beta. These values correspond closely to the values (1.4 for alpha and 0.7 for beta) reported by Bryant et al. (8). Exhaustive acid hydrolysis which approximately doubled their ninhydrin color produced glutamic and aspartic acids from both peptides. Hydrazinolysis of the synbhetic dipeptides, on the other hand, yielded only glutamic acid. Application of ion-exchange chromatography as described by Moore et al. (12) clearly differentiated the alpha and beta unacetylated dipeptides. Both peptides were eluted as single peaks; the beta emerged from the column early just prior to aspartic acid, and the alpha appeared much
rwhy,
Time
Time
I 100 FIG. 3. A portion of the elution pattern observed when a partial acid hydrolyzate of synthetic IV-acetyl-cu-L-aspartyl-L-glutamate is cochromatographed with a known mixture of amino acids.
30
26
OLSON, WADE,
AND AUDITORE
later positioning itself between proline and glycine (Figs. 1 and 2). The synthesized acetylated dipeptides and brain acetylated dipeptides were partially converted by acid hydrolysis to the unacetylated derivatives for analysis by ion-exchange chromatography. The acetylated peptides were hydrolyzed in 0.1 N HCl at 100” for 2 hours. Two distinct characteristic elution patterns were observed. Three prominent peaks were clearly evident in each of the three chromatograms (Figs. 3-5). The peaks corresponding to aspartic and glutamic acids were common to all of the hydrolyzates, but the third peak corresponded to the unacetylated dipeptide. It was apparent that the partial acid hydroly-
Time
P I !-I ii ;1
30
(Minutes)
FIG. 5. A portion of the elution pattern observed when a partial acid hydrolyzate of the acetylated brain peptide is cochromatographed with a known mixture of amino acids.
Time
( Minutes)
FIG. 4. A portion of the elution pattern observed when a partial acid hydrolyzate of synthetic N-acetyl-P-n-aspartyl-n-glutamate is cochromatographed with a known mixture of amino acids.
zate elution profile of the brain peptide was identical to the profile obtained with the alpha dipeptide hydrolyzate. That the brain dipeptide possessed the alpha linkage was further substantiated by the fact that cochromatography of the partial acid hydrolyzates of synthetic N-acetyl-ar-n-aspartyl-nglutamate and acetylated brain dipeptide gave the same chromatographic elution pattern. The possibility of the peptide being a tetra or higher homologue was discounted on the basis of its negative biuret test. Furthermore, the yields of glutamic acid obtained after hydrazinolysis, which liberates only C-terminal amino acids, were the same for N-acetyl-or-L-aspartyl-L-glutamate and the dipeptide when the glutamic acid obtained was expressed as the percentage of glutamic acid obtained after total acid
N-ACETYL-LU-L-ASPARTYL-L-GLUTAMATE TABLE I YIELDS OF GLUTAMIC ACID OBTAINED AFTER HYDRAZINOLYSIS AS COMP.4RED WITH YIELDS OF GLUTAMIC OBT.\INED .~FTEH EXHAUSTIVE ACID HYDROLYSIS Compound
N-Acetyl-a-L-aspartyI+glutamate Unknown
Per cent
50 44
hydrolysis (Table 1). Our yields of 50 % for the C-terminal glutamate were in keeping with previously observed yields of Cterminal amino acids from hydrazinolyzates (11). Moreover, the brain dipeptide yielded the same fundamental elution pattern (Fig. 5) when chromatographed after hydrolysis in 0.05 N HCl for 15, 30, and 70 minutes at 100”. Despite a tenfold increase in aspartic acid and a threefold increase in glutamic acid, additional peaks would have been in evidence if the brain peptide was a tetra or a higher polymer peptide. To determine the configuration of the amino acids of the brain dipeptide, the amino acid mixture obtained after total acid hydrolysis was acetylated and digested with hog kidney acylase, which hydrolyzes Nacetylated-D-amino acids. When a concentrated dialyzate of the digest was subjected to amino acid chromatography, a complex elution pattern was obtained probably because of trans peptidase reactions. However, the presence of aspartic and glutamic acids indicated that both amino acids of the acetylated brain dipeptide were of the L-configuration. It was also found that glutamic acid was not liberated when a mixture of N-acetyl-L-aspartic and Nacetyl-D-glutamic acid was subjected to the hog kidney acylase preparation. DISCUSSION Recently we reported the isolation of an acetylated peptide which yielded equimolar quantities of aspartic and glutamic acid on exhaustive acid hydrolysis (2). The absence of ammonia in the hydrolyzate ruled out the possibility of an amide derivative. Hywhich liberates C-terminal drazinolysis, amino acids, liberated glutamic acid and acetyl hydrazide. On the basis of t,his
IX IIUMAN BRAIN
27
evidence and the acetylated peptides negative biuret test, the brain peptide was considered to be either N-acetyl-ol-L-aspartyl-D-glutamate or N-acetyl+L-aspartylD-glutamate. Synthesized unacetylated alpha and beta peptides were easily differentiated by chromatography on polystyrenesulfonate resin with pH 3.28 citrate buffer as the eluting solution. The more rapid chromatographic mobility of the @-L-aspartyl-Dglutamate may be due to the fact that one of its free carboxyl groups is adjacent to the positively charged amino group. The amino group would enhance the ionization of the adjacent carboxyl group and make the molecule more negatively charged at) pH 3.2s. When partial acid hydrolyzat.es of the synthesized acetylated alpha peptide and brain dipeptide were subjected to amino acid chromatography the chromatogrnphic profiles were identical. Co-chromatography of the partial acid hydrolyzates of the synt’hesized acetylated alpha and the brain peptide also yielded the same pattern. However, the possibility that) the acet*ylated brain pept#ide was a larger pept,ide still existed. Therefore, a series of partial acid hydrolyzates of the brain dipeptide was subjected to amino acid chromatography. The elution pattern shown in Fig. 3 persisted regardless of the hydrolysis time, indicating that no other ninhydrin-positive peptides were released. 1/loreover, the ratio of glutamic acid obtained by hydrazinolysis to glutamic acid obtained by exhaustive acid hydrolysis was the same for N-acetyla+aspartyl-D-glutamate and the acetylated brain peptide. Therefore, w’c believe that the acetylated brain peptide is the dipeptide, N-acetyl-a+aspartyl-D-glutamate. Since human brain was used as a source of this peptide one might speculate that this compound is not a normal constituent of brain tissue but merely arises from postmortem catabolism, drug administration, or from a deteriorating physiological condition. This appears unlikely for several reasons. First, the peptide has been found in numerous human brains. Second, we have isolated a similar peptide from t.hc brains of
28
OLSON,
WADE,
healthy rats sacrificed by decapitation. The rat brains used in t’hese experiments were frozen in dry ice in less than one minute after the death of the animal. Furthermore, an apparent,ly similar acet’ylated aspartyl glutamate peptide has been isolated from horse brain and rabbit neurotaxis (3). It is noteworthy that Westall appears to have isolated a ninhydrin-positive peptide composed of aspartic and glutamic acid from human urine (14). Such a compound could arise from the deacylation of the brain dipeptide. A preliminary report of this work was given at the Federation meeting (15). REFERENCES 1. AUDITORE, J. V., AND Intern. J. Pharmacol. 3, 2. AUDITORE, J. V., OLSON, L. H., Arch. Biochem. (1966).
HENDRICKSON, Ii., 1 (1964). E. J., AND W.~DE, Biophys. 114, 452
AND
AUDITORE
3. CURATOLO, A., Abstr. IV Int. Cong. Biochem. N. Y., V. E. 98, (1964). 4. CURATOLO, A., D’ARC~NGELO, P., LINO, A., AND BKANC~TI, A., J. Neurochem. 12, 339 (1965). RYDON, II. N., .~ND SMITH, P. W. G., Nature 169, 922 (1952). FISCHER, It. F., J\~~ WHETSTONE, R. R., J. Am. Chem. Sot. 76, 750 (1955). IZ~MIY~, N., .UXD MAKISUMI, S., Nippon Kagaku Zasshi 76, 662 (1957). BRYANT, P. M., MOORE, R. H., PIMLOTT, P. J., BND YOUNG, G. T., J. Chem. Sot. 3858 (1959). 9. STADTMAN, E. R., .%NDBARKER, H. A., J. Biol. Chem. 184, 769 (1950). 10. BENOITON, L., Can. J. Chem. 40,570 (1962). H., J. 11. Nru, C. I., AND FRAENKEL-CONRAT, Am. Chem. Sot. 77, 5882 (1955). 12. MOORE, S., SPACKMSN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185, (1958). 13. FODOR, P. J., PRICE, V. E., AND GREENSTEIN, J. P., J. Biol. Chem. 178, 503 (1949). 14. WESTALL, R. G., Biochem. J. 60, 247 (1955). 15. OLSON, E. J., AND AUDITORE, J. V., Federation hoc. 26, 560 (1966).