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Isolation and quantitation of ubiquitin from rat brain
PROTEIN
EXPRESSION
AND
PURIFICATION
1,93-96
(19%))
Isolation and Quantitation of Ubiquitin from Rat Brain G. Liguri,
N. Taddei,
G. Manao,
P. Nassi,
C. Nediani,
U. K. Ikram,*
Department of Biochemical Sciences, University of Florence, Vi& Morgagni and *Department of Pharmucy, Gomal University, D. I. Khan, Pakistan
Received
June
15,1990,
and in revised
form
July
Press,
Inc.
Ubiquitin was first isolated by Goldstein et al. (1) during the purification of polypeptide hormones from bovine thymus. Schlesinger et al. (2) first reported the complete amino acid sequence of the polypeptide. Later, independently, Ciechanover et al. (3) identified a factor required for ATP-dependent proteolysis, which was subsequently identified as ubiquitin (4). Ubiquitin is an abundant, low-molecular-weight protein (76 amino acids), widely distributed in living organisms (1,5-7). Its primary structure has been found to be highly conserved from insects to man (8). Recently, the ubiquitin sequence has been found in the genome of a togavirus (9). Linked to histones H2A and H2B, ubiquitin is present in the nucleus where it appears to be involved in the regulation of gene transcription (10-12). In the cytoplasm, ubiquitin exists in free form and is required for the process of ATP-dependent proteolysis in eukaryotic cells (3). Ubiquitin has been isolated and studied from a variety of animal tissues, including red blood cells (13)) reticulocytes (3), and testes (14,15). Some of these isolation procedures yield an inactive polypeptide lacking the C1046~5928/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
50, 50134 Firenze,
Italy,
23, 1990
A fast and sensitive method for the isolation andquantitation of cytoplasmic ubiquitin from brain by reversed-phase high-performance liquid chromatography is described. Cytosol from brain tissue was obtained by differential centrifugation and, after perchloric acid treatment, the sample was concentrated and ubiquitin was quantitatively isolated by means of a single chromatographic run. The amino acid composition, molecular weight, and primary structure of the pure protein were identified. The addition of monoiodinated “‘Iubiquitin to the sample as an internal standard indicated high native ubiquitin recovery. Statistical analysis carried out on different preparations and standardization of the chromatographic system indicated both the accuracy and the reproducibility of the method. 0 1990 Academic
and G. Ramponi
terminal Gly-Gly residues. It has been suggested that those ubiquitin molecules lacking this dipeptide are nonphysiological proteolytic artifacts. In spite of its importance, a simple and rapid procedure for ubiquitin isolation and quantitation is not available, although a kinetic method for assaying free ubiquitin and ubiquitin-activating enzymes has been reported (16). Here we describe a reversed-phase high-performance liquid chromatography procedure which permits one-step isolation and quantitation of free ubiquitin from brain tissues. Data on structural properties are reported. MATERIALS
AND
METHODS
Materials HPLC-grade acetonitrile was purchased from E. Merck (Darmstadt, FRG). Water was purified with a MilliQ system. Sequencing grade trifluoroacetic acid was purchased from Aldrich Chemical Co. (Milwaukee, WI). 12’1 for ubiquitin iodination was purchased from Amersham (Buckinghamshire, UK). The protein sequencer and arylamine membrane were from Milligen (U.S.A.). All other chemicals were of analytical grade. Male Wistar rats, 4 months old, fed ad libitum, were purchased from S. Morini (S. Polo D’Enza, Italy). Methods High-performance liquid chromatography equipment. An LKB-2150 HPLC system with two pumps, equipped with a Bio-Rad variable-wavelength detector, and a lo-pm Aquapore RP-300 column (25 cm X 4.6 mm i.d.) were used with solvents and column at room temperature. Ubiquitin isolation. All the operations during preparation of the brain were carried out at 0-4°C. The brain of a decapitated rat was removed, rinsed in 0.154 M NaCl, weighed, minced, and homogenized with a Teflon/glass Potter homogenizer (8-9 strokes by hand) in 9 vol (w/v) of 0.1 M triethanolamine-HCl (TEA), pH 7.4, containing 10 mM sodium EDTA and 2 mM freshly pre93
Inc. reserved.
94
LIGURI
ET AL.
TABLE Summary
Total Step Homogenate 1st centrifugation 2nd centrifugation HClO, supernatant HPLC ubiquitin
supernatant supernatant peak
volume (ml)
1
of Purification
Procedure”
Protein concentration (mghnl)
11.0 16.4 17.6 42.0 2.5
’ Values refer to one rat brain, average weight 1.5 g. * Eight rat brains were processed separately in the same analysis, was 4.1. ’ Values are approximated to three significant figures.
Specific radioactivity (wnhdc
32.40 5.11 3.57 0.15 0.21
experiment.
pared p-chloromercurihenzoic acid (&MB). The homogenate was centrifuged at 3000g for 10 min in a Beckman Spinco centrifuge, and the precipitate was resuspended in 6 ml of 0.1 M TEA, pH 7.4. The resulting mixture was centrifuged again at 3000g for 15 min. The supernatants from the two centrifugations were then pooled and centrifuged at 50,OOOgfor 60 min. The pellet was resuspended in 2 ml of 0.1 M TEA, pH 7.4, and centrifuged at 50,OOOg for 60 min. The supernatants from the third and fourth centrifugations were then pooled, and 1 vol of cold 9% HClO, was added with stirring in order to precipitate most of the foreign proteins. The resulting mixture was centrifuged at 50,OOOg for 30 min. The pellet was suspended and homogenized in 9 ml of 4.5% HClO, and centrifuged at 50,OOOg for 30 min. The supernatants from the fifth and sixth centrifugations were pooled and neutralized to pH 7 by adding the required volume of 2 M Na,CO,. The solution was then concentrated to a final volume of 1 ml by ultrafiltration. Liquid chromatographic ubiquitin separation. The previously described extract was injected into the liquid chromatography system. Two solvents (A and B) were used to separate the fractions: solvent A contained 10 mM trifluoroacetic acid (TFA) in water and solvent B contained 10 mM TFA in acetonitrile. The flow rate was set at 1.5 ml/min with the initial composition of the solvent mixture at 85% A and 15% B. After 3 min under isocratic conditions, the proportion of solvent B was linearly increased to 32% over a period of 25 min. The proportion of solvent B was then raised to 35% over the following 10 min, to 40% over the following 2 min, and, finally, to 90% over the following 2 min, at which level it was maintained for 3 min to regenerate the resin. Starting conditions were established by decreasing solvent B to 15% over 2 min. An equilibration period of 5 min at initial composition was required before the next run. Effluent absorbance was monitored at 214 nm. The collected fractions were dried on a Uniequip vacuum centrifuge (Model Univapo 150 H) and were used for further analysis.
1,140 4,830 6,280 54,300 648,000
The
standard
deviation
Enrichment
Total radioactivity (cpm)
Yield* (%o)
1.0 4.2 5.5 47.6 568.4
407,000 404,670 389,990 342,360 340,110
100 99.4 95.8 84.1 83.5
of the
final
yield,
calculated
by statistical
Radioactive ubiquitin iodination. Ubiquitin was iodinated by the following method: 20 ~1 of 0.2 mg/ml ubiquitin was mixed with 5 ~1 of 100 mCi/ml Na12’I and 10 ~1 of 0.25 M sodium phosphate at pH 7.5. Then, 10 ~1 of 5 mg/ml chloramine-T and 100 ~1 of 1.2 mg/ml sodium bisulfite, freshly prepared, were added in rapid succession under continuous stirring. The volume of the sample was made up to 1 ml by adding a 2 mg/ml NaI solution. The sample was placed in a column (18 X 0.8 cm) that had been filled with Sephadex G-10 previously saturated with 1 ml of 10% bovine serum albumin solution. The resin was equilibrated with 0.25 M sodium phosphate at pH 7.5, containing 2% horse serum. Elution was achieved with 0.25 M phosphate-serum at pH 7.5, and the fractions were counted in a gamma counter. Fractions corresponding to the ubiquitin peak were collected, added to 40 ~1 of 0.2 mg/ml cold ubiquitin, and purified by HPLC under the same conditions as described above. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ubiquitin homogeneity was evaluated by SDS-PAGE analysis according to Laemmli (17). Ubiquitin amino acid analysis. The sample obtained from HPLC, dried by centrifugation under reduced pressure, was dissolved in 6 M HCI in the presence of 0.1% phenol and split into two equal samples; one sample was hydrolyzed at 1lO’C for 20 h and the other for 70 h, and both were subjected to amino acid analyses. The residue numbers for serine and threonine were calculated by extrapolation back to zero hydrolysis time. Ubiquitin amino acid sequence. Ubiquitin was covalently bound to an arylamine membrane by the following procedure: the polypeptide was dissolved in 30% acetonitrile, applied to the membrane, and dried at 55°C for 10 min. The membrane was wetted with a solution of 15 mg/ml 1-ethyl-3-(3dimethylaminopropyl)carbodiimide in 0.1 M sodium 4-morpholineethanesulfonate at
CHROMATOGRAPHIC
ASSAY
OF
RAT
BRAIN
95
UBIQUITIN TABLE
Amino
Acid
Amino
L I
0
1
10
‘0 ,
20 TIME (mid
FIG. 1.
Elution profile of the natant obtained from a perchloric peak is indicated by the arrow.
30
reversed-phase acid-treated
40
HPLC sample.
of the superThe ubiquitin
pH 5.0, containing 15% acetonitrile. After 20 min at room temperature, the membrane was placed in the protein sequencer, which was equipped with an on-line injection system. RESULTS
AND
DISCUSSION
The most critical step in the purification of ubiquitin appears to be homogenization, since partial proteolysis of its C-terminal Gly-Gly dipeptide has been noted (3). In order to ensure the integrity of the ubiquitin molecule, &MB and EDTA were added to the sample before homogenization as protease inhibitors. Table 1 summarizes the purification procedure for ubiquitin. In order to evaluate purification performance, about 5 X 10’ cpm of 12’1-ubiquitin, prepared and purified as described under Materials and Methods was added to the homogenate of each rat brain as an internal standard. Acid-deproteinized samples were neutralized by the addition of 2 M Na,CO, and further ultrafiltered to avoid any loss of ubiquitin. In order to speed up the whole procedure, this step can be replaced by neutralizing the sample with K,CO,, centrifuging the KClO, precipitate, and concluding with lyophilization. The yield of the whole procedure as determined by the radioactivity of the internal standard is high enough to allow almost complete recovery of the polypeptide. With eight different samples processed at the same time, the yield (expressed as % + SD of the ubiquitin content in the starting material) was 83.5 + 4.1%. Figure 1 shows the absorbance pattern at 214 nm of the effluent from reversed-phase HPLC. Ubiquitin corresponds to a single, homogeneous peak and represents about 7.9% of the proteins injected into the chromatographic system. Table 2 reports the results of amino acid composition analysis of ubiquitin isolated by HPLC. Calculated values demonstrate that the polypeptide contains the C-
of Ubiquitin”
acid
Residues
Asx Thr Ser Glx GUY Ala Val Met Ile Leu ‘br Phe LYS His Arg
_--_ L
Composition
2
7.0 5.9 3.7 12.4 6.0 2.1
(7) (6) (4) (12) (6) (2)
4.0 (4)
0.8 (1) 6.6 (7) 8.8 (9)
0.9 (1) 1.9 (2) 6.6 (7)
1.0 (1) 3.7 (4)
’ Acid hydrolyses were 70 h in duplicate samples. olation to zero hydrolysis tained from the primary
performed on ubiquitin at 110°C Thr and Ser values were obtained time. Numbers in parentheses structure of human ubiquitin.
for 20 and by extrapwere ob-
terminal Gly-Gly dipeptide. The residue numbers were attributed on the basis of the amino acid sequence of human erythrocyte ubiquitin. Figure 2 illustrates the SDS-polyacrylamide electropherogram of purified ubiquitin. The apparent M, of 7600 Da of rat brain ubiquitin, which is below the actual M, of 8600 for human ubiquitin, has been reported previously and is attributed to the anomalous migration of the polypeptide as a result of its incomplete unfolding in SDS-PAGE (3). Figure 3 reports the partial NH,-terminal amino acid sequence that corresponds to the first 50 residues of
17.0 14.4
8.2 6.2 2.5
A
FIG. 2.
B
15% SDS-polyacrylamide gel electrophoretic rified ubiquitin (A) and low-molecular-weight standards ues are X10e3.
pattern of pu(B). M, val-
96
LIGURI
5 H2N-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-
that has lymphocyte-differentiating represented universally in living
3.
Partial
amino
72,11-15. 2. Schlesinger,
25
30 35 Ala-Lys-Ile-Gln-Asp-Lys-Glu-Gly-Ile-Pro-Pro-Asp-Gln-Gln-
FIG.
complete stimulating
40
sequence
S., Heller, H., Ferber, S., and Hershko, A. (1980) Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes. J. Biol.
of the rat brain
ubiquitin.
ubiquitin. Sequence analysis was carried out for identification purposes. Data on partial sequence and amino acid composition confirm the identity of the primary structure of the rat brain ubiquitin with the known sequence of vertebrate ubiquitin. The responses in the analytical system to differing known amounts of ubiquitin are represented by the regression equation y = (4.048 X 10% + (8 X 103), where y = area values for the peaks obtained from HPLC and z = nanomoles of ubiquitin. Analyte amount was determined by amino acid assay. A high correlation coefficient (r = 0.98) was obtained, giving good accuracy on the linearity of the assay over the working range. The detection limit of the system can be evaluated to be around 0.05 nmol. The isolation and assay of brain ubiquitin described in this paper represent an improvement in terms of operative reliability and quantitative yield as compared to previously reported procedures (15,18). This procedure can be utilized in all circumstances in which ubiquitin must be isolated from brain areas or, after suitable adaptation, from other tissues, with the aim of further functional and structural studies; it can also be utilized in experiments that are designed to compare the levels of soluble ubiquitin in brain tissues. This last application could facilitate research on alterations in brain protein turnover, since ubiquitin-protein complexes have recently been found in neurofibrillary tangles and senile plaques of brains from Alzheimer’s disease patients (19,20). Preliminary studies in this laboratory indicate a significant increase, as compared to controls, in free ubiquitin in the brain areas that are most affected by degenerative processes (unpublished observation).
Chem.255,7525-7528. 4. Wilkinson, K. D., Urban,
The authors express their gratitude to Dr. A. Cagnini for his skillful technical assistance. This work was supported by grants from the Minister0 della Pubblica Istruzione and the Consiglio Nazionale delle Ricerche.
REFERENCES G., Scheid, M., Hammerling, D. H., and Niall, H. D. (1975)
M. K., and Hars, A. L. (1980) Ubiquitin proteolysis factor of rabbit reticulocytes. 7529-7532.
5. Schlesinger,
D. H., and Goldstein, G. (1975) tion of 74 amino acid sequence of ubiquitin man. Nature (London) 255,423-424.
Molecular between
conservacattle and
6. Vierstra,
R. D., Langan, S. M., and Haas, A. L. (1985) Purification and initial characterization of ubiquitin from the higher plant, Auena sntiua. J. Biol. Chem. 260, 12,015-12,021.
7. Ozkaynak, 8.
E., Finley, D., and Varshavsky, A. (1984) The yeast ubiquitin gene: Head-to-tail repeats encoding a polyubiquitin precursor protein. Nature (London) 312, 663-666. Gavilanes, J. G., Gonzales de Buitrago, G., Perez-Castelli, R., and Rodriguez, R. (1982) Isolation, characterization, and amino acid sequence of a ubiquitin-like protein from insect eggs. J. Biol. Chem. 257,10,267-10,270.
9. Meyers, G., Rumenappf, T., and Thiel, H. J. (1989) Ubiquitin in a togavirus. Nature (London) 341,491. 10. Goldknopf, I. L., and Busch, H. (1977) Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A-24. Proc. N&l. Acud. Sci. USA 74,864-868. 11. Levinger, L., and Varshavsky, A. (1982) ubiquitinated and Dl protein-containing Drosophila genome. Cell 28,375-385.
Selective arrangement nucleosomes within
of the
12. Watson, D. C., Levy, B. W., and Dixon, G. H. (1978) Free ubiquitin is a non-histone protein of trout testis chromatin. Nature (London) 276.196-198. 13. Haas, A. L., and Wilkinson, K. A. (1985) The large-scale purification of ubiquitin from human erythrocytes. Prep. Biochem. 15, 49-60. 14.
Loir, M., Caraty, A., Lanneau, M., Menozo, Y., Muh, J. P., and Sautiere, P. (1984) Purification and characterization of ubiquitin from mammalian testis. FEBS L&t. 169, 199-204. 15. Sun, N. E., Zhu, D. X., Han, K. K., Hemon, B., Belaiche, D., and Sautiere, P. (1988) Isolation and characterization of ATP-dependent proteolytically active ubiquitin in cock testis. Comp. Biothem. Physiol., B: Comp. Biochem. 91,777-781. 16. Rose, I. A., and Warms, J. V. B. (1987) A specific endpoint assay for ubiquitin. Proc. N&l. Acad. Sci. USA 84, 1477-1481. 17. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,
680.
ACKNOWLEDGMENTS
singer,
properties and is probably hoc. Nutl. Acad. Sci. USA
D. H., Goldstein, G., and Niall, H. D. (1975) The amino acid sequence of ubiquitin, an adenylate cyclase polypeptide probably universal in living cells. Bio-
is the ATP-dependent J. Biol. Chem. 255,
1. Goldstein,
cells.
chemistry14,2214-2218. 3. Ciechanover, A., Elias,
50
acid
AL.
10
15 20 Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-val-Lys-
45 Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu-....
ET
U., Boyse, E. A., SchleIsolation of a polypeptide
18. Zhu, D. X., Zhang, A., Zhu, N. Z., Xu, L. X., Deutsch, H. F., and Han, K. K. (1985) Investigation of primary and secondary structure of porcine ubiquitin and its acetylated lysine derivatives. Znt. J. Biochem. 17, 719-721. 19. Gallo, J. M., and Anderton, B. H. (1989) Brain diseases: Ubiquitous variations in nerves. Nature (London) 337, 687. 20. Mayer, R. J., Landon, M., G. P., Byrne, E. J., Lennox, ten, R. B. (1989) Ubiquitin 193.
Doherty, F. J., Lowe, J. S., Reynolds, G. G., Jefferson, D., and Godwin-Ausand dementia. Nature (London) 340,
EXPRESSION
AND
PURIFICATION
1,93-96
(19%))
Isolation and Quantitation of Ubiquitin from Rat Brain G. Liguri,
N. Taddei,
G. Manao,
P. Nassi,
C. Nediani,
U. K. Ikram,*
Department of Biochemical Sciences, University of Florence, Vi& Morgagni and *Department of Pharmucy, Gomal University, D. I. Khan, Pakistan
Received
June
15,1990,
and in revised
form
July
Press,
Inc.
Ubiquitin was first isolated by Goldstein et al. (1) during the purification of polypeptide hormones from bovine thymus. Schlesinger et al. (2) first reported the complete amino acid sequence of the polypeptide. Later, independently, Ciechanover et al. (3) identified a factor required for ATP-dependent proteolysis, which was subsequently identified as ubiquitin (4). Ubiquitin is an abundant, low-molecular-weight protein (76 amino acids), widely distributed in living organisms (1,5-7). Its primary structure has been found to be highly conserved from insects to man (8). Recently, the ubiquitin sequence has been found in the genome of a togavirus (9). Linked to histones H2A and H2B, ubiquitin is present in the nucleus where it appears to be involved in the regulation of gene transcription (10-12). In the cytoplasm, ubiquitin exists in free form and is required for the process of ATP-dependent proteolysis in eukaryotic cells (3). Ubiquitin has been isolated and studied from a variety of animal tissues, including red blood cells (13)) reticulocytes (3), and testes (14,15). Some of these isolation procedures yield an inactive polypeptide lacking the C1046~5928/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
50, 50134 Firenze,
Italy,
23, 1990
A fast and sensitive method for the isolation andquantitation of cytoplasmic ubiquitin from brain by reversed-phase high-performance liquid chromatography is described. Cytosol from brain tissue was obtained by differential centrifugation and, after perchloric acid treatment, the sample was concentrated and ubiquitin was quantitatively isolated by means of a single chromatographic run. The amino acid composition, molecular weight, and primary structure of the pure protein were identified. The addition of monoiodinated “‘Iubiquitin to the sample as an internal standard indicated high native ubiquitin recovery. Statistical analysis carried out on different preparations and standardization of the chromatographic system indicated both the accuracy and the reproducibility of the method. 0 1990 Academic
and G. Ramponi
terminal Gly-Gly residues. It has been suggested that those ubiquitin molecules lacking this dipeptide are nonphysiological proteolytic artifacts. In spite of its importance, a simple and rapid procedure for ubiquitin isolation and quantitation is not available, although a kinetic method for assaying free ubiquitin and ubiquitin-activating enzymes has been reported (16). Here we describe a reversed-phase high-performance liquid chromatography procedure which permits one-step isolation and quantitation of free ubiquitin from brain tissues. Data on structural properties are reported. MATERIALS
AND
METHODS
Materials HPLC-grade acetonitrile was purchased from E. Merck (Darmstadt, FRG). Water was purified with a MilliQ system. Sequencing grade trifluoroacetic acid was purchased from Aldrich Chemical Co. (Milwaukee, WI). 12’1 for ubiquitin iodination was purchased from Amersham (Buckinghamshire, UK). The protein sequencer and arylamine membrane were from Milligen (U.S.A.). All other chemicals were of analytical grade. Male Wistar rats, 4 months old, fed ad libitum, were purchased from S. Morini (S. Polo D’Enza, Italy). Methods High-performance liquid chromatography equipment. An LKB-2150 HPLC system with two pumps, equipped with a Bio-Rad variable-wavelength detector, and a lo-pm Aquapore RP-300 column (25 cm X 4.6 mm i.d.) were used with solvents and column at room temperature. Ubiquitin isolation. All the operations during preparation of the brain were carried out at 0-4°C. The brain of a decapitated rat was removed, rinsed in 0.154 M NaCl, weighed, minced, and homogenized with a Teflon/glass Potter homogenizer (8-9 strokes by hand) in 9 vol (w/v) of 0.1 M triethanolamine-HCl (TEA), pH 7.4, containing 10 mM sodium EDTA and 2 mM freshly pre93
Inc. reserved.
94
LIGURI
ET AL.
TABLE Summary
Total Step Homogenate 1st centrifugation 2nd centrifugation HClO, supernatant HPLC ubiquitin
supernatant supernatant peak
volume (ml)
1
of Purification
Procedure”
Protein concentration (mghnl)
11.0 16.4 17.6 42.0 2.5
’ Values refer to one rat brain, average weight 1.5 g. * Eight rat brains were processed separately in the same analysis, was 4.1. ’ Values are approximated to three significant figures.
Specific radioactivity (wnhdc
32.40 5.11 3.57 0.15 0.21
experiment.
pared p-chloromercurihenzoic acid (&MB). The homogenate was centrifuged at 3000g for 10 min in a Beckman Spinco centrifuge, and the precipitate was resuspended in 6 ml of 0.1 M TEA, pH 7.4. The resulting mixture was centrifuged again at 3000g for 15 min. The supernatants from the two centrifugations were then pooled and centrifuged at 50,OOOgfor 60 min. The pellet was resuspended in 2 ml of 0.1 M TEA, pH 7.4, and centrifuged at 50,OOOg for 60 min. The supernatants from the third and fourth centrifugations were then pooled, and 1 vol of cold 9% HClO, was added with stirring in order to precipitate most of the foreign proteins. The resulting mixture was centrifuged at 50,OOOg for 30 min. The pellet was suspended and homogenized in 9 ml of 4.5% HClO, and centrifuged at 50,OOOg for 30 min. The supernatants from the fifth and sixth centrifugations were pooled and neutralized to pH 7 by adding the required volume of 2 M Na,CO,. The solution was then concentrated to a final volume of 1 ml by ultrafiltration. Liquid chromatographic ubiquitin separation. The previously described extract was injected into the liquid chromatography system. Two solvents (A and B) were used to separate the fractions: solvent A contained 10 mM trifluoroacetic acid (TFA) in water and solvent B contained 10 mM TFA in acetonitrile. The flow rate was set at 1.5 ml/min with the initial composition of the solvent mixture at 85% A and 15% B. After 3 min under isocratic conditions, the proportion of solvent B was linearly increased to 32% over a period of 25 min. The proportion of solvent B was then raised to 35% over the following 10 min, to 40% over the following 2 min, and, finally, to 90% over the following 2 min, at which level it was maintained for 3 min to regenerate the resin. Starting conditions were established by decreasing solvent B to 15% over 2 min. An equilibration period of 5 min at initial composition was required before the next run. Effluent absorbance was monitored at 214 nm. The collected fractions were dried on a Uniequip vacuum centrifuge (Model Univapo 150 H) and were used for further analysis.
1,140 4,830 6,280 54,300 648,000
The
standard
deviation
Enrichment
Total radioactivity (cpm)
Yield* (%o)
1.0 4.2 5.5 47.6 568.4
407,000 404,670 389,990 342,360 340,110
100 99.4 95.8 84.1 83.5
of the
final
yield,
calculated
by statistical
Radioactive ubiquitin iodination. Ubiquitin was iodinated by the following method: 20 ~1 of 0.2 mg/ml ubiquitin was mixed with 5 ~1 of 100 mCi/ml Na12’I and 10 ~1 of 0.25 M sodium phosphate at pH 7.5. Then, 10 ~1 of 5 mg/ml chloramine-T and 100 ~1 of 1.2 mg/ml sodium bisulfite, freshly prepared, were added in rapid succession under continuous stirring. The volume of the sample was made up to 1 ml by adding a 2 mg/ml NaI solution. The sample was placed in a column (18 X 0.8 cm) that had been filled with Sephadex G-10 previously saturated with 1 ml of 10% bovine serum albumin solution. The resin was equilibrated with 0.25 M sodium phosphate at pH 7.5, containing 2% horse serum. Elution was achieved with 0.25 M phosphate-serum at pH 7.5, and the fractions were counted in a gamma counter. Fractions corresponding to the ubiquitin peak were collected, added to 40 ~1 of 0.2 mg/ml cold ubiquitin, and purified by HPLC under the same conditions as described above. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ubiquitin homogeneity was evaluated by SDS-PAGE analysis according to Laemmli (17). Ubiquitin amino acid analysis. The sample obtained from HPLC, dried by centrifugation under reduced pressure, was dissolved in 6 M HCI in the presence of 0.1% phenol and split into two equal samples; one sample was hydrolyzed at 1lO’C for 20 h and the other for 70 h, and both were subjected to amino acid analyses. The residue numbers for serine and threonine were calculated by extrapolation back to zero hydrolysis time. Ubiquitin amino acid sequence. Ubiquitin was covalently bound to an arylamine membrane by the following procedure: the polypeptide was dissolved in 30% acetonitrile, applied to the membrane, and dried at 55°C for 10 min. The membrane was wetted with a solution of 15 mg/ml 1-ethyl-3-(3dimethylaminopropyl)carbodiimide in 0.1 M sodium 4-morpholineethanesulfonate at
CHROMATOGRAPHIC
ASSAY
OF
RAT
BRAIN
95
UBIQUITIN TABLE
Amino
Acid
Amino
L I
0
1
10
‘0 ,
20 TIME (mid
FIG. 1.
Elution profile of the natant obtained from a perchloric peak is indicated by the arrow.
30
reversed-phase acid-treated
40
HPLC sample.
of the superThe ubiquitin
pH 5.0, containing 15% acetonitrile. After 20 min at room temperature, the membrane was placed in the protein sequencer, which was equipped with an on-line injection system. RESULTS
AND
DISCUSSION
The most critical step in the purification of ubiquitin appears to be homogenization, since partial proteolysis of its C-terminal Gly-Gly dipeptide has been noted (3). In order to ensure the integrity of the ubiquitin molecule, &MB and EDTA were added to the sample before homogenization as protease inhibitors. Table 1 summarizes the purification procedure for ubiquitin. In order to evaluate purification performance, about 5 X 10’ cpm of 12’1-ubiquitin, prepared and purified as described under Materials and Methods was added to the homogenate of each rat brain as an internal standard. Acid-deproteinized samples were neutralized by the addition of 2 M Na,CO, and further ultrafiltered to avoid any loss of ubiquitin. In order to speed up the whole procedure, this step can be replaced by neutralizing the sample with K,CO,, centrifuging the KClO, precipitate, and concluding with lyophilization. The yield of the whole procedure as determined by the radioactivity of the internal standard is high enough to allow almost complete recovery of the polypeptide. With eight different samples processed at the same time, the yield (expressed as % + SD of the ubiquitin content in the starting material) was 83.5 + 4.1%. Figure 1 shows the absorbance pattern at 214 nm of the effluent from reversed-phase HPLC. Ubiquitin corresponds to a single, homogeneous peak and represents about 7.9% of the proteins injected into the chromatographic system. Table 2 reports the results of amino acid composition analysis of ubiquitin isolated by HPLC. Calculated values demonstrate that the polypeptide contains the C-
of Ubiquitin”
acid
Residues
Asx Thr Ser Glx GUY Ala Val Met Ile Leu ‘br Phe LYS His Arg
_--_ L
Composition
2
7.0 5.9 3.7 12.4 6.0 2.1
(7) (6) (4) (12) (6) (2)
4.0 (4)
0.8 (1) 6.6 (7) 8.8 (9)
0.9 (1) 1.9 (2) 6.6 (7)
1.0 (1) 3.7 (4)
’ Acid hydrolyses were 70 h in duplicate samples. olation to zero hydrolysis tained from the primary
performed on ubiquitin at 110°C Thr and Ser values were obtained time. Numbers in parentheses structure of human ubiquitin.
for 20 and by extrapwere ob-
terminal Gly-Gly dipeptide. The residue numbers were attributed on the basis of the amino acid sequence of human erythrocyte ubiquitin. Figure 2 illustrates the SDS-polyacrylamide electropherogram of purified ubiquitin. The apparent M, of 7600 Da of rat brain ubiquitin, which is below the actual M, of 8600 for human ubiquitin, has been reported previously and is attributed to the anomalous migration of the polypeptide as a result of its incomplete unfolding in SDS-PAGE (3). Figure 3 reports the partial NH,-terminal amino acid sequence that corresponds to the first 50 residues of
17.0 14.4
8.2 6.2 2.5
A
FIG. 2.
B
15% SDS-polyacrylamide gel electrophoretic rified ubiquitin (A) and low-molecular-weight standards ues are X10e3.
pattern of pu(B). M, val-
96
LIGURI
5 H2N-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-
that has lymphocyte-differentiating represented universally in living
3.
Partial
amino
72,11-15. 2. Schlesinger,
25
30 35 Ala-Lys-Ile-Gln-Asp-Lys-Glu-Gly-Ile-Pro-Pro-Asp-Gln-Gln-
FIG.
complete stimulating
40
sequence
S., Heller, H., Ferber, S., and Hershko, A. (1980) Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes. J. Biol.
of the rat brain
ubiquitin.
ubiquitin. Sequence analysis was carried out for identification purposes. Data on partial sequence and amino acid composition confirm the identity of the primary structure of the rat brain ubiquitin with the known sequence of vertebrate ubiquitin. The responses in the analytical system to differing known amounts of ubiquitin are represented by the regression equation y = (4.048 X 10% + (8 X 103), where y = area values for the peaks obtained from HPLC and z = nanomoles of ubiquitin. Analyte amount was determined by amino acid assay. A high correlation coefficient (r = 0.98) was obtained, giving good accuracy on the linearity of the assay over the working range. The detection limit of the system can be evaluated to be around 0.05 nmol. The isolation and assay of brain ubiquitin described in this paper represent an improvement in terms of operative reliability and quantitative yield as compared to previously reported procedures (15,18). This procedure can be utilized in all circumstances in which ubiquitin must be isolated from brain areas or, after suitable adaptation, from other tissues, with the aim of further functional and structural studies; it can also be utilized in experiments that are designed to compare the levels of soluble ubiquitin in brain tissues. This last application could facilitate research on alterations in brain protein turnover, since ubiquitin-protein complexes have recently been found in neurofibrillary tangles and senile plaques of brains from Alzheimer’s disease patients (19,20). Preliminary studies in this laboratory indicate a significant increase, as compared to controls, in free ubiquitin in the brain areas that are most affected by degenerative processes (unpublished observation).
Chem.255,7525-7528. 4. Wilkinson, K. D., Urban,
The authors express their gratitude to Dr. A. Cagnini for his skillful technical assistance. This work was supported by grants from the Minister0 della Pubblica Istruzione and the Consiglio Nazionale delle Ricerche.
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AL.
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15 20 Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-val-Lys-
45 Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu-....
ET
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