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Characterisation of a high-molecular-weight developmentally regulated adult rat liver-specific protein
Biochhnh'a et t3iophysica Acta. ! I I~ 119q2) 2111-286 ,,~'~10t~2 Elsevier Science Publishers B.V. All rights reserved 11167-41138/92/$115.1111
281
BBAPRO 34128
Characterisation of a high-molecular-weight developmentally regulated adult rat liver-specific protein Usha K. Srinivas and C.J. Revathi ('~wtre ]'or Celluh#r and Mok'~'tthtr Bioh~A~.'. H t'~h'nthad (bulia)
(Received 18 September lqtJl)
Key wt~rds: Developmentalregulation; Liver pr~tcin: Protein characterization; (Rat liver) A major, hitherto unknown protein which is shown to be developmentally regulated has been isolated from rat liver. The protein has a molecular mass of 150 kDa and constitutes 8-10% of the adult liver protein, lmmunoblot experiments and immunofluorescence studies established its presence specifically in adult liver cells. This protein is not present in early embryonic liver cells, gets induced around day 18 of gestation and reaches near adult levels by ! day after birth and is not found in the cells of an ascitic hepatoma. Unlike albumin and a-fetoprotein, the two well known developmentally regulated liver proteins, the 150 kDa protein is not secreted. Some of the characteristics of this liver.specific protein are discussed.
Introduction
Materials and Methods
During animal development a pattern of differential gene expression is established resulting in cell populations that arc distinguishable from onc another [1]. Tissue-specific expression of genes coding for abundant proteins that are characteristic of differentiated cell type has been reported [2,3]. Rat liver providcs an excellent system for studying developmentally regulated tissue-specific expression of genes as many physiological, hormonal as well as morphological changes occur in liver during development, aFetoprotein and serum albumin, the two known major proteins characteristic of hepatic differentiation are secreted proteins and are differentially regulated during rat liver development [4,5]. In the course of our studies on the effect of heat shock on hepatic development [6], we have identified a hitherto unknown, developmentally regulated, high-molecular-weight, adultliver-specific protein which constitutes approx. 10% of the total liver protein. Tissue distribution, developmental regulation, amino acid coml~sition and N-terminal analysis indicate that the 150 kDa protein is a novel liver-specific protein which has not been isolated so far.
Materials Wistar rats and rat embryos of different developmental stages were used in this study. The rat livcr tumor, Zajdcla Ascitic Hcpatoma [7], was grown in the peritonial cavity of the animals by serial transplantation. All the chcmicals including FITC conjugated goat anti-rabbit lgG were obtained from Sigma (St Louis, MO, U.S.A.). Protein molecular weight markers were obtained from Pharmacia Fine Chemicals.
Preparation o]" tissue homogenates Small pieces of tissue or cells were suspended in 10 vols. of cold phosphate buffered saline (phosphatc, 2 mM (pH 7.2]: NaCi, 0.2 M: KCI, 2.5 mM) containing 0.1% NP40, I mM PMSF, 2 ~ g / m l trypsin inhibitor and homogenised in the cold using a polytron homogeniser. Samples were made with 3 × sample buffer as described by Lacmmli [8], (62.5 mM Tris (pH 6.8'1: 2.0% sodium dodecyl sulfate (SDS): 3.11%/~-mcrcaptoethanol: I|.t)1)1% bromophenol blue) before further analysis.
Protein attalysis
Correspondence: U.K. Srinivas. Centre fi~r Cellular and Molecular Biology, Hyderabad 5111111117.India.
Protein concentration of different homogenates was determined according to Lowry [9] and the protein concentration in all the samples was adjusted to 5 mg/ml. 100 # g of each sample was loaded on 7.5%
282 gels and SDS-PAGE was carried out as described by Laemmli [8]. After electrophoresis, gels were fixed and stained with Coomassie blue R-250, destained and photographed.
Purification of 150 kDa protein Taking advantage of the fact that the 150 kDa protein is a major constituent of adult liver, electroelution was used to purify the protein. Total protein of adult rat liver was run on 7.5% SDS-polyacrylamide gels, the gel portion corresponding to the 150 kDa protein was cut out, made into small pieces and subjected to electroelution as described by Hunkapiller et al. [10].
Ambm acM composition and N-termh~al analysis Amino acid composition of the electroeluted protein was determined by hydrolysing the protein with 6 M HCI under vacuum at 105°C for 24 h and analysing the hydrolysate on an LKB Alpha plus analyser. N-terminal analysis of 150 kDa protein was determined by the phenyl isothiocyanate procedure of Kuhn and Crabb [11] using a 1090 Lusi and Novapak Columns.
Antibodies to the 150 kDa protehl Antibodies to the 150 kDa protein were raised in rabbits by injecting 1 mg of protein along with complete adjuvant initially, and giving booster injections of 0.5 mg of protein in incomplete adjuvant at 1 week intervals. All the injections were given intradermally. The antisera were tested by Ouchterlony double diffusion method as described by Ouchterlony and Nilson [12]. Unfractionated antisera as well as the immunoglobulin fraction isolated on protein A Sepharose columns based on affinity were used in these studies.
mode. Adult liver sections incubated with 2nd antibody directly were included as controls. Results
Tissue specificity and devdopmental regulation of the 150 kDa protein Total proteins from different organs from the adult and from 14-15-day-old embryos of Wistar rats were analysed on 7.5% SDS-PAGE. In Figs. 1 and 2 Coomassie blue staining patterns are presented. A major protein band of 150 kDa was present in the adult liver (Fig. 1, lanes l and l0 and Fig. 2, lanes 3 and 12), but not in other adult or embryonic tissues or in the Z A H (Fig. l, lane I l). This protein could not be detected in the adult rat serum either, suggesting that it may not be a secreted protein (Fig. l, lane 12). These results indicate that the 150 kDa protein is adult rat liver specific. To study the developmental regulation of this 150 kDa protein, total liver proteins from l 1-, 13-, 14-, 15-, 18- and 20-day-old embryos, 1-day- and 1week-old pups and adult rat were analyzed by SDSPAGE. Results of this study, presented in Fig. 3, indicate that the 150 kDa proteins begins to appear in 18-day-old embryonic liver and reaches adult levels by day 1 after birth. The tissue-specific expression of the 150 kDa protein was further explored by immunoblot analysis of homogenates of various adult and embryonic tissues using antibody raised against electroeluted 150 kDa protein. Western blots of duplicate gels presented in Figs. 1 and 2 are presented in Figs. 4 and 5. Crossreactivity with antibody to 150 kDa was detected only in adult liver tissue (Fig. 4, lane 10 and Fig. 5, lane l), but
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lmmunoblotting Total protein (100 p.g) from different tissues was run on 7.5% SDS-PAGE and was transferred on to nitrocellulose paper in a Bio-Rad transfer apparatus [13l. The blot was incubated with antiserum to 150 kDa protein, washed and treated with iodinated protein A (iodination of protein A was done using iodogen as per suppliers instructions), again washed and exposed to X-ray film.
lmmunofluorescence Cryosections of embryonic and adult rat tissues, frozen immediately after excision from the animal, were incubated with a 1 : 100 dilution of antiserum to the 150 kDa protein for 1 h at room temperature. The sections were washed and further incubated with a 1:100 dilution of FITC conjugated goat anti-rabbit IgG, washed and examined in polyvar (Richeit) fluorescence microscope using B4 filters and epifluorescence
Fig. 1. SDS-PAGE profile of total protein from adult organs ot Wistar rats. Samples containing 100/zg of protein were separated on 7.5% SDS-PAGE and the gel was stained with Coomassie blue R-250 as described in Materials and Methods. Lane !, liver; 2, brain: 3, heart: 4, lung: 5. thymus; 6, spleen; 7, kidney; 8, intestine; 9. muscle: 10, liver: II, ZAH; 12, rat serum; 13, high and low tool. wt. markers 330, 220, 94, 67, 43, 30 and 20. Arrow indicates 150 kDa protein.
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Fig. 2. S D S - P A G E profile of proteins from embryonic organs of 14-15-day-old embryos. 101) jag of protein was separated on 7.55,; gel and the gel was stained with Coomassie blue R-2511 as described in Materials and Methods. Lane I, R N A polymerase core enzyme mol. wt. 151) and 38 kDa and low mol. wl. markers (Pharmacia) ~94, 67 and 43: 2, RNA polymerase (core enzyme) 1511 and 38 kDa: 3, A. liver; 4, fetal intestine: 5, F. muscle: 6, F. kidney: 7, F. spleen: 8. F. lung: 9. F. heart: II), F. brain: I I, F. liver and 12, adult liver, arrow on the right indicates the 1511kDa pn)tcin band.
not in any other tissue, either adult or early embryonic. These results strongly indicate that 151) kDa protein is an adult liver-specific protein. As indicated earlier, 150 kDa protein cannot be a secreted protein as the serum did not contain any proteins (either of 150 kDa or lower) that cross reacted with antibody to 150 kDa (Fig. 4 lane 12). Absence of the protein (cithcr in Coomassie blue pattern or in immunoblot) from a liver tumor, Zajdela Ascitic Hepatoma (Fig. 1, lane 11 and Fig. 4, lane 12) suggests the disappearance of the protein upon transformation.
Localization of the 150 kDa protein Tissue-specific expression and localization of this protein were further confirmed by immunofluorescence studies. Brain, liver, lung, spleen, kidney and pituitary of adult rats and livers of 13- and 19-day-old embryos were processed as described in Materials and Methods. In Fig. 6 the fluorescence photographs of adult liver and pituitory embedded in adult liver tissue are presented. Bright fluorescence was seen localized in the cytoplasm around the nucleus of adult liver cells. No such bright fluorescence was observed in any of the other, adult or embryonic tissues, or in the control slides. Tissue sections from 19-day-old embryonic liver and pancreas showed faint fluorescence, but it was too
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Fig. 3. S D S - P A G E profile of embryonic, new born and adult liver proteins. Sample containing I1111 ttg of protein was run on 7.5~; SDS-PAGE, gels were stained with (k)omassie bluc R-2511 as described in Materials and Methods. Lane I, I I days: 2. 13 days: 3, 14 days: 4. 15 days: 5. 18 days; 6. 2It-day-old embD'onic liver: lane 7 I-day-old pup: 8. I-week-old pup: 0, adult liver (arn)ws indicates 1511 kDa protein and albumin; IlL Pharmacia low m()l. wl. markers ~)4, 67, 43, 31t and 211.
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Fig. 4. lmmunoblot analysis of proteins from adult rat tissues: gels were run as described in Fig. 1, proteins were transferred onto nitrocellulose paper and processed for immunobJotting analysis as described in Materials and Methods. Lane 1, brain: 2. heart" 3, lung; 4, thymus; 5, spleen: fl, kidney: 7. intestine; 8, muscle: 9, pancreas: Irk liver; 11, Z A [ t : and 12, serum. Note: crossreuctivity with the antibudy could he detected only in adult liver hme but in no olher lane including the liver tumor Z A H and rat serum (hines I I and 12).
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Fig. 5. lmmtmoblot analysis of proteins from embryonic organs: Samples ~ere run on 7.5c; SDS-PAGE. transferred onto nitrocellulose paper and processed fi)r immunoblot analysis as described in Materials and Methods. Lane I. adult liver: 2, fetal liver: 3, F. brain; 4. F. heart: 5. F. lung: 6. F. spleen: 7, F. kidney: I-;,.F. muscle: and tL F. intestine.
Fig. 7. Determination of the molecular weight of the protein by SDS-PAGE: proteins were run on 7.5% gels and stained with Coomassie blue. Lane 1, high-molecular-weight markers (Pharmacia): 2. RNA polymerase core enzyme; 3. 150 kDa protein 15 ~g: 4. I11 ,ttg; 5, 5 #g; and 2.11~g.
faint to be p h o t o g r a p h e d . T h e f l u o r e s c e n c e seen in 19 day e m b r y o n i c liver is probably due to the induction o f this protein at that stage as seen in Fig. 3. T h e i m m u n o f l u o r e s c e n c e studies f u r t h e r c o n f i r m the tissue specific expression and d e v e l o p m e n t a l regulation o f 150 k D a protein.
Purification attd physical properties of 150 kDa protein
!
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Fig. 6. pr~iiein bY immun0flut,rescence. Cryosections of adult liver (A) and pituitary embedded in adult liver (B) were treated with antiserum to the 151) kDa protein and then with FITC-conjugated goat-antirabbit IgG, washed and observed under fluorescence microscope as described in Materials and Methods. Bar represents cme micrtm 150|}11× magnification).
As described in m e t h o d s , total p r o t e i n from liver was e [ e c t r o p h o r e s e d on 7.5% S D S - p o l y a c r y l a m i d e gel and the b a n d c o r r e s p o n d i n g to 150 k D a protein was cut out and electroeluted from the gel. Liver hom o g e n a t e c o n t a i n i n g 5 mg o f protein yielded as m u c h as 500 # g o f purified 150 k D a protein, in o r d e r to check the purity a n d estimate the m o l e c u l a r weight, the purified p r o t e i n was run on 7.5% SDS-polyacrylamide gels along with high m o l e c u l a r weight m a r k e r s and /3-subunit o f E. coli R N A p o l y m e r a s e . As seen in Fig. 7, the e l e c t r o e l u t e d protein gave a single b a n d migrating along with R N A p o l y m e r a s e /3-subunit confirming the m o l e c u l a r mass o f the p r o t e i n as 150 kDa.
Amino acid composition of the 150 kDa protein T h e a m i n o acid composition o f the p r o t e i n as c o m p a r e d to that o f the mitochondriai c a r b a m y l p h o s p h a t e
285 synthetase 1 is presented in Table I. in 150 kDa protein glutamic acid is the most predominant amino acid. The protein has no cysteine but has a small amount of methionine. N-terminal analysis revealed aspartic acid as the 1st amino acid and glutamic acid as the 2nd amino acid.
Discussion In this study we have identified a major liver-specific, developmentally regulated protein of 150 kDa. The protein constitutes as much as 8-10% of the liver protein. It appears to be more abundant than albumin, one of the most abundant proteins of liver. In spite of its abundance in the adult liver, our earlier studies indicated that 150 kDa protein cannot be labelled with [35S]methionine during 1 hour of incubation of liver cells with it [6]. This may suggest its long half-life similar to albumin, lmmunofluorescence studies confirmed the tissue specificity of the 150 kDa protein to liver, as none of the other tissues studied showcd bright fluorescence (Fig. 6). The faint fluorescence that was seen in pancreas could be due to .some nonspecific reaction. Since the antibody is polyclonal, it is possible that it interacts nonspecifieally with some other protein. Moreover, the pattern of fluorescence seen in pancreas appears amorphous whereas in adult liver it is very bright and distinct. We cannot, however, rule out the possibility that the 15(1 kDa protein is expressed at very low levels in the pancreas. Prealbumin, the precursor of albumin with a molecular weight of 105 kDa appears in fetal liver around the same time as 150 kDa protein does. In spite of this, 150 kDa protein cannot be a precursor of albumin because of the difference in molecular weight and the antibody to 150 kDa protein does not cross react with albumin (as there is no band in Fig. 4 or 5 at 67 kDa) nor does the antibody to albumin cross react with 150 kDa protein [6]. Amino acid composition and N-terminal amino acids of proteins that have similar developmental regulation and location were compared with 150 kDa protein (Table I). The results suggest that 150 kDa protein is a novel protein. Carbamyi phosphate synthetase 1 (CPS 1), a protein that has a molecular weight of 165 kDa, is specific to liver mitoehondria [14]. As shown in Table I, in 150 kDa protein, glutamic acid is the most predominent amino acid (13.5 mol%) followed by aspartic acid (10.9 mol%) and glycine (10.52 mol%) whereas in the mitochondrial CPS 1, aspartic acid appears to be the major amino acid (10.09 mol%) closely foliwed by glutamie acid (9..88 tool%) leueine (9.21 tool%) and alanine (8.28 mol%) (data for CPSI taken from Ref. 14). Comparison of the N-terminal two amino acids further confirmed the distinctness of 150 kDa protein.
TABLE I
Amh~o acid composition of the 150 kDa prolehl Amino acid
Aspartic acid Threonine Serene Glutamic acid Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine tlistidine Lysine
mol% 15(1 kDa
Carbamyl phosphate synthelase
10.9 6.3 7.7 13.5 10.52 9.4 8.3 1.5 6.0 9.6 2.5 4.8 2.3 6.9
10.08 5.8 I 6.54 9.88 7.81 8.28 7.88 2.53 6.2 I 9.2 I 2.34 3.8(I 2.0(I 6.94
*
* Data taken from Clarke [14].
The 1st and 2nd N-terminal amino acids of ~5(1 kDa protein are aspartic acid and glutamic acid whereas in CPSI they are methionine and threonine. Moreover, CPSI constitutes 15 to 20% of the total mitochondrial protein whereas 150 kDa is much more abundant and constitutes 8-1[1% of the total liver protein. Glutamine synthetase, a cytosolic protein that is involved in the fixation of ammonium is expressed in almost all the tissues of the adult. The first two Nterminal amino acids of glutamine synthetase arc mcthionine and alanine as compared to aspartic acid and glutamic acid of 150 kDa protein. Based on these results it can be concluded that 150 kDa protein is distinct from all hitherto known developmentally regulated liver specific proteins. It should be interesting to stody the function of the 150 kDa protein in view of its tissue-specific expression, developmental regulation and its abundance in liver. It is also interesting that the 150 kDa protein is not a secreted protein and is deregulated and not expressed in a chemically induced rat liver tumor, Zajdela Ascitic Hepatoma. Understanding the function of 150 kDa protein may give us an insight into the mechanism of tumorigenesis. Experiments are under way to isolate the protein in its native form and study its function.
Acknowledgements We thank Dr. M.R. Das for his encouragement and useful discussion we had while the work was in progress. We also thank Dr. P.D. Gupta for helping us in immunofluorescence studies and Mr. M.V. Jagannadham for helping us in amino acid analysis and Mrs. C. Subbalakshmi for the N-terminal analysis.
286 References 1 Davidson, E. (197~) Gene activity during early development, pp. 8-12. Academic Press. New York. 2 Hastie. N.D. and Bishop. J.O. (1976) Cell 9. 761-774. 3 Adamson, E.D. (1986) in Experimental approaches to mammalian development (Rossant, J. and Pederson. R.A., cds.), pp. 321-364. Cambridge University Press, Cambridge. 4 Sellum, C.H:, Frain. M., Erdos, T. and Sala Trepat, J.M. 119841 Devel. Biol. 102. 51-60. 5 Papa Constantinou, J., Rahek, J.P. and Dong-Er, Z. (1990) Develop. Growth and Differ. 32, 205-216. 6 Srinivas. U.K., Revathi. C.J. and Das. M.R. 119871 Mol. Cell Biol. 7, 4599-46112. 7 Zajdela, F. 11964)Colloque Frano-Sovietique (Quelques problems poses par la cellule cancereuse) p. 914. Gauthiers-Villars, Paris.
8 Laemmli, C.H.W., Hirs, S.N. and Timsheff, U.K. (1970) Nature 227, 6811-685. 9 Lowry, O.ti., Rosebrough, N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 10 Hunkapiller. M.W.. Lujan, E., Ostrander, F. and Hood, L.E. (19831 Methods Enzymol. 91,227-236. I I Kuhn, C.C. and Crabb. J.W. 11986) Modern manual microsequencing methods, in Advanced Methods in Protein Microsequence Analysis (Wil[man-Liebold, B., Salnikov, J. and Erdmann, V.A., eds.), pp. 64-76, Springer-Vcrlag, Berlin. 12 Ouchterlony, O, and Nilson, L.A. (1978) lmmunodiffusion and immunoelectrophoresis, Handbook of Experimental Immunology Vol. I, lmmunochemistry, 3rd Edn., (Wein, D.M., ed.) pp. 19.119.44, Blackwell Scientific Publication, Oxford. 13 Towbin, H., Stachelin, T. and Gordon, J. (19791 Proc. Natl. Acad. of Sci. USA 76, 4350-4354. 14 Clarke, S. 11976)J. Biol. Chem. 251,950-961.
281
BBAPRO 34128
Characterisation of a high-molecular-weight developmentally regulated adult rat liver-specific protein Usha K. Srinivas and C.J. Revathi ('~wtre ]'or Celluh#r and Mok'~'tthtr Bioh~A~.'. H t'~h'nthad (bulia)
(Received 18 September lqtJl)
Key wt~rds: Developmentalregulation; Liver pr~tcin: Protein characterization; (Rat liver) A major, hitherto unknown protein which is shown to be developmentally regulated has been isolated from rat liver. The protein has a molecular mass of 150 kDa and constitutes 8-10% of the adult liver protein, lmmunoblot experiments and immunofluorescence studies established its presence specifically in adult liver cells. This protein is not present in early embryonic liver cells, gets induced around day 18 of gestation and reaches near adult levels by ! day after birth and is not found in the cells of an ascitic hepatoma. Unlike albumin and a-fetoprotein, the two well known developmentally regulated liver proteins, the 150 kDa protein is not secreted. Some of the characteristics of this liver.specific protein are discussed.
Introduction
Materials and Methods
During animal development a pattern of differential gene expression is established resulting in cell populations that arc distinguishable from onc another [1]. Tissue-specific expression of genes coding for abundant proteins that are characteristic of differentiated cell type has been reported [2,3]. Rat liver providcs an excellent system for studying developmentally regulated tissue-specific expression of genes as many physiological, hormonal as well as morphological changes occur in liver during development, aFetoprotein and serum albumin, the two known major proteins characteristic of hepatic differentiation are secreted proteins and are differentially regulated during rat liver development [4,5]. In the course of our studies on the effect of heat shock on hepatic development [6], we have identified a hitherto unknown, developmentally regulated, high-molecular-weight, adultliver-specific protein which constitutes approx. 10% of the total liver protein. Tissue distribution, developmental regulation, amino acid coml~sition and N-terminal analysis indicate that the 150 kDa protein is a novel liver-specific protein which has not been isolated so far.
Materials Wistar rats and rat embryos of different developmental stages were used in this study. The rat livcr tumor, Zajdcla Ascitic Hcpatoma [7], was grown in the peritonial cavity of the animals by serial transplantation. All the chcmicals including FITC conjugated goat anti-rabbit lgG were obtained from Sigma (St Louis, MO, U.S.A.). Protein molecular weight markers were obtained from Pharmacia Fine Chemicals.
Preparation o]" tissue homogenates Small pieces of tissue or cells were suspended in 10 vols. of cold phosphate buffered saline (phosphatc, 2 mM (pH 7.2]: NaCi, 0.2 M: KCI, 2.5 mM) containing 0.1% NP40, I mM PMSF, 2 ~ g / m l trypsin inhibitor and homogenised in the cold using a polytron homogeniser. Samples were made with 3 × sample buffer as described by Lacmmli [8], (62.5 mM Tris (pH 6.8'1: 2.0% sodium dodecyl sulfate (SDS): 3.11%/~-mcrcaptoethanol: I|.t)1)1% bromophenol blue) before further analysis.
Protein attalysis
Correspondence: U.K. Srinivas. Centre fi~r Cellular and Molecular Biology, Hyderabad 5111111117.India.
Protein concentration of different homogenates was determined according to Lowry [9] and the protein concentration in all the samples was adjusted to 5 mg/ml. 100 # g of each sample was loaded on 7.5%
282 gels and SDS-PAGE was carried out as described by Laemmli [8]. After electrophoresis, gels were fixed and stained with Coomassie blue R-250, destained and photographed.
Purification of 150 kDa protein Taking advantage of the fact that the 150 kDa protein is a major constituent of adult liver, electroelution was used to purify the protein. Total protein of adult rat liver was run on 7.5% SDS-polyacrylamide gels, the gel portion corresponding to the 150 kDa protein was cut out, made into small pieces and subjected to electroelution as described by Hunkapiller et al. [10].
Ambm acM composition and N-termh~al analysis Amino acid composition of the electroeluted protein was determined by hydrolysing the protein with 6 M HCI under vacuum at 105°C for 24 h and analysing the hydrolysate on an LKB Alpha plus analyser. N-terminal analysis of 150 kDa protein was determined by the phenyl isothiocyanate procedure of Kuhn and Crabb [11] using a 1090 Lusi and Novapak Columns.
Antibodies to the 150 kDa protehl Antibodies to the 150 kDa protein were raised in rabbits by injecting 1 mg of protein along with complete adjuvant initially, and giving booster injections of 0.5 mg of protein in incomplete adjuvant at 1 week intervals. All the injections were given intradermally. The antisera were tested by Ouchterlony double diffusion method as described by Ouchterlony and Nilson [12]. Unfractionated antisera as well as the immunoglobulin fraction isolated on protein A Sepharose columns based on affinity were used in these studies.
mode. Adult liver sections incubated with 2nd antibody directly were included as controls. Results
Tissue specificity and devdopmental regulation of the 150 kDa protein Total proteins from different organs from the adult and from 14-15-day-old embryos of Wistar rats were analysed on 7.5% SDS-PAGE. In Figs. 1 and 2 Coomassie blue staining patterns are presented. A major protein band of 150 kDa was present in the adult liver (Fig. 1, lanes l and l0 and Fig. 2, lanes 3 and 12), but not in other adult or embryonic tissues or in the Z A H (Fig. l, lane I l). This protein could not be detected in the adult rat serum either, suggesting that it may not be a secreted protein (Fig. l, lane 12). These results indicate that the 150 kDa protein is adult rat liver specific. To study the developmental regulation of this 150 kDa protein, total liver proteins from l 1-, 13-, 14-, 15-, 18- and 20-day-old embryos, 1-day- and 1week-old pups and adult rat were analyzed by SDSPAGE. Results of this study, presented in Fig. 3, indicate that the 150 kDa proteins begins to appear in 18-day-old embryonic liver and reaches adult levels by day 1 after birth. The tissue-specific expression of the 150 kDa protein was further explored by immunoblot analysis of homogenates of various adult and embryonic tissues using antibody raised against electroeluted 150 kDa protein. Western blots of duplicate gels presented in Figs. 1 and 2 are presented in Figs. 4 and 5. Crossreactivity with antibody to 150 kDa was detected only in adult liver tissue (Fig. 4, lane 10 and Fig. 5, lane l), but
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lmmunoblotting Total protein (100 p.g) from different tissues was run on 7.5% SDS-PAGE and was transferred on to nitrocellulose paper in a Bio-Rad transfer apparatus [13l. The blot was incubated with antiserum to 150 kDa protein, washed and treated with iodinated protein A (iodination of protein A was done using iodogen as per suppliers instructions), again washed and exposed to X-ray film.
lmmunofluorescence Cryosections of embryonic and adult rat tissues, frozen immediately after excision from the animal, were incubated with a 1 : 100 dilution of antiserum to the 150 kDa protein for 1 h at room temperature. The sections were washed and further incubated with a 1:100 dilution of FITC conjugated goat anti-rabbit IgG, washed and examined in polyvar (Richeit) fluorescence microscope using B4 filters and epifluorescence
Fig. 1. SDS-PAGE profile of total protein from adult organs ot Wistar rats. Samples containing 100/zg of protein were separated on 7.5% SDS-PAGE and the gel was stained with Coomassie blue R-250 as described in Materials and Methods. Lane !, liver; 2, brain: 3, heart: 4, lung: 5. thymus; 6, spleen; 7, kidney; 8, intestine; 9. muscle: 10, liver: II, ZAH; 12, rat serum; 13, high and low tool. wt. markers 330, 220, 94, 67, 43, 30 and 20. Arrow indicates 150 kDa protein.
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Fig. 2. S D S - P A G E profile of proteins from embryonic organs of 14-15-day-old embryos. 101) jag of protein was separated on 7.55,; gel and the gel was stained with Coomassie blue R-2511 as described in Materials and Methods. Lane I, R N A polymerase core enzyme mol. wt. 151) and 38 kDa and low mol. wl. markers (Pharmacia) ~94, 67 and 43: 2, RNA polymerase (core enzyme) 1511 and 38 kDa: 3, A. liver; 4, fetal intestine: 5, F. muscle: 6, F. kidney: 7, F. spleen: 8. F. lung: 9. F. heart: II), F. brain: I I, F. liver and 12, adult liver, arrow on the right indicates the 1511kDa pn)tcin band.
not in any other tissue, either adult or early embryonic. These results strongly indicate that 151) kDa protein is an adult liver-specific protein. As indicated earlier, 150 kDa protein cannot be a secreted protein as the serum did not contain any proteins (either of 150 kDa or lower) that cross reacted with antibody to 150 kDa (Fig. 4 lane 12). Absence of the protein (cithcr in Coomassie blue pattern or in immunoblot) from a liver tumor, Zajdela Ascitic Hepatoma (Fig. 1, lane 11 and Fig. 4, lane 12) suggests the disappearance of the protein upon transformation.
Localization of the 150 kDa protein Tissue-specific expression and localization of this protein were further confirmed by immunofluorescence studies. Brain, liver, lung, spleen, kidney and pituitary of adult rats and livers of 13- and 19-day-old embryos were processed as described in Materials and Methods. In Fig. 6 the fluorescence photographs of adult liver and pituitory embedded in adult liver tissue are presented. Bright fluorescence was seen localized in the cytoplasm around the nucleus of adult liver cells. No such bright fluorescence was observed in any of the other, adult or embryonic tissues, or in the control slides. Tissue sections from 19-day-old embryonic liver and pancreas showed faint fluorescence, but it was too
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Fig. 3. S D S - P A G E profile of embryonic, new born and adult liver proteins. Sample containing I1111 ttg of protein was run on 7.5~; SDS-PAGE, gels were stained with (k)omassie bluc R-2511 as described in Materials and Methods. Lane I, I I days: 2. 13 days: 3, 14 days: 4. 15 days: 5. 18 days; 6. 2It-day-old embD'onic liver: lane 7 I-day-old pup: 8. I-week-old pup: 0, adult liver (arn)ws indicates 1511 kDa protein and albumin; IlL Pharmacia low m()l. wl. markers ~)4, 67, 43, 31t and 211.
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Fig. 4. lmmunoblot analysis of proteins from adult rat tissues: gels were run as described in Fig. 1, proteins were transferred onto nitrocellulose paper and processed for immunobJotting analysis as described in Materials and Methods. Lane 1, brain: 2. heart" 3, lung; 4, thymus; 5, spleen: fl, kidney: 7. intestine; 8, muscle: 9, pancreas: Irk liver; 11, Z A [ t : and 12, serum. Note: crossreuctivity with the antibudy could he detected only in adult liver hme but in no olher lane including the liver tumor Z A H and rat serum (hines I I and 12).
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3
4
5
6
7
8
9
!
2
3
4
5
6
N
Fig. 5. lmmtmoblot analysis of proteins from embryonic organs: Samples ~ere run on 7.5c; SDS-PAGE. transferred onto nitrocellulose paper and processed fi)r immunoblot analysis as described in Materials and Methods. Lane I. adult liver: 2, fetal liver: 3, F. brain; 4. F. heart: 5. F. lung: 6. F. spleen: 7, F. kidney: I-;,.F. muscle: and tL F. intestine.
Fig. 7. Determination of the molecular weight of the protein by SDS-PAGE: proteins were run on 7.5% gels and stained with Coomassie blue. Lane 1, high-molecular-weight markers (Pharmacia): 2. RNA polymerase core enzyme; 3. 150 kDa protein 15 ~g: 4. I11 ,ttg; 5, 5 #g; and 2.11~g.
faint to be p h o t o g r a p h e d . T h e f l u o r e s c e n c e seen in 19 day e m b r y o n i c liver is probably due to the induction o f this protein at that stage as seen in Fig. 3. T h e i m m u n o f l u o r e s c e n c e studies f u r t h e r c o n f i r m the tissue specific expression and d e v e l o p m e n t a l regulation o f 150 k D a protein.
Purification attd physical properties of 150 kDa protein
!
!:
Fig. 6. pr~iiein bY immun0flut,rescence. Cryosections of adult liver (A) and pituitary embedded in adult liver (B) were treated with antiserum to the 151) kDa protein and then with FITC-conjugated goat-antirabbit IgG, washed and observed under fluorescence microscope as described in Materials and Methods. Bar represents cme micrtm 150|}11× magnification).
As described in m e t h o d s , total p r o t e i n from liver was e [ e c t r o p h o r e s e d on 7.5% S D S - p o l y a c r y l a m i d e gel and the b a n d c o r r e s p o n d i n g to 150 k D a protein was cut out and electroeluted from the gel. Liver hom o g e n a t e c o n t a i n i n g 5 mg o f protein yielded as m u c h as 500 # g o f purified 150 k D a protein, in o r d e r to check the purity a n d estimate the m o l e c u l a r weight, the purified p r o t e i n was run on 7.5% SDS-polyacrylamide gels along with high m o l e c u l a r weight m a r k e r s and /3-subunit o f E. coli R N A p o l y m e r a s e . As seen in Fig. 7, the e l e c t r o e l u t e d protein gave a single b a n d migrating along with R N A p o l y m e r a s e /3-subunit confirming the m o l e c u l a r mass o f the p r o t e i n as 150 kDa.
Amino acid composition of the 150 kDa protein T h e a m i n o acid composition o f the p r o t e i n as c o m p a r e d to that o f the mitochondriai c a r b a m y l p h o s p h a t e
285 synthetase 1 is presented in Table I. in 150 kDa protein glutamic acid is the most predominant amino acid. The protein has no cysteine but has a small amount of methionine. N-terminal analysis revealed aspartic acid as the 1st amino acid and glutamic acid as the 2nd amino acid.
Discussion In this study we have identified a major liver-specific, developmentally regulated protein of 150 kDa. The protein constitutes as much as 8-10% of the liver protein. It appears to be more abundant than albumin, one of the most abundant proteins of liver. In spite of its abundance in the adult liver, our earlier studies indicated that 150 kDa protein cannot be labelled with [35S]methionine during 1 hour of incubation of liver cells with it [6]. This may suggest its long half-life similar to albumin, lmmunofluorescence studies confirmed the tissue specificity of the 150 kDa protein to liver, as none of the other tissues studied showcd bright fluorescence (Fig. 6). The faint fluorescence that was seen in pancreas could be due to .some nonspecific reaction. Since the antibody is polyclonal, it is possible that it interacts nonspecifieally with some other protein. Moreover, the pattern of fluorescence seen in pancreas appears amorphous whereas in adult liver it is very bright and distinct. We cannot, however, rule out the possibility that the 15(1 kDa protein is expressed at very low levels in the pancreas. Prealbumin, the precursor of albumin with a molecular weight of 105 kDa appears in fetal liver around the same time as 150 kDa protein does. In spite of this, 150 kDa protein cannot be a precursor of albumin because of the difference in molecular weight and the antibody to 150 kDa protein does not cross react with albumin (as there is no band in Fig. 4 or 5 at 67 kDa) nor does the antibody to albumin cross react with 150 kDa protein [6]. Amino acid composition and N-terminal amino acids of proteins that have similar developmental regulation and location were compared with 150 kDa protein (Table I). The results suggest that 150 kDa protein is a novel protein. Carbamyi phosphate synthetase 1 (CPS 1), a protein that has a molecular weight of 165 kDa, is specific to liver mitoehondria [14]. As shown in Table I, in 150 kDa protein, glutamic acid is the most predominent amino acid (13.5 mol%) followed by aspartic acid (10.9 mol%) and glycine (10.52 mol%) whereas in the mitochondrial CPS 1, aspartic acid appears to be the major amino acid (10.09 mol%) closely foliwed by glutamie acid (9..88 tool%) leueine (9.21 tool%) and alanine (8.28 mol%) (data for CPSI taken from Ref. 14). Comparison of the N-terminal two amino acids further confirmed the distinctness of 150 kDa protein.
TABLE I
Amh~o acid composition of the 150 kDa prolehl Amino acid
Aspartic acid Threonine Serene Glutamic acid Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine tlistidine Lysine
mol% 15(1 kDa
Carbamyl phosphate synthelase
10.9 6.3 7.7 13.5 10.52 9.4 8.3 1.5 6.0 9.6 2.5 4.8 2.3 6.9
10.08 5.8 I 6.54 9.88 7.81 8.28 7.88 2.53 6.2 I 9.2 I 2.34 3.8(I 2.0(I 6.94
*
* Data taken from Clarke [14].
The 1st and 2nd N-terminal amino acids of ~5(1 kDa protein are aspartic acid and glutamic acid whereas in CPSI they are methionine and threonine. Moreover, CPSI constitutes 15 to 20% of the total mitochondrial protein whereas 150 kDa is much more abundant and constitutes 8-1[1% of the total liver protein. Glutamine synthetase, a cytosolic protein that is involved in the fixation of ammonium is expressed in almost all the tissues of the adult. The first two Nterminal amino acids of glutamine synthetase arc mcthionine and alanine as compared to aspartic acid and glutamic acid of 150 kDa protein. Based on these results it can be concluded that 150 kDa protein is distinct from all hitherto known developmentally regulated liver specific proteins. It should be interesting to stody the function of the 150 kDa protein in view of its tissue-specific expression, developmental regulation and its abundance in liver. It is also interesting that the 150 kDa protein is not a secreted protein and is deregulated and not expressed in a chemically induced rat liver tumor, Zajdela Ascitic Hepatoma. Understanding the function of 150 kDa protein may give us an insight into the mechanism of tumorigenesis. Experiments are under way to isolate the protein in its native form and study its function.
Acknowledgements We thank Dr. M.R. Das for his encouragement and useful discussion we had while the work was in progress. We also thank Dr. P.D. Gupta for helping us in immunofluorescence studies and Mr. M.V. Jagannadham for helping us in amino acid analysis and Mrs. C. Subbalakshmi for the N-terminal analysis.
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