/. Biochem. 82, 1297-1305 (1977)
Takako KATO, 1 Yukio KATO,' Hisataka KASAI, 4 and Tsuneo OKUYAMA Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158 Received for publication, May 30, 1977
Highly acidic proteins were extracted from bovine liver with 5 5 % saturated ammonium sulfate and purified by DEAE-Sephadex A-50 column chromatography and Sephadex G-75 column chromatography. Four acidic protein fractions (BLA I-IV) were obtained in yields of 150, 100, 50, and 450 mg, respectively, from 6 kg of bovine liver. When analyzed by 7.5% and 15% polyacrylamide gel disc electrophoresis, BLA I and BLA II fractions were still heterogeneous, and BLA III fraction was composed of two main and one minor protein components. On the other hand, BLA IV fraction was homogeneous after rechromatography on DEAESephadex A-50. Gel electrophoresis also showed that the four acidic protein fractions were clearly different each other. The molecular weights were estimated to be 12,500 and 14,500 for the two main protein components of BLA III fraction, and 16,000 for BLA IV, as determined by SDS-polyacrylamide gel electrophoresis. Three N-terminal amino acids for BLA III fraction were identified by a dansylation method, but none was found for BLA IV. BLA IV showed an unusual UV spectrum, while BLA I—III fractions gave usual protein spectra. BLA III and BLA IV fractions were compared with highly acidic proteins of bovine brain (PAP I and PAP II). BLA III fraction was different from PAP I (bovine brain S-100 protein) and BLA IV fraction was virtually identical with PAP II, on the basis of amino acid compositions and peptide maps of tryptic digests. Immunodiffusion tests with anti-S-100 serum demonstrated that S-100 protein could not be detected in bovine liver; the content is less than one-thousandth of that in bovine brain. Thus, S-100 protein was not detected in bovine liver by chemical analysis or by immunochemical tests. However, it appears that almost identical acidic proteins are present in bovine liver and brain (BLA IV and PAP II) as well as in chick brain (CBA II).
1
A preliminary account of this study has appeared in Seikagaku, 44, 708 (1972) (in Japanese). * Present address: Department of Microbiology, School of Medicine, Gunma University, Maebashi, Gunma 371. 1 Present address: Departmnt of Protein Chemistry, Institute of Endocrinology, Gunma University, Maebashi, Gunma 371. 4 To whom all inquiries should be addressed. Abbreviations: BPB, bromphenol blue; TPCK, L-l-tosylamido-2-phenylchIoromethyl ketone; dansyl, 1-dimethylaminonaphthalene-5-sulfonyl; SDS, sodium dodecyl sulfate. Vol. 82, No. 5, 1977
1297
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
Highly Acidic Proteins in Bovine Liver1
1298
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
MATERIALS AND METHODS Materials—DEAE-Sephadex A-50 (particle size 40-120 ftm) and Sephadex G-75 (particle size 40-120 fim) were purchased from Pharmacia Fine Chem., Sweden. Thin-layer plates, Silica gel Fjti, were obtained from E. Merck, Germany. Pepsin was purchased from Nutritional Biochem. Co., and lysozyme and trypsin from Worthington Biochem. Co. Ribonuclease A was purified by the method of Klee (8). Trypsin was treated with L-l-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) according to the procedure of Carpenter (9). An antiserum against bovine S-100 protein fraction was kindly supplied by Dr. Levine of Brandeis University, Massachusetts, U.S.A. Bovine livers were obtained from a local slaughterhouse. Preparation of Acidic Proteins from Bovine Liver—Preparation of acidic proteins from bovine
liver was carried out essentially according to the method of Isobe and Okuyama (5), as described in the previous paper (6). Briefly, a total of 6 kg of bovine liver was homogenized in 4 volumes of 0 . 1 M potassium phosphate buffer, p H 7 . 1 , containing 1 mM EDTA and 55 % saturated ammonium sulfate (325 g per liter). Extracted soluble proteins were dialyzed and subjected to chromatography on a DEAE-Sephadex A-50 column (4.6x40 cm) equilibrated with 0.1 M potassium phosphate buffer, p H 7 . 1 , containing 1 mM EDTA and 0.1 M NaCI. The column was washed with 3 column volumes of the equilibrating buffer and eluted in a stepwise manner with the buffer containing 0 . 1 7 M, 0.22 M, or 0 . 4 0 M NaCI (3.5, 4, or 6 column volumes, respectively). The eluted acidic protein fractions were collected and subjected to further fractionation, mainly on Sephadex G-75. Determination of Protein Concentration—Protein concentration was determined in terms of the absorbance at 280 nm in a Hitachi 101 spectrophotometer equipped with an auto-sampler or by the method of Lowry (10) using bovine serum albumin as a standard. The UV spectrum was measured in a Hitachi EPS-3T automatic spectrophotometer. Polyacrylamide Gel Disc Electrophoresis— Disc electrophoresis was performed in a glass tube ( 5 x 8 0 mm) with 7.5% and 15% polyacrylamide gel as described by Ornstein (11) and Davis (12). Bromphenol blue (BPB) was used as a marker dye. Protein was stained with 1 % Amido black in 7% acetic acid and destained in 7% acetic acid. Molecular Weight Determination—Molecular weight was determined by SDS-polyacrylamide gel electrophoresis after dansylation of proteins, essentially according to the method of Talbot and Yphantis (13). Protein samples (each 0.5 mg) were treated in sodium phosphate buffer containing SDS and 2-mercaptoethanol. Electrophoresis was performed in a gel containing 15% polyacrylamide, 0.75% N,N'-methylenebisacrylamide, 0 . 1 % SDS, and 4 M urea at 6-7 milliamperes per tube for about 6 h (14). Fluorescent protein bands were detected at 365 nm with a UV lamp. Pepsin, trypsin, lysozyme, and ribonuclease A were used as standard proteins. Amino Acid Analysis—Each sample (about 1 mg) was hydrolyzed at 110°C for 24 h in an evacuated, sealed tube with 1.0 ml of constant/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
More than ten years ago, Moore and McGreger (/) successfully applied ion-exchange chromatography and electrophoresis for the purification of acidic proteins in the brain to obtain brain-specific proteins such as S-100 protein and 14-3-2 protein, the function of which still remain to be determined {2-4). Recently, Isobe and Okuyama purified two highly acidic proteins (PAP I and PAP II) from bovine brain on a large scale by DEAESephadex A-50 chromatography and Sephadex G-75 gel chromatography (5, 6); PAP I crossreacted with antiserum against bovine S-100 protein and PAP II did not (6). This procedure also permitted the separation of two highly acidic proteins (CBA I and CBA II) from chick brain, as described in the previous paper (6). It was found that S-100 protein (CBA I) was present in low content in chick brain but was very different from that of bovine brain in chemical properties. Furthermore, it was found that PAP II and CBA II were very similar proteins. In the present study, the purification and characterization of highly acidic proteins in bovine liver were attempted by the procedure described above, using a large amount of the tissue. S-100 protein could not be detected either chemically or immunologically in bovine liver, in contrast with the results of Moore (7), whereas a protein similar to PAP II in bovine brain and to CBA II in chick brain was obtained in high yield.
1299
boiling HC1 (Wako, analytical grade). Amino acid analysis was performed with a Hitachi KLA3B automatic amino acid analyzer. Analysis of N-Terminal Amino Acid—The N-terminal amino acid was determined according to the method of Gros and Labouesses (75). Each protein sample (about 1 mg) was reacted with dansyl chloride and hydrolyzed at 110°C for 4 h or 16 h with constant-boiling HC1. Dansyl-amino acid was identified on a thin-layer plate of Silica gel F m (5x10 cm). Two solvent systems, benzene/ pyridine/acetic acid (80:20:2, v/v) (76") and chloroform/methanol/acetic acid (75 : 20 : 5, v/v) (77), were used separately. Trypsin Digestion—Protein samples were aminoethylated according to the method of Cole (18). The modified protein (2 to 3 mg) was digested at 37°C for 24 h with one-fiftieth, portion of TPCKtrypsin (w/w) in 0.1 M N-ethylmorpholine-acetic acid buffer, pH 7.0 or 8.0. The digest was then quickly lyophilized. Peptide Map—Peptide mapping was performed on Whatman 3MM paper. About 2 mg of the tryptic digest was subjected to descending chromatography for 16 h to 20 h in the shorter dimension using 1-butanol/pyridine/acetic acid/ water (15 : 10 : 3 : 12, v/v). Electrophoresis was carried out in the second dimension at pH 3.6, using pyridine/acetic acid/water ( 1 : 1 0 : 289, v/v) at a potential of 50 volt per cm for about 1 h.
Peptides on paper were stained with 0.2% ninhydrin in acetone. Immunodiffusion—Double diffusion (Ouchterlony's method) was performed in 0.84% Difco Bacto Agar gel containing 0.85% NaCl, 0.01% merzonin, and 0.01 % trypan blue according to the standard method (79). Antiserum against bovine S-100 protein fraction was dissolved at a concentration of 40 mg'per ml in 0.85% NaCl, and each sample was dissolved in the same saline solution. Immunoreaction was performed overnight at room temperature. RESyLTS Purification of Highly Acidic Proteins—From a total of 6 kg of bovine liver, 123 g of soluble proteins was extracted and fractionated on a DEAE-Sephadex A-50 column under the conditions described above. The elution pattern is shown in Fig. 1. Seventy percent of the soluble proteins passed through from the column. The acidic proteins, which migrated with BPB marker dye on 7.5% polyacrylamide gel disc electrophoresis, were detected in the fractions eluted at the concentrations of 0.10, 0.17, 0.22, and 0.40 M NaCl (Fractions I-IV). The shaded fractions shown in Fig. 1 were collected and further fractionated, mainly on Sephadex G-75 as shown in Fig. 2A-D. Highly acidic proteins which migrated with BPB
HACL CONCENTRATION (
0,10
5.0
•0.17 — |
0,40
0.22
2.5
0
'
100
200
100
500
700
800
FRACTION HUHBER
Fig. 1. DEAE-Sephadex A-50 column chromatography of bovine liver extract. The soluble protein fraction (123 g of proteins/3.2 1 from 6 kg of bovine liver) was applied to a DEAE-Sephadex A-50 column (4.6x40 cm) and eluted stepwise with 0.10, 0.17, 0.22, and 0.40M N a d , as described in " MATERIALS AND METHODS." Fractions of 14.5 ml per tube were collected. The flow rate was 40 ml/h. Absorbance at 280 nm was measured. The shadowed fractions in the figure (Fractions I, II, III, and IV) were collected and subjected to the further fractionation. Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
1300
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
on 7.5% gel were detected in the underlined fractions and designated as BLA I, BLA n , BLA m , and BLA IV as indicated. The yields of BLA I-IV fractions at this stage were 150, 100, 50,
0.2 -
50
30
70
90
110
FRACtlON NUWER
E 0.1OI W l
A Ml
/
0.50H bCI \
|
NA—J 1 50
100
ISO FRACTION KU»EK
Fig. 2
200
250
300
Fig. 2. Chromatography of fractions I-IV on Sephadex G-75 and DEAE-Sephadex A-50. (A) and (B), the patterns of rechromatography on Sephadex G-75 of Fractions I and II from Fig. 1; Fractions I and II were subjected to chromatography on Sephadex G-75 and the acidic protein fractions were collected and subjected to rechromatography on Sephadex G-75. (C) and (D), the patterns of final gel chromatography on Sephadex G-75 of Fractions III and IV from Fig. I; Fractions III and IV were subjected to chromatography on Sephadex G-75, and the acidic protein fractions were chromatographed successively on Sephadex G-50 and Sephadex G-75. The fractions underlined in Figs. 2A, B, C, and D were named BLA I, BLA II, BLA III, and BLA IV fractions, respectively. Column sizes were 1.7x85 cm (A), 4.5x86 cm (B), 1.7x85 cm (C), and 4.8x96 cm (D). The flow rates were 30, 50, 25, and 66 ml/h and fractions of 3.7, 10.5, 2.8, and 11.8 ml were collected, respectively. (E), The patterns of rechromatography on DEAE-Sephadex A-50 of BLA IV fraction (Fig. 2D); BLA IV fraction was applied to a DEAE-Sephadex A-50 column (2.0x25 cm) equilibrated with 0.1 M potassium phosphate, pH 7.1, containing 0.10 M NaCl and 1 min EDTA, and eluted stepwise with 0.10, 0.30, and 0.50 M NaCl in the same buffer. The flow rate was 22 ml/h. Fractions of 4.0 ml were collected. The resulting acidic protein, BLA IV (underlined in the figure), was homogeneous. The yield was low, presumably due to aggregation or degradation during storage.
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
0.1 -
and 450 mg, respectively, from 6 kg of bovine liver. BLA I, II, and HI fractions were used for characterization without further purification. BLA IV fraction was subjected to further chromatography on DEAE-Sephadex A-50 as shown in Fig. 2E and eluted at a concentration of 0.30 M NaCl in the underlined fractions. BLA IV was thus obtained in a homogeneous form free from nucleic acid. Electrophoretic Analysis of Acidic Proteins— The electrophoretic patterns of BLA I—III fractions and BLA IV on 7.5% and 15% polyacrylamide gels are shown in Fig. 3. On 7.5% gel electrophoresis, the BLA I and BLA II fractions showed protein bands in addition to a main protein band (BLA I and BLA II, respectively) at the BPB marker dye, whereas BLA III fraction and BLA IV each gave a single band at the BPB marker dye (Fig. 3A). On the other hand, BLA I-III fractions and BLA IV all showed different mobilities on 15% gel electrophoresis (Fig. 3B). The mobilities
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
1301
BLA I FRACTION
MIGRATION DISTANCE
0.39 0.65
»0.70
ni-3 in-? m-i
BPE
0.86 0.91 1.00
BLA I
BLA II
BLA I I I
FRACTION
FRACTION
FRACTION
(CM)
Fig. 4. Estimation of the molecular weights of BLA I-II1 main components and BLA IV. After dansylation, the proteins were electrophoresed on 15% polyacrylamide gel containing 0.1% SDS and 4 M urea, pH 7.2. Pepsin (1), trypsin (2), lysozyme (3), and ribonuclease A (4) were used as standard proteins.
BLA IV
Fig. 3. Disc electrophoresis of purified acidic proteins on 7.5% (A) and on 15% (B) polyacrylamide gels. The mobility of each main component relative to BPB is shown in (B). of the main components relative to BPB (Rf) were 0.65 for BLA I and 0.39 for BLA II fractions. The three components of BLA III fraction were named BLA III-l (R, = \.O), BLA ni-2 (/{,= 0.91), and BLA III-3 (R,=0.86). The rechromatographed BLA IV showed a single band (^/=0.70), indicating it to be homogeneous. Some Properties of the Acidic Proteins—The molecular weights were determined by SDSpolyacrylamide gel electrohoresis after dansylation (13). BLA I and BLA II fractions gave several fluorescence bands. BLA III fraction gave two fluorescence bands, but BLA IV showed only one. The molecular weights were estimated to be 9,800 for BLA I main component, 17,000 for BLA II main component, 12,500 for BLA III-2, 14,500 for BLA m-3, and 16,000 for BLA IV (Fig. 4). N-Terminal amino acids were determined for BLA III fraction and BLA IV by a dansylation method (75). After acid hydrolysis of the dansylated BLA III fraction for-4 or 17 h, three dansyl amino acids (dansyl-Thr and -Tyr as main, and Vol. 82, No. 5, 1977
210
250
260
270
280
WAVELENGTH (NH)
Fig. 5. Ultraviolet absorption spectrum of BLA IV. The protein was dissolved in 0.1 M potassium phosphate buffer, pH 7.1, containing 1 mM EDTA at a concentration of 2.7 mg/ml. dansyl-Ile as a minor components) were identified on a thin-layer plate. This suggests that BLA III fraction contains at least three components, as expected from the results described above (Figs. 3B and 4). Under the same conditions, BLA IV gave no a-NH,-dansylated amino acid, indicating that the N-terminal amino group of BLA IV may be masked. BLA I-UI fractions showed typical protein absorption spectra (data not shown), while BLA IV showed an unusual ultraviolet absorption spectrum
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
BPB
1302
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA of histidine, 2 residues each of proline and tyrosine, and 7 residues of phenylalanine per molecule of protein, with no cysteine or tryptophan; these results indicate that BLA IV is very similar to PAP II obtained in the same way. The minimal molecular weight determined by amino acid analysis, assuming 10 residues of alanine per molecule of protein, was 15,500, corresponding well with that estimated by SDS-po!yacrylamide gel electrophoresis. The maps of tryptic peptides of BLA m fraction and IV are shown in Fig. 6, together with those of bovine brain acidic proteins (PAP I and II). Twenty-one ninhydrin-positive spots were detected for BLA i n fraction, 18 for PAP I, 16 for BLA IV, and 16 for PAP H. The number of peptides on the map of BLA i n fraction was greater than expected from the amino acid composition, probably
TABLE I. Amino acid compositions of highly acidic proteins obtained from bovine liver and brain. Amino acid compositions are expressed as molar %, but values in parentheses correspond to the numbers of amino acid residues in the protein molecule. No correction has been made for destruction during acid hydrolysis. 1
BLAI»
i
Lys His
:
ATB
Asp Thr 'Ser : — Glu 1 ' Pro Gly Ala Cys Val Met He Leu Tyr Phe Trp»
9.98 2.10 3.42 10.13 4.07 5.79 14.43 •
4.75 -
6.83 6.30 1.78 7.34 2.55 3.56 8.38 3.68 4.87 n. d.f
1
J 7.81
1
i
VBLAm
6.40 2.63 • 1.24 3.94 . . 3.51 10.92 • 13.09 4.84 . • 4.82 5.74 5.49 14.17' 20.07 -4.49 3.26 6.32 7.33 6.32 7.81 1.24 1.20 6.70 5.99 2.14 3.96 4.42 4.50 8.85 7.10 2.60 2.49 4.13 4.49 n.d. f n.d.'
BLA IV" 5.86 0.96 4.05 16.43 7.63 2.62 18.65 1.70 7.66 7.48 0.00 3.79 5.64 5.34 6.11 1.34 5.21 0.00
(7. 82) (0.92) (6. 36) (21.90) (10.18) (3.47) (24.95) (2.27) (10.22) (10.00) (0) (5.07) (7.54) (7.00) (8.16) (1.74) (6.95) (0)
PAPIc 9.11 5.35 1.04 10.65 3.38 5.46 20.17 0.00 4.95 6.15 1.041 . 6.35 3.19 3.87 9.74 1.35 7.16 0.39
PAP IID.c
(7.48) (0. 88) (5.43) (22.05) (10.24) (3.46) (20.60) (2. 21) (9.80) (10.00) (0)" (6.04) (7.74) (6.93) (8.01) (1.80) (7-10) (0)
• BLA I and BLA II fractions were analyzed. b The ratios of amino acid residues are indicated, taking the content of alanine as 10.00 residues per molecule of protein. = Taken from the previous paper (6). PAP I and PAP II ' were obtained in the fractions eluted with 0.22 M and 0.35 M NaCl from DEAE-Sephade*A-50. d Determined after performic acid oxidation of proteins. • Determined spectrophotometrically or after alkaline hydrolysis of the proteins. f Not determined. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
witrT 5' maxima at 254, 260, 266, 27O, and 278 nm (Fig. 5). The fine structure may be due to a high content of phenylalanine residues and a low content of tyrosine residues (Table I ) . ' The molar absorbance of BLA IV was 2.8x10* at 280 nm in deionized water, assuming the molecular weight to be 16,000. Chemical Comparisons of Acidic Proteins—The amino acid compositions of BLA I-IV. fra'ctions are given in Table I, together with those : of bovine brain acidic proteins (PAP I and PAP tl). BLA i n f r a c t i o n is distinctly different from PAP I as regards amino acid composition, especially in proline, histidine and arginine contents, though both were obtained in the same way by DEAE'-Sephadex A-50 chromatography. On the other hand, BLA IV as well as PAP II was characterized by 1 residue
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
o o o Asp
Glu Gly
o
1303
c
Lys
o
o
Glu Gly
Lys
o o Picric acid
Asp
•
8
Gly
O Val
O
Val
OLIU
0
Leu
0
nd
Electrophoresis
°
Picric «dd
OGly
, Asp
o o Glu ' Gly
0
Ala
4 •
/
o
Ptwnol red
Electrophoresis
+ D
o Picric acid
ao o o
Aipl
Glu
Lys
Gly
OGly OAI.
Oval
Fig. 6. Peptide maps of tryptic peptides of BLA III fraction (A) and IV (B), and bovine brain acidic proteins, PAP I (Q and II (D). The reduced sample was aminoethylated, digested with trypsin and subjected to two-dimensional peptide mapping as described in " MATERIALS AND METHODS." Peptides were visualized with ninhydrin, and the color intensity is shown in increasing order as follows; dotted circle, open circle, shadowed circle, and solid circle. because this fraction was a mixture of two main against bovine S-100 protein. Only PAP I crosscomponents (BLA IH-2 and -3). No correspond- reacted, as shown in Fig. 7. Under the conditions ence between the peptide maps of BLA III fraction used, PAP I could react at a minimum concentraand PAP I was observed. Thus it was concluded tion of 1.2 /ig per ml. Even when the protein conthat BLA III fraction and PAP I are different pro- centration was increased up to 2 mg per ml, BLA tein species. On the other hand, the peptide map I-III fractions, BLA IV and PAP II did not crossof BLA IV showed good correspondence with that react. The bovine liver extract applied to a of PAP II, except for two weak spots, but was DEAE-Sephadex A-50 column also did not react. clearly different from those of BLA III fraction These results clearly indicate that bovine liver conand PAP I. These results suggest that BLA IV in tains at most less than one-thousandth as much bovine liver is virtually identical with PAP II in S-100 protein as bovine brain. bovine brain. Immunological Properties of Acidic Proteins— DISCUSSION To confirm the brain specificity of S-100 protein, immunoreaction was performed on BLA I-IJJ frac- In the present study, bovine liver acidic proteins, tions, BLA IV, and PAP I and Il.with an antiserum which migrated with BPB on 7.5% polyacrylamide Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
OGly OAla
1304
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
/. Biochem,
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
Sephadex A-50 chromatography; both were eluted with 0.22 M NaCl. However, each of the three components of BLA,'Infraction migrated faster than PAP I on -i-5-% gel disi^ electrophoresisr while BLA III fraction and_PAP I migrated with BPB marker dye on 7.5% gel,. The molecular weights of BLA III components were larger than that of PAP I (8,000)'(5) on SDS-polyacrylamide gel electrophoresis. B&A»' III fraction showed a typical UV spectnfm, whereas PAP • I showed a characteristic spectrum with maxima in the region of 250 to 290 nm. Three N-terminal arnino acid residues were detected for BLA III. fraction byji dansylation method, but none for PAP L_ ._ . * The amino acid analysis and peptide mapping also showed that^BLA^III fraction and PAP I were quite different fcom^*ach! other. Furthermore, "PAPT gave a pfecipifirfline with*antiserum against bovine S-100 protein, but BLA III fraction did not. As the liver extract did not react with antiserum against S-100 protein, the brain specificity of S-100 protein was again confirmed in the present study. However, the physiological function of BLA III components remains unknown. BLA IV in bovine liver was essentially identical with PAP II in bovine brain on the basis of several criteria, as described below; both proteins were Fig. 7. Immunodiffusion profiles against anti S-100 obtained in the fraction eluted with 0.40 M (or serum-of crude extracts of liver and brain (A), and of 0.30-0.35 M) NaCl from a DEAE-Sephadex A-50 purified acidic proteins from liver and brain (B). Im- column, and they were different from S-100 protein, munodiffusion was performed as described in " MA- judging from their chemical and immunological TERIALS AND METHODS." A: (1) Liver extract (45 mg/ml); (2) brain extract (3.9 mg/ml); B: (1) BLA I, properties. The electrophoretic mobilities of the (2) BLA II, (3) BLA III, (4) BLA IV, (5) PAP I, and (6) two proteins were the same both on 7.5 % gel and PAP U. The protein concentration in B was 2 mg/ml on 15% gel. In fact, a mixture of BLA IV and PAP II (1 : 1, by weight) gave a single band on except for PAP I (1.2 /ig/ml). 15% gel (data not shown). Both proteins showed characteristic UV spectra with 5 maxima, due to gel electrophoresis, were separated in four fraca high ratio of phenylalanine to tyrosine residues; tions (BLA I-IV) by DEAE-Sephadex A-50 ionother proteins with similar UV spectra were reexchange chromatography. Some of them were still heterogeneous, but each fraction was different ported recently (20, 21). The molecular weight when analyzed by gel electrophoresis, and by amino was estimated to be 16,000 for both BLA IV and acid analysis. In this section, BLA III fraction is PAP II by SDS-polyacrylamide gel electrophoresis compared in more detail with PAP I, and BLA IV after dansylation. No N-terminal amino acid with PAP II. PAP I and PAP II were previously was detected in the dansylated proteins (BLA obtained from bovine brain (5,6") by the procedure IV and PAP II) even after prolonged hydrolysis (16 h). The results of amino acid analysis and used for bovine liver. mapping of tryptic peptides clearly also suggest Several lines of evidence suggested that BLA that the two proteins are identical. III fraction from bovine liver is different from PAP I in bovine brain, as described below. Both As described in the previous paper (6), one of proteins showed the same behavior on DEAE- the two highly acidic proteins from chick brain,
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
REFERENCES 1. Moore, B.W. & McGreger, D. (1965) /. Biol. Chem. 240, 1647-] 653 2. Hyden, H. & Lange, P.W. (1970) Proc. Nail. Acad. Sci. U.S. 67, 1959-1966 3. Hyden, H. (1974) Proc. Nail. Acad. Sci. U.S. 71, 2965-2968 4. Marangos, P.J., Zomzely-Neurath, C , Luk, D.C., & York, C. (1975) J. Biol. Chem. 250, 1884-1891 5. Isobe, T. & Okuyama, T. (1971) Seikagaku (in Japanese) 43, 479 6. Kato, Y., Kato, T., Kasai, H., Okuyama, T., & Uyemura, K. (1977) J. Biochem. 82, 43-51 7. Moore, B.W. (1965) Biochem. Biophys. Res. Commun. 19, 739-744 8. Klee, W.A. (1966) in Procedures in Nucleic Acid Research (Contoni, G.L. & Davies, D.R., eds.) pp. 20-30, Hapen and Row, New York 9. Carpenter, F.H. (1967) in Methods in Enzymology (Hire, C.H.W., ed.) Vol. 11, p. 237, Academic Press, New York
Vol. 82, No. 5, 1977
10. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 11. Ornstein, L. (1964) Ann. New York Acad. Sci. 121, 321-349 12. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, 404-427 13. Talbot, D.N. & Yphantis, D.A. (1971) Anal. Biochem. 44, 246-253 14. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412 15. Gros, C. & Labouesse, B. (1969) Eur. J. Biochem. 7, 463t7O 16. Morse, D. & Horecker, B.L. (1966) Anal. Biochem. 14, 429^33 17. Seiler, N. & Wiechmann, M. (1964) Experientia 20, 559-560 18. Cole, R.D. (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 315-317, Academic Press, New York 19. Clausen, M.J. (1969) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T.S. & Work, E., eds.) Vol. 1, pp. 397-572, North-Holland Pub. Co., Amsterdam 20. Dorrington, K.J., Hui, A., Hofmann, T., Hitchman, A.J.W., & Harrison, J.E. (1974) /. Biol. Chem. 249, 199-204 21. Lehky, P., Blum, H.E., Stein, E.A., & Fischer, E.H. (1974) J. Biol. Chem. 249, 4332^334 22. Isobe, T., Kato, Y., Kato, T., Kasai, H., Arai, T., & Okuyama, T. (1972) Seikagaku (in Japanese) 44, 439 23. Aochi, M., Suzuki, M., Kasai, H., & Okuyama, T. (1973) Seikagaku (in Japanese) 45, 468 24. Coffee, C.J. & Bradshaw, R.A. (1973) /. Biol. Chem. 248, 3305-3312 25. Kretsinger, R.H. & Nockolds, C.E. (1973) /. Biol. Chem. 248, 3313-3326 26. Enfiels, D.L., Ericsson, L.H., Blum, H.E., Fischer, E.H. & Neurath, H. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1309-1313 27. Collins, J.H., Potter, J.D., Horn, M.J., Wilshire, G., & Jackman, N. (1973) FEBS Lett. 36, 268-272 28. van Eerd, J-P. & Takahashi, K. (1976) Biochemistry 15, 1171-1180 29. Wolff, D.J. & Siege!, F.L. (1972) / . Biol. Chem. 2A1, 4180-^185 30. Brooks, J.C. & Siegel, F.L. (1973) /. Biol. Chem. 248,4189-4193 31. Huang, W-Y., Cohen, D.V., Hamilton, J.W., Fullmer, C. & Wasserman, R.H. (1975) /. Biol. Chem. 250, 7647-7655 32. Isobe, T., Kato, Y., Kasai, H., & Okuyama, T. (1975) 1SN Barcelona Meeting (abstr.) p. 303 33. Kasai, H., Yamagata, Y., Kato, Y., & Okuyama, T. (1976) 10th Internal. Congr. Biochem. (abstr.) p. 571 34. Teo, T.S., Wang, T.H., & Wang, J.H. (1973) /. Biol. Chem. 248, 588-595 35. Lin, Y.M., Liu, Y.P., & Cheung, W.Y. (1974) /. Biol. Chem. 249, 4943-4954 36. Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., & Vanaman, T.C. (1976) / . Biol. Chem. 251, 4501^513
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
CBA II, which was eluted with 0.35 M NaCl on DEAE-Sephadex A-50 chromatography, was almost identical with PAP II as regards chemical properties. The relevant yields were 450 mg from 6 kg of bovine liver (BLA IV), 700 mg from 8 kg of bovine brain (PAP II), and 700 mg from 9 kg of chick brain (CBA II). Thus, it appears that essentially the same protein was present in high content in bovine liver and brain, and in chick brain. In our laboratory, a similar protein was recently obtained from human brain and bovine erythrocytes (22, 23). These results suggest that this highly acidic protein has no organ or species specificity and is distributed widely in cells with little change in its primary structure. Recently, several acidic proteins with Cabinding activity have been reported, such as parvalbumin (24-26), troponin C (27, 28), Cabinding phosphoprotein (29, 30), and vitamin D-dependent Ca-binding protein (20, 31), most of which were characterized by unusual UV spectra, resembling that of PAP II. Investigation of the structural similarity between troponin C and PAP II is now in progress (32). PAP II was identified as an activator of cyclic AMP phosphodiesterase and as a Ca-binding protein (33). The Cadependent activator proteins of phosphodiesterase already reported (34-36) are very similar to PAP II, especially in chemical properties and in their mode of activation of the enzyme (6, 33).
1305
Takako KATO, 1 Yukio KATO,' Hisataka KASAI, 4 and Tsuneo OKUYAMA Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158 Received for publication, May 30, 1977
Highly acidic proteins were extracted from bovine liver with 5 5 % saturated ammonium sulfate and purified by DEAE-Sephadex A-50 column chromatography and Sephadex G-75 column chromatography. Four acidic protein fractions (BLA I-IV) were obtained in yields of 150, 100, 50, and 450 mg, respectively, from 6 kg of bovine liver. When analyzed by 7.5% and 15% polyacrylamide gel disc electrophoresis, BLA I and BLA II fractions were still heterogeneous, and BLA III fraction was composed of two main and one minor protein components. On the other hand, BLA IV fraction was homogeneous after rechromatography on DEAESephadex A-50. Gel electrophoresis also showed that the four acidic protein fractions were clearly different each other. The molecular weights were estimated to be 12,500 and 14,500 for the two main protein components of BLA III fraction, and 16,000 for BLA IV, as determined by SDS-polyacrylamide gel electrophoresis. Three N-terminal amino acids for BLA III fraction were identified by a dansylation method, but none was found for BLA IV. BLA IV showed an unusual UV spectrum, while BLA I—III fractions gave usual protein spectra. BLA III and BLA IV fractions were compared with highly acidic proteins of bovine brain (PAP I and PAP II). BLA III fraction was different from PAP I (bovine brain S-100 protein) and BLA IV fraction was virtually identical with PAP II, on the basis of amino acid compositions and peptide maps of tryptic digests. Immunodiffusion tests with anti-S-100 serum demonstrated that S-100 protein could not be detected in bovine liver; the content is less than one-thousandth of that in bovine brain. Thus, S-100 protein was not detected in bovine liver by chemical analysis or by immunochemical tests. However, it appears that almost identical acidic proteins are present in bovine liver and brain (BLA IV and PAP II) as well as in chick brain (CBA II).
1
A preliminary account of this study has appeared in Seikagaku, 44, 708 (1972) (in Japanese). * Present address: Department of Microbiology, School of Medicine, Gunma University, Maebashi, Gunma 371. 1 Present address: Departmnt of Protein Chemistry, Institute of Endocrinology, Gunma University, Maebashi, Gunma 371. 4 To whom all inquiries should be addressed. Abbreviations: BPB, bromphenol blue; TPCK, L-l-tosylamido-2-phenylchIoromethyl ketone; dansyl, 1-dimethylaminonaphthalene-5-sulfonyl; SDS, sodium dodecyl sulfate. Vol. 82, No. 5, 1977
1297
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
Highly Acidic Proteins in Bovine Liver1
1298
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
MATERIALS AND METHODS Materials—DEAE-Sephadex A-50 (particle size 40-120 ftm) and Sephadex G-75 (particle size 40-120 fim) were purchased from Pharmacia Fine Chem., Sweden. Thin-layer plates, Silica gel Fjti, were obtained from E. Merck, Germany. Pepsin was purchased from Nutritional Biochem. Co., and lysozyme and trypsin from Worthington Biochem. Co. Ribonuclease A was purified by the method of Klee (8). Trypsin was treated with L-l-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) according to the procedure of Carpenter (9). An antiserum against bovine S-100 protein fraction was kindly supplied by Dr. Levine of Brandeis University, Massachusetts, U.S.A. Bovine livers were obtained from a local slaughterhouse. Preparation of Acidic Proteins from Bovine Liver—Preparation of acidic proteins from bovine
liver was carried out essentially according to the method of Isobe and Okuyama (5), as described in the previous paper (6). Briefly, a total of 6 kg of bovine liver was homogenized in 4 volumes of 0 . 1 M potassium phosphate buffer, p H 7 . 1 , containing 1 mM EDTA and 55 % saturated ammonium sulfate (325 g per liter). Extracted soluble proteins were dialyzed and subjected to chromatography on a DEAE-Sephadex A-50 column (4.6x40 cm) equilibrated with 0.1 M potassium phosphate buffer, p H 7 . 1 , containing 1 mM EDTA and 0.1 M NaCI. The column was washed with 3 column volumes of the equilibrating buffer and eluted in a stepwise manner with the buffer containing 0 . 1 7 M, 0.22 M, or 0 . 4 0 M NaCI (3.5, 4, or 6 column volumes, respectively). The eluted acidic protein fractions were collected and subjected to further fractionation, mainly on Sephadex G-75. Determination of Protein Concentration—Protein concentration was determined in terms of the absorbance at 280 nm in a Hitachi 101 spectrophotometer equipped with an auto-sampler or by the method of Lowry (10) using bovine serum albumin as a standard. The UV spectrum was measured in a Hitachi EPS-3T automatic spectrophotometer. Polyacrylamide Gel Disc Electrophoresis— Disc electrophoresis was performed in a glass tube ( 5 x 8 0 mm) with 7.5% and 15% polyacrylamide gel as described by Ornstein (11) and Davis (12). Bromphenol blue (BPB) was used as a marker dye. Protein was stained with 1 % Amido black in 7% acetic acid and destained in 7% acetic acid. Molecular Weight Determination—Molecular weight was determined by SDS-polyacrylamide gel electrophoresis after dansylation of proteins, essentially according to the method of Talbot and Yphantis (13). Protein samples (each 0.5 mg) were treated in sodium phosphate buffer containing SDS and 2-mercaptoethanol. Electrophoresis was performed in a gel containing 15% polyacrylamide, 0.75% N,N'-methylenebisacrylamide, 0 . 1 % SDS, and 4 M urea at 6-7 milliamperes per tube for about 6 h (14). Fluorescent protein bands were detected at 365 nm with a UV lamp. Pepsin, trypsin, lysozyme, and ribonuclease A were used as standard proteins. Amino Acid Analysis—Each sample (about 1 mg) was hydrolyzed at 110°C for 24 h in an evacuated, sealed tube with 1.0 ml of constant/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
More than ten years ago, Moore and McGreger (/) successfully applied ion-exchange chromatography and electrophoresis for the purification of acidic proteins in the brain to obtain brain-specific proteins such as S-100 protein and 14-3-2 protein, the function of which still remain to be determined {2-4). Recently, Isobe and Okuyama purified two highly acidic proteins (PAP I and PAP II) from bovine brain on a large scale by DEAESephadex A-50 chromatography and Sephadex G-75 gel chromatography (5, 6); PAP I crossreacted with antiserum against bovine S-100 protein and PAP II did not (6). This procedure also permitted the separation of two highly acidic proteins (CBA I and CBA II) from chick brain, as described in the previous paper (6). It was found that S-100 protein (CBA I) was present in low content in chick brain but was very different from that of bovine brain in chemical properties. Furthermore, it was found that PAP II and CBA II were very similar proteins. In the present study, the purification and characterization of highly acidic proteins in bovine liver were attempted by the procedure described above, using a large amount of the tissue. S-100 protein could not be detected either chemically or immunologically in bovine liver, in contrast with the results of Moore (7), whereas a protein similar to PAP II in bovine brain and to CBA II in chick brain was obtained in high yield.
1299
boiling HC1 (Wako, analytical grade). Amino acid analysis was performed with a Hitachi KLA3B automatic amino acid analyzer. Analysis of N-Terminal Amino Acid—The N-terminal amino acid was determined according to the method of Gros and Labouesses (75). Each protein sample (about 1 mg) was reacted with dansyl chloride and hydrolyzed at 110°C for 4 h or 16 h with constant-boiling HC1. Dansyl-amino acid was identified on a thin-layer plate of Silica gel F m (5x10 cm). Two solvent systems, benzene/ pyridine/acetic acid (80:20:2, v/v) (76") and chloroform/methanol/acetic acid (75 : 20 : 5, v/v) (77), were used separately. Trypsin Digestion—Protein samples were aminoethylated according to the method of Cole (18). The modified protein (2 to 3 mg) was digested at 37°C for 24 h with one-fiftieth, portion of TPCKtrypsin (w/w) in 0.1 M N-ethylmorpholine-acetic acid buffer, pH 7.0 or 8.0. The digest was then quickly lyophilized. Peptide Map—Peptide mapping was performed on Whatman 3MM paper. About 2 mg of the tryptic digest was subjected to descending chromatography for 16 h to 20 h in the shorter dimension using 1-butanol/pyridine/acetic acid/ water (15 : 10 : 3 : 12, v/v). Electrophoresis was carried out in the second dimension at pH 3.6, using pyridine/acetic acid/water ( 1 : 1 0 : 289, v/v) at a potential of 50 volt per cm for about 1 h.
Peptides on paper were stained with 0.2% ninhydrin in acetone. Immunodiffusion—Double diffusion (Ouchterlony's method) was performed in 0.84% Difco Bacto Agar gel containing 0.85% NaCl, 0.01% merzonin, and 0.01 % trypan blue according to the standard method (79). Antiserum against bovine S-100 protein fraction was dissolved at a concentration of 40 mg'per ml in 0.85% NaCl, and each sample was dissolved in the same saline solution. Immunoreaction was performed overnight at room temperature. RESyLTS Purification of Highly Acidic Proteins—From a total of 6 kg of bovine liver, 123 g of soluble proteins was extracted and fractionated on a DEAE-Sephadex A-50 column under the conditions described above. The elution pattern is shown in Fig. 1. Seventy percent of the soluble proteins passed through from the column. The acidic proteins, which migrated with BPB marker dye on 7.5% polyacrylamide gel disc electrophoresis, were detected in the fractions eluted at the concentrations of 0.10, 0.17, 0.22, and 0.40 M NaCl (Fractions I-IV). The shaded fractions shown in Fig. 1 were collected and further fractionated, mainly on Sephadex G-75 as shown in Fig. 2A-D. Highly acidic proteins which migrated with BPB
HACL CONCENTRATION (
0,10
5.0
•0.17 — |
0,40
0.22
2.5
0
'
100
200
100
500
700
800
FRACTION HUHBER
Fig. 1. DEAE-Sephadex A-50 column chromatography of bovine liver extract. The soluble protein fraction (123 g of proteins/3.2 1 from 6 kg of bovine liver) was applied to a DEAE-Sephadex A-50 column (4.6x40 cm) and eluted stepwise with 0.10, 0.17, 0.22, and 0.40M N a d , as described in " MATERIALS AND METHODS." Fractions of 14.5 ml per tube were collected. The flow rate was 40 ml/h. Absorbance at 280 nm was measured. The shadowed fractions in the figure (Fractions I, II, III, and IV) were collected and subjected to the further fractionation. Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
1300
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
on 7.5% gel were detected in the underlined fractions and designated as BLA I, BLA n , BLA m , and BLA IV as indicated. The yields of BLA I-IV fractions at this stage were 150, 100, 50,
0.2 -
50
30
70
90
110
FRACtlON NUWER
E 0.1OI W l
A Ml
/
0.50H bCI \
|
NA—J 1 50
100
ISO FRACTION KU»EK
Fig. 2
200
250
300
Fig. 2. Chromatography of fractions I-IV on Sephadex G-75 and DEAE-Sephadex A-50. (A) and (B), the patterns of rechromatography on Sephadex G-75 of Fractions I and II from Fig. 1; Fractions I and II were subjected to chromatography on Sephadex G-75 and the acidic protein fractions were collected and subjected to rechromatography on Sephadex G-75. (C) and (D), the patterns of final gel chromatography on Sephadex G-75 of Fractions III and IV from Fig. I; Fractions III and IV were subjected to chromatography on Sephadex G-75, and the acidic protein fractions were chromatographed successively on Sephadex G-50 and Sephadex G-75. The fractions underlined in Figs. 2A, B, C, and D were named BLA I, BLA II, BLA III, and BLA IV fractions, respectively. Column sizes were 1.7x85 cm (A), 4.5x86 cm (B), 1.7x85 cm (C), and 4.8x96 cm (D). The flow rates were 30, 50, 25, and 66 ml/h and fractions of 3.7, 10.5, 2.8, and 11.8 ml were collected, respectively. (E), The patterns of rechromatography on DEAE-Sephadex A-50 of BLA IV fraction (Fig. 2D); BLA IV fraction was applied to a DEAE-Sephadex A-50 column (2.0x25 cm) equilibrated with 0.1 M potassium phosphate, pH 7.1, containing 0.10 M NaCl and 1 min EDTA, and eluted stepwise with 0.10, 0.30, and 0.50 M NaCl in the same buffer. The flow rate was 22 ml/h. Fractions of 4.0 ml were collected. The resulting acidic protein, BLA IV (underlined in the figure), was homogeneous. The yield was low, presumably due to aggregation or degradation during storage.
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
0.1 -
and 450 mg, respectively, from 6 kg of bovine liver. BLA I, II, and HI fractions were used for characterization without further purification. BLA IV fraction was subjected to further chromatography on DEAE-Sephadex A-50 as shown in Fig. 2E and eluted at a concentration of 0.30 M NaCl in the underlined fractions. BLA IV was thus obtained in a homogeneous form free from nucleic acid. Electrophoretic Analysis of Acidic Proteins— The electrophoretic patterns of BLA I—III fractions and BLA IV on 7.5% and 15% polyacrylamide gels are shown in Fig. 3. On 7.5% gel electrophoresis, the BLA I and BLA II fractions showed protein bands in addition to a main protein band (BLA I and BLA II, respectively) at the BPB marker dye, whereas BLA III fraction and BLA IV each gave a single band at the BPB marker dye (Fig. 3A). On the other hand, BLA I-III fractions and BLA IV all showed different mobilities on 15% gel electrophoresis (Fig. 3B). The mobilities
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
1301
BLA I FRACTION
MIGRATION DISTANCE
0.39 0.65
»0.70
ni-3 in-? m-i
BPE
0.86 0.91 1.00
BLA I
BLA II
BLA I I I
FRACTION
FRACTION
FRACTION
(CM)
Fig. 4. Estimation of the molecular weights of BLA I-II1 main components and BLA IV. After dansylation, the proteins were electrophoresed on 15% polyacrylamide gel containing 0.1% SDS and 4 M urea, pH 7.2. Pepsin (1), trypsin (2), lysozyme (3), and ribonuclease A (4) were used as standard proteins.
BLA IV
Fig. 3. Disc electrophoresis of purified acidic proteins on 7.5% (A) and on 15% (B) polyacrylamide gels. The mobility of each main component relative to BPB is shown in (B). of the main components relative to BPB (Rf) were 0.65 for BLA I and 0.39 for BLA II fractions. The three components of BLA III fraction were named BLA III-l (R, = \.O), BLA ni-2 (/{,= 0.91), and BLA III-3 (R,=0.86). The rechromatographed BLA IV showed a single band (^/=0.70), indicating it to be homogeneous. Some Properties of the Acidic Proteins—The molecular weights were determined by SDSpolyacrylamide gel electrohoresis after dansylation (13). BLA I and BLA II fractions gave several fluorescence bands. BLA III fraction gave two fluorescence bands, but BLA IV showed only one. The molecular weights were estimated to be 9,800 for BLA I main component, 17,000 for BLA II main component, 12,500 for BLA III-2, 14,500 for BLA m-3, and 16,000 for BLA IV (Fig. 4). N-Terminal amino acids were determined for BLA III fraction and BLA IV by a dansylation method (75). After acid hydrolysis of the dansylated BLA III fraction for-4 or 17 h, three dansyl amino acids (dansyl-Thr and -Tyr as main, and Vol. 82, No. 5, 1977
210
250
260
270
280
WAVELENGTH (NH)
Fig. 5. Ultraviolet absorption spectrum of BLA IV. The protein was dissolved in 0.1 M potassium phosphate buffer, pH 7.1, containing 1 mM EDTA at a concentration of 2.7 mg/ml. dansyl-Ile as a minor components) were identified on a thin-layer plate. This suggests that BLA III fraction contains at least three components, as expected from the results described above (Figs. 3B and 4). Under the same conditions, BLA IV gave no a-NH,-dansylated amino acid, indicating that the N-terminal amino group of BLA IV may be masked. BLA I-UI fractions showed typical protein absorption spectra (data not shown), while BLA IV showed an unusual ultraviolet absorption spectrum
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
BPB
1302
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA of histidine, 2 residues each of proline and tyrosine, and 7 residues of phenylalanine per molecule of protein, with no cysteine or tryptophan; these results indicate that BLA IV is very similar to PAP II obtained in the same way. The minimal molecular weight determined by amino acid analysis, assuming 10 residues of alanine per molecule of protein, was 15,500, corresponding well with that estimated by SDS-po!yacrylamide gel electrophoresis. The maps of tryptic peptides of BLA m fraction and IV are shown in Fig. 6, together with those of bovine brain acidic proteins (PAP I and II). Twenty-one ninhydrin-positive spots were detected for BLA i n fraction, 18 for PAP I, 16 for BLA IV, and 16 for PAP H. The number of peptides on the map of BLA i n fraction was greater than expected from the amino acid composition, probably
TABLE I. Amino acid compositions of highly acidic proteins obtained from bovine liver and brain. Amino acid compositions are expressed as molar %, but values in parentheses correspond to the numbers of amino acid residues in the protein molecule. No correction has been made for destruction during acid hydrolysis. 1
BLAI»
i
Lys His
:
ATB
Asp Thr 'Ser : — Glu 1 ' Pro Gly Ala Cys Val Met He Leu Tyr Phe Trp»
9.98 2.10 3.42 10.13 4.07 5.79 14.43 •
4.75 -
6.83 6.30 1.78 7.34 2.55 3.56 8.38 3.68 4.87 n. d.f
1
J 7.81
1
i
VBLAm
6.40 2.63 • 1.24 3.94 . . 3.51 10.92 • 13.09 4.84 . • 4.82 5.74 5.49 14.17' 20.07 -4.49 3.26 6.32 7.33 6.32 7.81 1.24 1.20 6.70 5.99 2.14 3.96 4.42 4.50 8.85 7.10 2.60 2.49 4.13 4.49 n.d. f n.d.'
BLA IV" 5.86 0.96 4.05 16.43 7.63 2.62 18.65 1.70 7.66 7.48 0.00 3.79 5.64 5.34 6.11 1.34 5.21 0.00
(7. 82) (0.92) (6. 36) (21.90) (10.18) (3.47) (24.95) (2.27) (10.22) (10.00) (0) (5.07) (7.54) (7.00) (8.16) (1.74) (6.95) (0)
PAPIc 9.11 5.35 1.04 10.65 3.38 5.46 20.17 0.00 4.95 6.15 1.041 . 6.35 3.19 3.87 9.74 1.35 7.16 0.39
PAP IID.c
(7.48) (0. 88) (5.43) (22.05) (10.24) (3.46) (20.60) (2. 21) (9.80) (10.00) (0)" (6.04) (7.74) (6.93) (8.01) (1.80) (7-10) (0)
• BLA I and BLA II fractions were analyzed. b The ratios of amino acid residues are indicated, taking the content of alanine as 10.00 residues per molecule of protein. = Taken from the previous paper (6). PAP I and PAP II ' were obtained in the fractions eluted with 0.22 M and 0.35 M NaCl from DEAE-Sephade*A-50. d Determined after performic acid oxidation of proteins. • Determined spectrophotometrically or after alkaline hydrolysis of the proteins. f Not determined. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
witrT 5' maxima at 254, 260, 266, 27O, and 278 nm (Fig. 5). The fine structure may be due to a high content of phenylalanine residues and a low content of tyrosine residues (Table I ) . ' The molar absorbance of BLA IV was 2.8x10* at 280 nm in deionized water, assuming the molecular weight to be 16,000. Chemical Comparisons of Acidic Proteins—The amino acid compositions of BLA I-IV. fra'ctions are given in Table I, together with those : of bovine brain acidic proteins (PAP I and PAP tl). BLA i n f r a c t i o n is distinctly different from PAP I as regards amino acid composition, especially in proline, histidine and arginine contents, though both were obtained in the same way by DEAE'-Sephadex A-50 chromatography. On the other hand, BLA IV as well as PAP II was characterized by 1 residue
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
o o o Asp
Glu Gly
o
1303
c
Lys
o
o
Glu Gly
Lys
o o Picric acid
Asp
•
8
Gly
O Val
O
Val
OLIU
0
Leu
0
nd
Electrophoresis
°
Picric «dd
OGly
, Asp
o o Glu ' Gly
0
Ala
4 •
/
o
Ptwnol red
Electrophoresis
+ D
o Picric acid
ao o o
Aipl
Glu
Lys
Gly
OGly OAI.
Oval
Fig. 6. Peptide maps of tryptic peptides of BLA III fraction (A) and IV (B), and bovine brain acidic proteins, PAP I (Q and II (D). The reduced sample was aminoethylated, digested with trypsin and subjected to two-dimensional peptide mapping as described in " MATERIALS AND METHODS." Peptides were visualized with ninhydrin, and the color intensity is shown in increasing order as follows; dotted circle, open circle, shadowed circle, and solid circle. because this fraction was a mixture of two main against bovine S-100 protein. Only PAP I crosscomponents (BLA IH-2 and -3). No correspond- reacted, as shown in Fig. 7. Under the conditions ence between the peptide maps of BLA III fraction used, PAP I could react at a minimum concentraand PAP I was observed. Thus it was concluded tion of 1.2 /ig per ml. Even when the protein conthat BLA III fraction and PAP I are different pro- centration was increased up to 2 mg per ml, BLA tein species. On the other hand, the peptide map I-III fractions, BLA IV and PAP II did not crossof BLA IV showed good correspondence with that react. The bovine liver extract applied to a of PAP II, except for two weak spots, but was DEAE-Sephadex A-50 column also did not react. clearly different from those of BLA III fraction These results clearly indicate that bovine liver conand PAP I. These results suggest that BLA IV in tains at most less than one-thousandth as much bovine liver is virtually identical with PAP II in S-100 protein as bovine brain. bovine brain. Immunological Properties of Acidic Proteins— DISCUSSION To confirm the brain specificity of S-100 protein, immunoreaction was performed on BLA I-IJJ frac- In the present study, bovine liver acidic proteins, tions, BLA IV, and PAP I and Il.with an antiserum which migrated with BPB on 7.5% polyacrylamide Vol. 82, No. 5, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
OGly OAla
1304
T. KATO, Y. KATO, H. KASAI, and T. OKUYAMA
/. Biochem,
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
Sephadex A-50 chromatography; both were eluted with 0.22 M NaCl. However, each of the three components of BLA,'Infraction migrated faster than PAP I on -i-5-% gel disi^ electrophoresisr while BLA III fraction and_PAP I migrated with BPB marker dye on 7.5% gel,. The molecular weights of BLA III components were larger than that of PAP I (8,000)'(5) on SDS-polyacrylamide gel electrophoresis. B&A»' III fraction showed a typical UV spectnfm, whereas PAP • I showed a characteristic spectrum with maxima in the region of 250 to 290 nm. Three N-terminal arnino acid residues were detected for BLA III. fraction byji dansylation method, but none for PAP L_ ._ . * The amino acid analysis and peptide mapping also showed that^BLA^III fraction and PAP I were quite different fcom^*ach! other. Furthermore, "PAPT gave a pfecipifirfline with*antiserum against bovine S-100 protein, but BLA III fraction did not. As the liver extract did not react with antiserum against S-100 protein, the brain specificity of S-100 protein was again confirmed in the present study. However, the physiological function of BLA III components remains unknown. BLA IV in bovine liver was essentially identical with PAP II in bovine brain on the basis of several criteria, as described below; both proteins were Fig. 7. Immunodiffusion profiles against anti S-100 obtained in the fraction eluted with 0.40 M (or serum-of crude extracts of liver and brain (A), and of 0.30-0.35 M) NaCl from a DEAE-Sephadex A-50 purified acidic proteins from liver and brain (B). Im- column, and they were different from S-100 protein, munodiffusion was performed as described in " MA- judging from their chemical and immunological TERIALS AND METHODS." A: (1) Liver extract (45 mg/ml); (2) brain extract (3.9 mg/ml); B: (1) BLA I, properties. The electrophoretic mobilities of the (2) BLA II, (3) BLA III, (4) BLA IV, (5) PAP I, and (6) two proteins were the same both on 7.5 % gel and PAP U. The protein concentration in B was 2 mg/ml on 15% gel. In fact, a mixture of BLA IV and PAP II (1 : 1, by weight) gave a single band on except for PAP I (1.2 /ig/ml). 15% gel (data not shown). Both proteins showed characteristic UV spectra with 5 maxima, due to gel electrophoresis, were separated in four fraca high ratio of phenylalanine to tyrosine residues; tions (BLA I-IV) by DEAE-Sephadex A-50 ionother proteins with similar UV spectra were reexchange chromatography. Some of them were still heterogeneous, but each fraction was different ported recently (20, 21). The molecular weight when analyzed by gel electrophoresis, and by amino was estimated to be 16,000 for both BLA IV and acid analysis. In this section, BLA III fraction is PAP II by SDS-polyacrylamide gel electrophoresis compared in more detail with PAP I, and BLA IV after dansylation. No N-terminal amino acid with PAP II. PAP I and PAP II were previously was detected in the dansylated proteins (BLA obtained from bovine brain (5,6") by the procedure IV and PAP II) even after prolonged hydrolysis (16 h). The results of amino acid analysis and used for bovine liver. mapping of tryptic peptides clearly also suggest Several lines of evidence suggested that BLA that the two proteins are identical. III fraction from bovine liver is different from PAP I in bovine brain, as described below. Both As described in the previous paper (6), one of proteins showed the same behavior on DEAE- the two highly acidic proteins from chick brain,
HIGHLY ACIDIC PROTEINS IN BOVINE LIVER
REFERENCES 1. Moore, B.W. & McGreger, D. (1965) /. Biol. Chem. 240, 1647-] 653 2. Hyden, H. & Lange, P.W. (1970) Proc. Nail. Acad. Sci. U.S. 67, 1959-1966 3. Hyden, H. (1974) Proc. Nail. Acad. Sci. U.S. 71, 2965-2968 4. Marangos, P.J., Zomzely-Neurath, C , Luk, D.C., & York, C. (1975) J. Biol. Chem. 250, 1884-1891 5. Isobe, T. & Okuyama, T. (1971) Seikagaku (in Japanese) 43, 479 6. Kato, Y., Kato, T., Kasai, H., Okuyama, T., & Uyemura, K. (1977) J. Biochem. 82, 43-51 7. Moore, B.W. (1965) Biochem. Biophys. Res. Commun. 19, 739-744 8. Klee, W.A. (1966) in Procedures in Nucleic Acid Research (Contoni, G.L. & Davies, D.R., eds.) pp. 20-30, Hapen and Row, New York 9. Carpenter, F.H. (1967) in Methods in Enzymology (Hire, C.H.W., ed.) Vol. 11, p. 237, Academic Press, New York
Vol. 82, No. 5, 1977
10. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 11. Ornstein, L. (1964) Ann. New York Acad. Sci. 121, 321-349 12. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, 404-427 13. Talbot, D.N. & Yphantis, D.A. (1971) Anal. Biochem. 44, 246-253 14. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412 15. Gros, C. & Labouesse, B. (1969) Eur. J. Biochem. 7, 463t7O 16. Morse, D. & Horecker, B.L. (1966) Anal. Biochem. 14, 429^33 17. Seiler, N. & Wiechmann, M. (1964) Experientia 20, 559-560 18. Cole, R.D. (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 315-317, Academic Press, New York 19. Clausen, M.J. (1969) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T.S. & Work, E., eds.) Vol. 1, pp. 397-572, North-Holland Pub. Co., Amsterdam 20. Dorrington, K.J., Hui, A., Hofmann, T., Hitchman, A.J.W., & Harrison, J.E. (1974) /. Biol. Chem. 249, 199-204 21. Lehky, P., Blum, H.E., Stein, E.A., & Fischer, E.H. (1974) J. Biol. Chem. 249, 4332^334 22. Isobe, T., Kato, Y., Kato, T., Kasai, H., Arai, T., & Okuyama, T. (1972) Seikagaku (in Japanese) 44, 439 23. Aochi, M., Suzuki, M., Kasai, H., & Okuyama, T. (1973) Seikagaku (in Japanese) 45, 468 24. Coffee, C.J. & Bradshaw, R.A. (1973) /. Biol. Chem. 248, 3305-3312 25. Kretsinger, R.H. & Nockolds, C.E. (1973) /. Biol. Chem. 248, 3313-3326 26. Enfiels, D.L., Ericsson, L.H., Blum, H.E., Fischer, E.H. & Neurath, H. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1309-1313 27. Collins, J.H., Potter, J.D., Horn, M.J., Wilshire, G., & Jackman, N. (1973) FEBS Lett. 36, 268-272 28. van Eerd, J-P. & Takahashi, K. (1976) Biochemistry 15, 1171-1180 29. Wolff, D.J. & Siege!, F.L. (1972) / . Biol. Chem. 2A1, 4180-^185 30. Brooks, J.C. & Siegel, F.L. (1973) /. Biol. Chem. 248,4189-4193 31. Huang, W-Y., Cohen, D.V., Hamilton, J.W., Fullmer, C. & Wasserman, R.H. (1975) /. Biol. Chem. 250, 7647-7655 32. Isobe, T., Kato, Y., Kasai, H., & Okuyama, T. (1975) 1SN Barcelona Meeting (abstr.) p. 303 33. Kasai, H., Yamagata, Y., Kato, Y., & Okuyama, T. (1976) 10th Internal. Congr. Biochem. (abstr.) p. 571 34. Teo, T.S., Wang, T.H., & Wang, J.H. (1973) /. Biol. Chem. 248, 588-595 35. Lin, Y.M., Liu, Y.P., & Cheung, W.Y. (1974) /. Biol. Chem. 249, 4943-4954 36. Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., & Vanaman, T.C. (1976) / . Biol. Chem. 251, 4501^513
Downloaded from https://academic.oup.com/jb/article-abstract/82/5/1297/771437 by University of Otago user on 18 January 2019
CBA II, which was eluted with 0.35 M NaCl on DEAE-Sephadex A-50 chromatography, was almost identical with PAP II as regards chemical properties. The relevant yields were 450 mg from 6 kg of bovine liver (BLA IV), 700 mg from 8 kg of bovine brain (PAP II), and 700 mg from 9 kg of chick brain (CBA II). Thus, it appears that essentially the same protein was present in high content in bovine liver and brain, and in chick brain. In our laboratory, a similar protein was recently obtained from human brain and bovine erythrocytes (22, 23). These results suggest that this highly acidic protein has no organ or species specificity and is distributed widely in cells with little change in its primary structure. Recently, several acidic proteins with Cabinding activity have been reported, such as parvalbumin (24-26), troponin C (27, 28), Cabinding phosphoprotein (29, 30), and vitamin D-dependent Ca-binding protein (20, 31), most of which were characterized by unusual UV spectra, resembling that of PAP II. Investigation of the structural similarity between troponin C and PAP II is now in progress (32). PAP II was identified as an activator of cyclic AMP phosphodiesterase and as a Ca-binding protein (33). The Cadependent activator proteins of phosphodiesterase already reported (34-36) are very similar to PAP II, especially in chemical properties and in their mode of activation of the enzyme (6, 33).
1305
