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Isolation of a 65-kDa protein from white muscle of warm temperature-acclimated goldfish (Carassius auratus)
Comparative Biochemistry and Physiology Part B 120 (1998) 385 – 391
Isolation of a 65-kDa protein from white muscle of warm temperature-acclimated goldfish (Carassius auratus) Kiyoshi Kikuchi a, Shugo Watabe a,*, Katsumi Aida b a
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Bunkyo, Tokyo 113, Japan b Laboratory of Aquatic Animal Physiology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Bunkyo, Tokyo 113, Japan Received 7 May 1997; received in revised form 20 February 1998; accepted 24 March 1998
Abstract A 65-kDa protein expressed in association with warm temperature acclimation of goldfish (Carassius auratus) was purified from epaxial muscle by successive ion-exchange, gel filtration, and reversed-phase columns while monitoring immuno-reaction with a specific antibody. A total of 517 mg of the 65-kDa protein was obtained from 23.4 g of the muscle of 30°C-acclimated fish. The purified 65-kDa protein gave one band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was determined to be 65000 by gel filtration and SDS-PAGE, demonstrating that it consists of a single polypeptide chain; 44 amino acid residues were determined by N-terminal amino acid sequencing. The amino acid stretch was comparatively rich in histidine and phenylalanine. Homology search in the National Biomedical Research Foundation and Swiss-Prot bank did not identify any known amino acid sequence with significant homology to the 44-amino acid stretch of the 65-kDa protein, suggesting it to be a novel protein. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Acclimation; Adaptation; Goldfish; Muscle; N-terminal amino acids; Ectotherms; 65-kDa protein; Warm temperature
1. Introduction Temperature fluctuation, acute or long-term, is a critical factor for ectotherms including fish [5]. The heat shock response, which is triggered by a brief exposure to high temperature, involves induction of synthesis of heat shock proteins [2,11,13]. These appear to have repair and protective functions at the cellular level [1,18,22]. Some environmental fluctuations take place over weeks or months, inducing various types of physiological reorganization. Adaptation to such longer-term temperature fluctuation is often referred to as temperature acclimation and is one of the strategies of eurythermal fish such as carp and goldfish to compensate for * Corresponding author. Tel.: +81 3 38122111 ext. 5297; fax: +81 3 56840622. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10045-7
seasonally changing environmental temperatures. Acclimatory responses involve the regulation of protein synthetic systems, for example, in the contractile apparatus of goldfish and carp [3,8,9,20]. Different isoforms of myosin are expressed at differing acclimation temperatures. Expression of different proteins in association with acclimation temperature is not limited to contractile proteins. We have recently observed increased abundance of a 65-kDa protein in various tissues of 30°Cacclimated goldfish comparing 16 individuals each from 30 and 10°C-acclimated groups [10,21]. We used twodimensional electrophoresis to separate this 65-kDa protein from others and we obtained sufficient quantities for developing a specific antibody [10]. Changes in levels of this protein in response to environmental temperature were clearly demonstrated using immunoblot analysis. When water temperature was raised
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from 20 to 30°C over a duration of 20 h, the 65kDa protein increased in concentration after 2 days, and maintained maximal levels for at least 9 days [10]. Thus, this protein can be considered as one of the best candidates for monitoring the acclimation process at the protein synthetic level. We report here purification of the 65-kDa protein from the muscle of 30°C-acclimated goldfish by a series of chromatographic steps. The N-terminal amino acid sequence of this protein was also determined to predict its possible physiological functions.
2. Materials and methods
2.1. Materials Goldfish (36–45 g) were acclimated in laboratory aquariums to either 10 or 30°C for a minimum of 5 weeks. The acclimation period was determined in reference to the data of Heap et al. [6] for carp. All fish were fed commercial pellets daily ad libitum. After acclimation, fish from each temperature group were sacrificed and samples of white epaxial muscle were dissected out. Resins used in HPLC and conventional chromatography included TSKgel DEAE-5PW, TSKgel G3000 SWG, TSKgel Phenyl-5PW and DEAE-Toyopearl, and were purchased from Tosoh (Tokyo, Japan). Standard proteins used to establish a calibration curve for molecular weight determination on gel filtration were purchased from Sigma (St. Louis, MO) and included blue dextran (2000 kDa), b-amylase from sweet potato (200 kDa), alcohol dehydrogenase from yeast (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase from bovine erythrocytes (29 kDa), and cytochrome c from horse heart (12 kDa). Molecular weight markers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (Sigma) included myosin heavy chain from rabbit muscle (205 kDa), b-galactosidase from Escherichia coli (116 kDa), phosphorylase b from rabbit muscle (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).
2.3. Isolation of the 65 -kDa protein The fraction containing the 65-kDa component was precipitated from the muscle extract at 50–80% saturation of (NH4)2SO4. The precipitate was dissolved in and extensively dialyzed against buffer A containing 20 mM Tris-acetate, pH 7.5, 20 mM NaCl, 0.5 mM dithiotheritol (DTT), 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), and 0.05% NaN3. The dialysate was then centrifuged at 33000×g for 20 min at 4°C. The supernatant containing the 65-kDa protein was applied to HPLC using a TSKgel DEAE-5PW column (0.75× 7.5 cm) equilibrated with buffer A. Proteins were eluted with a linear gradient of 20–500 mM NaCl. Fractions containing the 65-kDa protein were pooled and applied to high speed gel filtration using a TSKgel G3000 SWG column (2.15× 60 cm) equilibrated with buffer B containing 50 mM potassium phosphate, pH 6.9, 0.5 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, and 0.05% NaN3. Fractions containing the 65-kDa protein were pooled and dialyzed against buffer C containing 25 mM Tris–HCl, pH 8.0, 7 M urea, 15 mM b-mercaptoethanol, and applied to a DEAE-Toyopearl column (1.6× 8.0 cm) equilibrated with buffer C. A linear gradient with 300 ml of buffer C and 300 ml of buffer C plus 0.1 M KCl was used. The flow rate was 60 ml h − 1. Fractions containing the 65-kDa protein were exhaustively dialyzed against 0.05% DTT for 24 h and lyophilized. The sample was dissolved in 0.1% trifluoroacetate (TFA) containing 8 M urea and then ap-
2.2. Preparation of cytosolic protein fraction Muscle tissues were homogenized with a Polytron PT10-35 in an equal volume of an ice-cold 1:1 mixture of 0.06 M barbital buffer (pH 8.6) and glycerol (v/v) and centrifuged at 33000×g for 15 min as described previously [21]. The resulting supernatants were used for electrophoretic analyses or (NH4)2SO4 precipitation.
Fig. 1. SDS-PAGE (A) and immunoblotting (B) patterns of goldfish sarcoplasmic proteins. Proteins were extracted from white muscle of goldfish acclimated to either 30 (lane 1) or 10°C (lane 2). An arrowhead in panel B indicates a protein band reactive with antibody specific to the 65-kDa protein appearing in 30°C-acclimated fish. Sample volumes used were 10 ml for SDS-PAGE.
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Fig. 2. TSKgel DEAE-5PW HPLC of the sample preparation after ammonium sulfate precipitation. The precipitate between 50 and 80% (NH4)2SO4 saturation was dissolved in and dialyzed against buffer A. The dialysate was then centrifuged at 33000×g for 20 min at 4°C. The supernatant containing 285 mg proteins was applied to a TSKgel DEAE-5PW column equilibrated with buffer A. Proteins were eluted with a linear gradient of 20 – 500 mM NaCl at a flow rate of 1.0 ml min − 1. Fractions of 1.0 ml were collected and analyzed for their absorbance at 280 nm (). Panel A shows Coomassie blue-stained 12.5% SDS-PAGE patterns of selected fractions. Panel B shows their immuno-staining patterns. The sample before chromatography was loaded in lane A. Sample volumes used were 10 ml except for lane A and fraction 5 where 1 ml of samples were loaded. In this and following figures, molecular weight markers (kDa) are indicated at the left.
plied to HPLC using a TSKgel Phenyl-5PW reversed phase column (0.75 × 7.5 cm) equilibrated with 0.1% TFA. Absorbed proteins were eluted with a liner gradient of 0.7–70% acetonitrile. Absorbance at 280 nm of the column effluent was monitored with a Shimazu UV-160 spectrophotometer. Protein concentrations were determined with Bradford (Bio-Rad, Hercules, CA) procedures using bovine serum albumin as a standard.
electrophoresis was carried out by the method of O’Farrell [17], using 4% polyacrylamide gels in the presence of 8 M urea and 1% Ampholine (composed of 0.8% pH range 5–8 and 0.2% pH range 3.5–10) for isoelectric focusing and slab gels for SDS-PAGE. Gels were stained with 0.1% Coomassie brilliant blue R250 or silver nitrate [16] after electrophoresis.
2.4. Electrophoretic analyses
Immunoblotting was carried out on an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) after electrophoresis [19] using the antibody against the 65-kDa protein [10]. Biotin labeling of immunoglobulin G and immuno-staining of the
SDS-PAGE was carried out by the method of Laemmli [12] using 7.5 – 20% polyacrylamide gradient slab gels containing 0.1% SDS. Two-dimensional
2.5. Immunoblotting
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Fig. 3. Gel filtration of the 65-kDa protein fraction. The 65-kDa protein fractions (25 mg proteins) eluted from a TSKgel DEAE-5PW column (fractions 31 – 34 in Fig. 2) were pooled and applied to a TSKgel G3000 SWG column equilibrated with buffer B. The flow rate was 3.0 ml min − 1. Fractions of 3.0 ml were collected and analyzed for their absorbance at 280 nm (). Panel A: staining with Coomassie blue 12.5% SDS-PAGE patterns for every three fractions. Panel B: immuno-staining. Sample volumes used were 10 ml for SDS-PAGE. V0 is the void volume.
membrane were carried out according to the Vectastain manual (Vecter, Burlingame, CA).
3. Results and discussion
2.6. N-terminal amino acid sequencing
3.1. SDS-PAGE and immunoblotting
An Applied Biosystems model 473A gas-phase sequencer was used for amino acid sequencing. The protein homology search was carried out by using the SWISS-PROT and National Biomedical Research Foundation (NBRF) 34 database.
When proteins extracted from muscle were subjected to SDS-PAGE and subsequent immunoblotting analysis using the antibody the 65-kDa protein [10], a strong reactivity was observed with the 65-kDa protein of the 30°C-acclimated goldfish (Fig. 1).
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Fig. 4. DEAE-Toyopearl column chromatography of the 65-kDa protein fraction. The 65-kDa protein fractions (1.6 mg proteins) eluted from a TSKgel G3000 SWG column (fraction 18–21 in Fig. 3) were pooled, dialyzed against buffer C and applied to a DEAE-Toyopearl column equilibrated in the same buffer. A linear gradient with 300 ml of buffer C and 300 ml of buffer C plus 0.1 M KCl was used. The flow rate was 60 ml h − 1. Fractions of 5.0 ml were collected and analyzed for their absorbance at 280 nm (). In the photograph are shown silver nitrate-stained 12.5% SDS-PAGE patterns of selected fractions. Sample volumes used were 10 ml for SDS-PAGE.
3.2. Ion-exchange HPLC Most of the 65-kDa protein in the crude extract was precipitated by (NH4)2SO4 ranging between 50 and 80% saturation. The precipitate was dissolved in buffer A and applied to a TSKgel DEAE-5PW column. The solution contained various components including a major component of about 45 kDa, determined to be creatine kinase [15]. Washing with buffer A removed
most proteins from the column. The 65-kDa protein was eluted at about 320 mM NaCl at the leading edge of the major peak (Fig. 2).
3.3. High-speed gel filtration Fractions containing the 65-kDa protein were pooled and applied to a gel filtration column of TSKgel G3000 SWG. As shown in Fig. 3, the 65-kDa protein was
Fig. 5. TSKgel Phenyl-5PW reversed-phase HPLC of the 65-kDa protein. The 65-kDa proteins fractions eluted from a DEAE-Toyopearl (fraction 34–36 in Fig. 4) were dialyzed against 0.05% DTT for 24 h and lyophilized. The lyophilized materials were dissolved in 0.1% TFA containing 8 M urea, then applied to a TSKgel Phenyl-5PW reversed-phase column equilibrated with 0.1% TFA. Proteins were eluted with a linear gradient of 0.7– 70% acetonitrile containing 0.1% TFA. The flow rate was 1.0 ml min − 1. In panel A are shown Coomassie blue-stained 12.5% SDS-PAGE patterns and in panel B, their immuno-staining patterns for peak 1 (lane 1) and peak 2 (lane 2). One-third of the fractions obtained after changing the eluting buffer to buffer A were loaded onto SDS-PAGE.
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Fig. 6. Two-dimensional electrophoretic patterns of the 65-kDa protein (A) and proteins extracted from fast skeletal muscle tissues of goldfish acclimated to either 30 (B) or 10°C (C). The 65-kDa protein was obtained as peak 2 in TSKgel Phenyl-5PW HPLC of Fig. 5. The arrow in panel B indicates the 65-kDa protein dominating in the 30°C-acclimated fish. 100 ml lysate were used in panels B and C.
eluted around fraction 20. High speed gel filtration with the extract gave a molecular weight of 65000 for the immuno-reactive peak, suggesting that this protein is not composed of subunits.
3.4. Ion-exchange chromatography in the presence of urea Considerable amounts of a 53-kDa protein co-eluted with the 65-kDa protein from gel filtration column. To remove these impurities, the fractions containing the 65-kDa protein were dialyzed against buffer C containing 7 M urea and subjected to a DEAE-Toyopearl column equilibrated with the same buffer. SDS-PAGE of fraction 35 eluted at about 420 mM KCl gave a single band on silver nitrate staining. The 65-kDa protein was successfully separated from the 53-kDa protein on this column (Fig. 4).
3.5. Re6ersed phase HPLC To determine amino acid sequence of any peptide, it is desirable to exclude any contamination as much as possible. Therefore, the 65-kDa protein separated on the DEAE-Toyopearl column was subjected to HPLC with a Phenyl-5PW reversed phase column. The second peak eluted at 41% acetonitrile and contained the 65kDa protein. This preparation on SDS-PAGE gave a single band (Fig. 5). The first peak possibly included an isoform of the 65-kDa protein, since its N-terminal amino acid sequence was identical to that of the second peak as described in the following section. Other minor impurities in Fig. 5 were successfully removed. The component in the second peak was subjected to two-dimensional electrophoresis, showing the same isoelectric point and molecular weight as those of the 65-kDa protein in the muscle extract (Fig. 6). With these methods, 517 mg of the 65-kDa protein was obtained from 23.4 g of epaxial muscle of 30°C-acclimated goldfish.
3.6. N-terminal amino acid sequence The N-terminal amino acid sequence of the 65-kDa protein is shown in Fig. 7. Though the 41st amino acid from the N-terminus could not be identified, a total of 44 amino acid residues were determined. Homology search by NBRF34 revealed that the 44 amino acid stretch of the 65-kDa protein was 29 and 26% homologous to spinach DNA polymerase b% subunit of 78 kDa and human tyrosine hydroxylase of 50 kDa, respectively (Fig. 7) (personal communication from T. Ooi, Kyoto Women’s University). Use of SWISS-PROT gave similar results. RNA polymerase b% of 677 amino acids is a subunit of RNA polymerase which is implicated in the transcription of spinach chloroplast [7]. The subunit is thought to carry the binding site for DNA template. The homologous region is located in the C-terminal part of the b% subunit, whereas DNA binding site is thought to be in the N-terminal portion. Tyrosine hydroxylase (497 aa residues) catalyzes the formation of 3,4-dihydroxyphenylalanine from tyrosine [4,14]. The homologous region is in the center part which is assumed to contribute to substrate specificity and regulation of enzyme activity, but has no catalytic activity. Despite such similarities, there are important differences between the 65-kDa protein and these two proteins. Most notable is the location of a homologous structure which is the N-terminal sequence of the 65-kDa protein, whereas it is not in the N-terminus of RNA polymerase b% and tyrosine hydroxylase. In addition, no cysteine residue is conserved among these proteins. Thus, these homologies do not allow us to make predictions concerning the function of the 65-kDa protein, but suggest that it might be a novel protein. The 65-kDa protein expressed during warm temperature acclimation was similar to heat shock proteins in terms of their increasing concentration after temperature stress. However, the amino acid sequence of the 65-kDa protein was clearly different from those of any
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Fig. 7. N-terminal amino acid sequence of the 65-kDa protein in comparison with sequences of spinach RNA polymerase b% subunit and tyrosine hydroxylase. The numbers start from the N-terminus. An x at position 41 from the N-terminus of the 65-kDa protein indicates an unidentified amino acid. Identical and conserved amino acids between the 65-kDa protein and RNA polymerase b% subunit (RNApase b%) or tyrosine hydroxylase (TH) are indicated by asterisks and pluses, respectively. A dash denotes a gap introduced to maximize homology.
other heat shock proteins (data not shown). We have already shown evidence that the 65-kDa protein does not belong to the known family of heat shock proteins with a molecular weight of about 70 kDa, since the 65-kDa protein did not bind to an ATP-agarose column and was not immediately expressed after temperature increase [10]. It is requisite to carry out biochemical and physiological experiments with chromatographically-purified 65kDa protein to elucidate its function at the cellular level. The establishment of the method for its purification in this report will lead to further studies which will address the question. Additionally, cDNA cloning of the 65-kDa protein is now being undertaken in our laboratory to obtain more information on its primary structure.
Acknowledgements We thank Dr M.N. Wilder of Japan International Research Center for Agricultural Sciences for reading the manuscript. This study was funded in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. K. Kikuchi was supported by a fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.
References [1] Arai A, Mitani H, Naruse K, Shima A. Relationship between the induction of proteins in the HSP70 family and thermosensitivity in two species of Oryzias (Pisces). Comp Biochem Physiol B 1994;109:647 – 54. [2] Dietz TJ, Somero GN. Species and tissue-specific synthesis patterns for heat-shock proteins HSP70 and HSP90 in several marine teleost fishes. Physiol Zool 1993;66:863–80. [3] Gerlach G, Turay L, Malik TA, Lida J, Scutt A, Goldspink G. Mechanisms of temperature acclimation in the carp: A molecular approach. Am J Physiol 1990;259:R237–44. [4] Grima B, Lamouroux A, Boni C, Julien J, Javoy-Agid F. A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature 1987;326:707 – 11. [5] Hazel JR, Prosser CL. Molecular mechanisms of temperature .
compensation in poikilotherms. Physiol Rev 1974;54:620–77. [6] Heap SP, Goldspink G. Consequences of thermal change on the myofibrillar ATPase of five freshwater teleosts. J Fish Biol 1985;26:733 – 8. [7] Hudson GS, Holton TA, Whitfeld R, Bottomley W. Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase. J Mol Biol 1988;200:639 – 54. [8] Hwang GC, Watabe S, Hashimoto K. Changes in carp myosin ATPase induced by temperature acclimation. J Comp Physiol B 1990;160:233 – 9. [9] Johnston IA, Davison W, Goldspink G. Adaptation in Mg2 + activated myofibrillar ATPase activity induced by temperature acclimation. FEBS Lett 1975;50:293 – 5. [10] Kikuchi K, Watabe S, Suzuki Y, Aida K, Nakajima H. The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus. J Comp Physiol B 1993;163:349 – 54. [11] Koban M, Yup AA, Agellon LB, Powers DA. Molecular adaptation to environmental temperature: Heat-shock response of the eurythermal teleost Fundulus heteroclitus. Mol Marine Biol Biotech 1991;1:1 – 17. [12] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [13] Lindquist S. The heat shock response. Annu Rev Biochem 1986;55:1156 – 91. [14] Lodley FD, DiLella AG, Kwol SCM, Woo SLC. Homology between phenylalanine and tyrosine hydroxylase reveals common structural and functional domains. Biochemistry 1985;24:3390– 4. [15] Nakagawa T, Watabe S, Hashimoto K. Identification of three major components in fish sarcoplasmic proteins. Nippon Suisan Gakkaishi 1988;54:999 – 1004. [16] Oakley BR, Kirsch DR, Morris ML. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361 – 5. [17] O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007– 21. [18] Schlesinger MJ. How the cell copes with stress and the function of heat shock proteins. Pediatr Res 1994;36:1 – 6. [19] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gel to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350 – 4. [20] Watabe S, Imai J, Nakaya M, Hirayama H, Okamoto Y, Masaki H, Uozumi T, Hirono I, Aoki T. Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle. Biochem Biophys Res Commun 1995;208:118 – 25. [21] Watabe S, Kikuchi K, Aida K. Cold- and warm-temperature acclimation induces specific cytosolic protein in goldfish and carp. Nippon Suisan Gakkaishi 1993;59:151 – 6. [22] Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins and implications for medicine and disease. Physiol Rev 1992;72:1063 – 81.
Isolation of a 65-kDa protein from white muscle of warm temperature-acclimated goldfish (Carassius auratus) Kiyoshi Kikuchi a, Shugo Watabe a,*, Katsumi Aida b a
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Bunkyo, Tokyo 113, Japan b Laboratory of Aquatic Animal Physiology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Bunkyo, Tokyo 113, Japan Received 7 May 1997; received in revised form 20 February 1998; accepted 24 March 1998
Abstract A 65-kDa protein expressed in association with warm temperature acclimation of goldfish (Carassius auratus) was purified from epaxial muscle by successive ion-exchange, gel filtration, and reversed-phase columns while monitoring immuno-reaction with a specific antibody. A total of 517 mg of the 65-kDa protein was obtained from 23.4 g of the muscle of 30°C-acclimated fish. The purified 65-kDa protein gave one band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was determined to be 65000 by gel filtration and SDS-PAGE, demonstrating that it consists of a single polypeptide chain; 44 amino acid residues were determined by N-terminal amino acid sequencing. The amino acid stretch was comparatively rich in histidine and phenylalanine. Homology search in the National Biomedical Research Foundation and Swiss-Prot bank did not identify any known amino acid sequence with significant homology to the 44-amino acid stretch of the 65-kDa protein, suggesting it to be a novel protein. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Acclimation; Adaptation; Goldfish; Muscle; N-terminal amino acids; Ectotherms; 65-kDa protein; Warm temperature
1. Introduction Temperature fluctuation, acute or long-term, is a critical factor for ectotherms including fish [5]. The heat shock response, which is triggered by a brief exposure to high temperature, involves induction of synthesis of heat shock proteins [2,11,13]. These appear to have repair and protective functions at the cellular level [1,18,22]. Some environmental fluctuations take place over weeks or months, inducing various types of physiological reorganization. Adaptation to such longer-term temperature fluctuation is often referred to as temperature acclimation and is one of the strategies of eurythermal fish such as carp and goldfish to compensate for * Corresponding author. Tel.: +81 3 38122111 ext. 5297; fax: +81 3 56840622. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10045-7
seasonally changing environmental temperatures. Acclimatory responses involve the regulation of protein synthetic systems, for example, in the contractile apparatus of goldfish and carp [3,8,9,20]. Different isoforms of myosin are expressed at differing acclimation temperatures. Expression of different proteins in association with acclimation temperature is not limited to contractile proteins. We have recently observed increased abundance of a 65-kDa protein in various tissues of 30°Cacclimated goldfish comparing 16 individuals each from 30 and 10°C-acclimated groups [10,21]. We used twodimensional electrophoresis to separate this 65-kDa protein from others and we obtained sufficient quantities for developing a specific antibody [10]. Changes in levels of this protein in response to environmental temperature were clearly demonstrated using immunoblot analysis. When water temperature was raised
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from 20 to 30°C over a duration of 20 h, the 65kDa protein increased in concentration after 2 days, and maintained maximal levels for at least 9 days [10]. Thus, this protein can be considered as one of the best candidates for monitoring the acclimation process at the protein synthetic level. We report here purification of the 65-kDa protein from the muscle of 30°C-acclimated goldfish by a series of chromatographic steps. The N-terminal amino acid sequence of this protein was also determined to predict its possible physiological functions.
2. Materials and methods
2.1. Materials Goldfish (36–45 g) were acclimated in laboratory aquariums to either 10 or 30°C for a minimum of 5 weeks. The acclimation period was determined in reference to the data of Heap et al. [6] for carp. All fish were fed commercial pellets daily ad libitum. After acclimation, fish from each temperature group were sacrificed and samples of white epaxial muscle were dissected out. Resins used in HPLC and conventional chromatography included TSKgel DEAE-5PW, TSKgel G3000 SWG, TSKgel Phenyl-5PW and DEAE-Toyopearl, and were purchased from Tosoh (Tokyo, Japan). Standard proteins used to establish a calibration curve for molecular weight determination on gel filtration were purchased from Sigma (St. Louis, MO) and included blue dextran (2000 kDa), b-amylase from sweet potato (200 kDa), alcohol dehydrogenase from yeast (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase from bovine erythrocytes (29 kDa), and cytochrome c from horse heart (12 kDa). Molecular weight markers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (Sigma) included myosin heavy chain from rabbit muscle (205 kDa), b-galactosidase from Escherichia coli (116 kDa), phosphorylase b from rabbit muscle (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).
2.3. Isolation of the 65 -kDa protein The fraction containing the 65-kDa component was precipitated from the muscle extract at 50–80% saturation of (NH4)2SO4. The precipitate was dissolved in and extensively dialyzed against buffer A containing 20 mM Tris-acetate, pH 7.5, 20 mM NaCl, 0.5 mM dithiotheritol (DTT), 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), and 0.05% NaN3. The dialysate was then centrifuged at 33000×g for 20 min at 4°C. The supernatant containing the 65-kDa protein was applied to HPLC using a TSKgel DEAE-5PW column (0.75× 7.5 cm) equilibrated with buffer A. Proteins were eluted with a linear gradient of 20–500 mM NaCl. Fractions containing the 65-kDa protein were pooled and applied to high speed gel filtration using a TSKgel G3000 SWG column (2.15× 60 cm) equilibrated with buffer B containing 50 mM potassium phosphate, pH 6.9, 0.5 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, and 0.05% NaN3. Fractions containing the 65-kDa protein were pooled and dialyzed against buffer C containing 25 mM Tris–HCl, pH 8.0, 7 M urea, 15 mM b-mercaptoethanol, and applied to a DEAE-Toyopearl column (1.6× 8.0 cm) equilibrated with buffer C. A linear gradient with 300 ml of buffer C and 300 ml of buffer C plus 0.1 M KCl was used. The flow rate was 60 ml h − 1. Fractions containing the 65-kDa protein were exhaustively dialyzed against 0.05% DTT for 24 h and lyophilized. The sample was dissolved in 0.1% trifluoroacetate (TFA) containing 8 M urea and then ap-
2.2. Preparation of cytosolic protein fraction Muscle tissues were homogenized with a Polytron PT10-35 in an equal volume of an ice-cold 1:1 mixture of 0.06 M barbital buffer (pH 8.6) and glycerol (v/v) and centrifuged at 33000×g for 15 min as described previously [21]. The resulting supernatants were used for electrophoretic analyses or (NH4)2SO4 precipitation.
Fig. 1. SDS-PAGE (A) and immunoblotting (B) patterns of goldfish sarcoplasmic proteins. Proteins were extracted from white muscle of goldfish acclimated to either 30 (lane 1) or 10°C (lane 2). An arrowhead in panel B indicates a protein band reactive with antibody specific to the 65-kDa protein appearing in 30°C-acclimated fish. Sample volumes used were 10 ml for SDS-PAGE.
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Fig. 2. TSKgel DEAE-5PW HPLC of the sample preparation after ammonium sulfate precipitation. The precipitate between 50 and 80% (NH4)2SO4 saturation was dissolved in and dialyzed against buffer A. The dialysate was then centrifuged at 33000×g for 20 min at 4°C. The supernatant containing 285 mg proteins was applied to a TSKgel DEAE-5PW column equilibrated with buffer A. Proteins were eluted with a linear gradient of 20 – 500 mM NaCl at a flow rate of 1.0 ml min − 1. Fractions of 1.0 ml were collected and analyzed for their absorbance at 280 nm (). Panel A shows Coomassie blue-stained 12.5% SDS-PAGE patterns of selected fractions. Panel B shows their immuno-staining patterns. The sample before chromatography was loaded in lane A. Sample volumes used were 10 ml except for lane A and fraction 5 where 1 ml of samples were loaded. In this and following figures, molecular weight markers (kDa) are indicated at the left.
plied to HPLC using a TSKgel Phenyl-5PW reversed phase column (0.75 × 7.5 cm) equilibrated with 0.1% TFA. Absorbed proteins were eluted with a liner gradient of 0.7–70% acetonitrile. Absorbance at 280 nm of the column effluent was monitored with a Shimazu UV-160 spectrophotometer. Protein concentrations were determined with Bradford (Bio-Rad, Hercules, CA) procedures using bovine serum albumin as a standard.
electrophoresis was carried out by the method of O’Farrell [17], using 4% polyacrylamide gels in the presence of 8 M urea and 1% Ampholine (composed of 0.8% pH range 5–8 and 0.2% pH range 3.5–10) for isoelectric focusing and slab gels for SDS-PAGE. Gels were stained with 0.1% Coomassie brilliant blue R250 or silver nitrate [16] after electrophoresis.
2.4. Electrophoretic analyses
Immunoblotting was carried out on an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) after electrophoresis [19] using the antibody against the 65-kDa protein [10]. Biotin labeling of immunoglobulin G and immuno-staining of the
SDS-PAGE was carried out by the method of Laemmli [12] using 7.5 – 20% polyacrylamide gradient slab gels containing 0.1% SDS. Two-dimensional
2.5. Immunoblotting
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Fig. 3. Gel filtration of the 65-kDa protein fraction. The 65-kDa protein fractions (25 mg proteins) eluted from a TSKgel DEAE-5PW column (fractions 31 – 34 in Fig. 2) were pooled and applied to a TSKgel G3000 SWG column equilibrated with buffer B. The flow rate was 3.0 ml min − 1. Fractions of 3.0 ml were collected and analyzed for their absorbance at 280 nm (). Panel A: staining with Coomassie blue 12.5% SDS-PAGE patterns for every three fractions. Panel B: immuno-staining. Sample volumes used were 10 ml for SDS-PAGE. V0 is the void volume.
membrane were carried out according to the Vectastain manual (Vecter, Burlingame, CA).
3. Results and discussion
2.6. N-terminal amino acid sequencing
3.1. SDS-PAGE and immunoblotting
An Applied Biosystems model 473A gas-phase sequencer was used for amino acid sequencing. The protein homology search was carried out by using the SWISS-PROT and National Biomedical Research Foundation (NBRF) 34 database.
When proteins extracted from muscle were subjected to SDS-PAGE and subsequent immunoblotting analysis using the antibody the 65-kDa protein [10], a strong reactivity was observed with the 65-kDa protein of the 30°C-acclimated goldfish (Fig. 1).
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Fig. 4. DEAE-Toyopearl column chromatography of the 65-kDa protein fraction. The 65-kDa protein fractions (1.6 mg proteins) eluted from a TSKgel G3000 SWG column (fraction 18–21 in Fig. 3) were pooled, dialyzed against buffer C and applied to a DEAE-Toyopearl column equilibrated in the same buffer. A linear gradient with 300 ml of buffer C and 300 ml of buffer C plus 0.1 M KCl was used. The flow rate was 60 ml h − 1. Fractions of 5.0 ml were collected and analyzed for their absorbance at 280 nm (). In the photograph are shown silver nitrate-stained 12.5% SDS-PAGE patterns of selected fractions. Sample volumes used were 10 ml for SDS-PAGE.
3.2. Ion-exchange HPLC Most of the 65-kDa protein in the crude extract was precipitated by (NH4)2SO4 ranging between 50 and 80% saturation. The precipitate was dissolved in buffer A and applied to a TSKgel DEAE-5PW column. The solution contained various components including a major component of about 45 kDa, determined to be creatine kinase [15]. Washing with buffer A removed
most proteins from the column. The 65-kDa protein was eluted at about 320 mM NaCl at the leading edge of the major peak (Fig. 2).
3.3. High-speed gel filtration Fractions containing the 65-kDa protein were pooled and applied to a gel filtration column of TSKgel G3000 SWG. As shown in Fig. 3, the 65-kDa protein was
Fig. 5. TSKgel Phenyl-5PW reversed-phase HPLC of the 65-kDa protein. The 65-kDa proteins fractions eluted from a DEAE-Toyopearl (fraction 34–36 in Fig. 4) were dialyzed against 0.05% DTT for 24 h and lyophilized. The lyophilized materials were dissolved in 0.1% TFA containing 8 M urea, then applied to a TSKgel Phenyl-5PW reversed-phase column equilibrated with 0.1% TFA. Proteins were eluted with a linear gradient of 0.7– 70% acetonitrile containing 0.1% TFA. The flow rate was 1.0 ml min − 1. In panel A are shown Coomassie blue-stained 12.5% SDS-PAGE patterns and in panel B, their immuno-staining patterns for peak 1 (lane 1) and peak 2 (lane 2). One-third of the fractions obtained after changing the eluting buffer to buffer A were loaded onto SDS-PAGE.
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Fig. 6. Two-dimensional electrophoretic patterns of the 65-kDa protein (A) and proteins extracted from fast skeletal muscle tissues of goldfish acclimated to either 30 (B) or 10°C (C). The 65-kDa protein was obtained as peak 2 in TSKgel Phenyl-5PW HPLC of Fig. 5. The arrow in panel B indicates the 65-kDa protein dominating in the 30°C-acclimated fish. 100 ml lysate were used in panels B and C.
eluted around fraction 20. High speed gel filtration with the extract gave a molecular weight of 65000 for the immuno-reactive peak, suggesting that this protein is not composed of subunits.
3.4. Ion-exchange chromatography in the presence of urea Considerable amounts of a 53-kDa protein co-eluted with the 65-kDa protein from gel filtration column. To remove these impurities, the fractions containing the 65-kDa protein were dialyzed against buffer C containing 7 M urea and subjected to a DEAE-Toyopearl column equilibrated with the same buffer. SDS-PAGE of fraction 35 eluted at about 420 mM KCl gave a single band on silver nitrate staining. The 65-kDa protein was successfully separated from the 53-kDa protein on this column (Fig. 4).
3.5. Re6ersed phase HPLC To determine amino acid sequence of any peptide, it is desirable to exclude any contamination as much as possible. Therefore, the 65-kDa protein separated on the DEAE-Toyopearl column was subjected to HPLC with a Phenyl-5PW reversed phase column. The second peak eluted at 41% acetonitrile and contained the 65kDa protein. This preparation on SDS-PAGE gave a single band (Fig. 5). The first peak possibly included an isoform of the 65-kDa protein, since its N-terminal amino acid sequence was identical to that of the second peak as described in the following section. Other minor impurities in Fig. 5 were successfully removed. The component in the second peak was subjected to two-dimensional electrophoresis, showing the same isoelectric point and molecular weight as those of the 65-kDa protein in the muscle extract (Fig. 6). With these methods, 517 mg of the 65-kDa protein was obtained from 23.4 g of epaxial muscle of 30°C-acclimated goldfish.
3.6. N-terminal amino acid sequence The N-terminal amino acid sequence of the 65-kDa protein is shown in Fig. 7. Though the 41st amino acid from the N-terminus could not be identified, a total of 44 amino acid residues were determined. Homology search by NBRF34 revealed that the 44 amino acid stretch of the 65-kDa protein was 29 and 26% homologous to spinach DNA polymerase b% subunit of 78 kDa and human tyrosine hydroxylase of 50 kDa, respectively (Fig. 7) (personal communication from T. Ooi, Kyoto Women’s University). Use of SWISS-PROT gave similar results. RNA polymerase b% of 677 amino acids is a subunit of RNA polymerase which is implicated in the transcription of spinach chloroplast [7]. The subunit is thought to carry the binding site for DNA template. The homologous region is located in the C-terminal part of the b% subunit, whereas DNA binding site is thought to be in the N-terminal portion. Tyrosine hydroxylase (497 aa residues) catalyzes the formation of 3,4-dihydroxyphenylalanine from tyrosine [4,14]. The homologous region is in the center part which is assumed to contribute to substrate specificity and regulation of enzyme activity, but has no catalytic activity. Despite such similarities, there are important differences between the 65-kDa protein and these two proteins. Most notable is the location of a homologous structure which is the N-terminal sequence of the 65-kDa protein, whereas it is not in the N-terminus of RNA polymerase b% and tyrosine hydroxylase. In addition, no cysteine residue is conserved among these proteins. Thus, these homologies do not allow us to make predictions concerning the function of the 65-kDa protein, but suggest that it might be a novel protein. The 65-kDa protein expressed during warm temperature acclimation was similar to heat shock proteins in terms of their increasing concentration after temperature stress. However, the amino acid sequence of the 65-kDa protein was clearly different from those of any
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Fig. 7. N-terminal amino acid sequence of the 65-kDa protein in comparison with sequences of spinach RNA polymerase b% subunit and tyrosine hydroxylase. The numbers start from the N-terminus. An x at position 41 from the N-terminus of the 65-kDa protein indicates an unidentified amino acid. Identical and conserved amino acids between the 65-kDa protein and RNA polymerase b% subunit (RNApase b%) or tyrosine hydroxylase (TH) are indicated by asterisks and pluses, respectively. A dash denotes a gap introduced to maximize homology.
other heat shock proteins (data not shown). We have already shown evidence that the 65-kDa protein does not belong to the known family of heat shock proteins with a molecular weight of about 70 kDa, since the 65-kDa protein did not bind to an ATP-agarose column and was not immediately expressed after temperature increase [10]. It is requisite to carry out biochemical and physiological experiments with chromatographically-purified 65kDa protein to elucidate its function at the cellular level. The establishment of the method for its purification in this report will lead to further studies which will address the question. Additionally, cDNA cloning of the 65-kDa protein is now being undertaken in our laboratory to obtain more information on its primary structure.
Acknowledgements We thank Dr M.N. Wilder of Japan International Research Center for Agricultural Sciences for reading the manuscript. This study was funded in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. K. Kikuchi was supported by a fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.
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