ISOLATION A N D P A R T I A L P U R I F I C A T I O N OF C E R U L O P L A S M I N M E S S E N G E R RNA FROM RAT LIVER
S.A. NEIFAKH0V.S. GAITSKHOKI,N.A. KLIMOV,L.V. PUCHKOVA. M.M. SHAVLOVSKI,and A.L. SCHWARTZMAN Laboratory of Biochemical Genetics of the Institute of Experimental Medicine, Leningrad, U.S.S.R.
(Received 29 November, 1976) Abstract. Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1• 106 daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cellfree translation had molecular weights in the range 4.5-5.4X 104 daltons as estimated by gelelectrophoresis under denaturating conditions. These values approach the weight of the halfmolecule of native ceruloplasmin.
I. INTRODUCTION Ceruloplasmin (CP)* is a copper-containing glycoprotein of the o~2-globulin fraction of human and other mammalian plasma proteins [1-4]. CP molecular structure is not completely understood. It is known that the molecular weight of human CP is about 130 000 and the protein consists of either two equal size subunits [5, 6] or even a single polypeptide chain [7]. CP synthesis is a liver-specific process [ 1, 2]. The study of the synthesis of this protein and the mechanisms controlling different stages of CP gene expression is of interest in view of the fact that hepatolenticular degeneration, a human hereditary disease, is known to be a result of the deficiency of CP synthesis. This defect is apparently based on the mutation in either a CP structural gene or some other genes involved in the control of the CP synthesis [1,8, 9]. The isolation and cell-free translation of CP messenger RNA seems to be a valuable approach to these unsolved problems. We report here our data on the isolation of partially purified preparations of CP mRNA from rat liver.
* Unusual abbreviations: CP - ceruloplasmin,TCA - trichloroaceticacid. 235 Molecular Biology Reports 3 (1977) 235-242. All Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
II. MATERIALS ANDMETHODS Rabbits were immunized with homogeneous CP preparations isolated from rat serum as described earlier [ 10]. The immunoglobulin fraction from the sera of immunized animals was monospecific to CP in agar gel immunodiffusion tests. Commercially available preparations of anti-rabbit donkey immunoglobulin served as the 'second' antibodies. Total polysome preparations were isolated from rat liver. The tissue was homogenized in 0.25 M sucrose containing 25 mM potassium chloride, 5 mM magnesium chloride, 0.05 m trisHC1, pH 7.6 and 50/ag rn1-1 heparin. The postmitochondrial supematant was lysed with 2% Triton X- 100. Polysomes were precipitated with 100 mM magnesium chloride [ 11] and then pelleted through 2 M sucrose (39 000 rpm, 18 hr, Spinco rotor Ti 42). CP polysomes were obtained from the total polysome preparations by means of indirect immunoprecipitation [ 12, 13]. 50 A26 o-units m1-1 polysomes were incubated with 100/~g ml-~ rabbit anti-CP immunoglobulin for 1 hr at 37 ~ and for 6 - 8 hr at 4 ~ Then the 'second' antibodies (200/ag mY 1) were added and the mixture was incubated again at 37 ~ for one hour and at 4 ~ for 6 hr. The immunoprecipitate formed during the incubation was collected by centrifugation at 6000 rpm for 40 min. Total high molecular weight polysomal RNA was isolated with 4 M urea-4 M lithium chloride according to Rhoads [14] and then desalted by 3 - 4 cycles of ethanol precipitation. Poly(A)-containing RNA was isolated using poly(U)-Sepharose columns according to Lindberg and Persson [ 15 ]. RNA preparations were iodinated with potassium [12 s I]-iodide as reported earlier [ 16]. Poly(U) hybridization with subsequent hydroxyapatite fractionation [17] was used for the analytical estimation of the proportions of poly(A)-containing RNA in the preparations. Electrophoresis of RNA was performed in 2.7% polyacrylamide gels containing 1% sodium dodecyl sulfate in the buffer system described by Loening [ 18]. The methylene blue-stained gels were traced at 580 nm in the densitometric device adapted to a Hitachi-356 spectrophotometer. Total polysomal RNA [14] served as a source of molecular weight markers (28 S and 18 S ribosomal RNAs). The cell-free submitochondrial system described earlier [ 19 ] was used for the translation of the RNA preparations. Mitochondria were isolated from the livers of fasting rats. The newly formed CP polypeptides were isolated with the aid of indirect immunoprecipitation. The molecular weight distribution of the immunoprecipitable radioactive material was analyzed by gel electrophoresis under denaturing conditions [20] using 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. The molecular weight markers ('Serva') were run in parallel tubes.
Reagents: Poly(U)-Sepharose (Pharmacia, Sweden), heparin, poly(U), ATP, GTP, phosphoenol pyruvate, pyruvate kinase (Sigma, U.S.A.), 14C.amino acid mixture (sp. act. 45 mCi/matom C, Amersham, England), K 12 s I (carrier-free, Isotop, U.S.S.R.), donkey anti-rabbit immunoglobulins (N. F. Gamaleya Institute for Epidemiology and Microbiology, Moscow, U.S.S.R.). III. RESULTS AND DISCUSSION The data of a series of experiments on the quantitation of immunoprecipitated CP polysomes and the determination of the poly(U)-retained poly(A)-containing RNA in the precipitated material are summarized in the Table I. It is seen that CP-synthesizing polysomes contain about 236
TABLE I: Yield of CP-synthesizing polysomes and CP mRNA from the total polysomal preparations of rat liver No. Expt.
Proportion of CP polysomes,% A26o a
RNA b"
1
2.1
0.7 c
2 3 4 5
2.6 3.2 3.3 1.6
0.75 0.72 0.66 0.56
Proportion of poly(A)-containing RNA in CP polysomes, % d 0.7 0.6 0.3 -
A260 of immunoprecipitate A260 of total polysomes b RNA from CP polysomes
x 100.
x 100. RNA from total polysomes The amount of RNA in CP polysomeswas measured after deproteinization.The RNA content in the total polysomeswas calculatedassumingthat I A260 unit of polysomalmaterial corresponds to 50/dg of polysomalRNA. c The differencebetween values in these columnsseems to be due to significantcontribution of immunoglobulins to the measured absorption of the irnmunoprecipitateat 260 rim. d poly(U)-Sepharoseretained RNA x 100. total RNA
0.5-0.7% of the total polysomal RNA from the rat liver. These data are consistent with the results of our previous determinations on the proportion of CP polysomes in liver biopsy specimens from patients with non-hereditary diseases [9]. The proportion of poly(A)-containing RNA was 0.3-0.7% of the total high molecular weight polysomal RNA recovered from the immunoprecipitate. After in vitro [12 s I]-labelling the poly(A)-containing RNA from CP polysomes annealed almost quantitatively with poly(U) (Figure 1). Thus, these RNA preparations were not contaminated with measurable amounts of ribosomal RNA and other poly(A)-free RNAs. Electrophoretic analysis showed that poly(A)-containing RNA from immunoprecipitated polysomes migrated as a homogeneous major component between 28 S and 18 S ribosomal RNA markers (Figure 2A). The radioactivity peak of [12 s I]-RNA followed closely the peak of the material stained with methylene blue (Figure 2B). The densitometric recording revealed also a minor, somewhat lighter, component. It could be suggested therefore that CP mRNA preparations isolated from immunoprecipitated polysomes are represented by two mRNA species of similar though not identical molecular weights. Minor contamination with other (nonCP) mRNAs cannot be excluded. The asymmetric distribution of [12 s I]-RNA in the gels with a shoulder of the same mobility as the minor peak in the stained gels is also suggestive for the presence of a minor component differing in electrophoretic mobility from the major RNA fraction. The apparent molecular weight of the major poly(A)-containing RNA from CP polysomes is as high as 1.0• 106 daltons assuming an inverse relationship between the rates of RNA migration and the logarithms of their molecular weights. 237
0,5 M
o,25 u I0
I0
20
3O
40
Fig. 1. Poly(U) hybridization of poly(A)-containing RNA from immunoprecipitated polyribosomes. [125i].labelle d RNA (30 000 cpm) was incubated with 100 #g m1-1 poly(U) in 1 M sodium chloride - 10 mM sodium phosphate buffer, pH 6.8 - 0.1% sodium dodecyl sulfate for 1 hr at 45 ~ The incubated samples were diluted 10 times with 0.2 M sodium chloride - 0.01 M phosphate buffer, pH 6.8 - 0.1% sodium dodecyl sulfate and chromatographed on hydroxyapatite columns IX 1.5 cm at 45 ~ [17]. Abscissa - number of eluate fractions (1.5 ml each). Ordinate - [ 1 2 5 I] radioactivity, cpm• 10-3 . The arrows indicate the change in the molarity of the eluting phosphate buffer, e - - e - $ RNA incubated without poly(U), o-o--o RNA annealed with poly(U). Cell-free translation of various RNA preparations from rat liver polysomes in the submitochondrial system was performed in order to identify CP-mRNA functionally. The results of these studies (Table II) demonstrated that non-fractionated RNA from precipitated polysomes is three times enriched (in the terms of specific template activity) in CP mRNA content as compared to the total polysomal RNA preparation from rat liver. Further enrichment (more than tenfold) was achieved when poly(A)-containing RNA was isolated from CP polysomes. On the other hand, poly(A)-free RNA from these polysomes was characterized by very low specific template activity in the cell-free CP synthesis. The electrophoretic analysis of radioactive newly formed polypeptides recovered from the immunoprecipitates (Figure 3) showed a rather broad distribution in sodium dodecyl sulfate-containing gels. Two unresolved peaks were observed at 238
28S
ISS
02
t0
o,,
,
iO
I
I
20
30
B
~0
Fig. 2. Polyacrylamide gel electrophoresis of poly(A)-containing RNA from immunoprecipitated polysomes. Electrophoresis was performed in 2.7% polyacrylamide gel containing 1% sodium dodecyl sulfate. A. Photographs of the gels stained with methylene blue. 1. Total RNA from polysomes (100 #g tube-1). 2. Poly(A)-containing RNA from immunoprecipitated polysomes (15 #g tube-1). B. Densitometric tracing of the gel A-I and the distribution of [125i].labelle d poly(A)-containing RNA from CP polysomes. Abscissa: Slice number. Ordinate: Asso (right, - -- ) and radioactivity of 1 2 5 I-RNA, cpm• 10-2 (left, 0__0_@). The arrows indicate the position of markers (28 S and 18 S ribosomal RNAs from rat liver cytoplasm).
positions corresponding to the apparent molecular weights of 54 000 and 45 000, respectively. The electrophoretic distribution of CP immunoreactive material from the samples containing the total RNA of CP polysomes (Figure 3A) and its poly(A)-containing fraction (Figure 3B) was similar. The molecular size heterogeneity of the radioactive polypeptides could be a result 239
TABLE II: Synthesis of immunoreactive CP in the submitochondrial system programmed with various preparations of polysomal RNA from rat liver C.amin o acid incorporation, cpm/mg RNA 14
Enrichment factor
RNA preparation
Total polysomal RNA Total RNA from CP polysomes Poly(A).containing RNA from CP polysomes Poly(A)-free RNA from CP polysomes
TCA-insoluble material
immunoprecipitate
350 000 280 000 1 400 000
2 500 8 250 100 000
250 000
4 000
x 1.0 x 3.3 x 40.0 x
1.6
of contribution of polysome-bound uncompleted CP chains because the immunoprecipitation of CP polypeptides was performed directly from incubation mixtures without removal of polysomes. The existence of two electrophoretic components (mol. wts 45 000 and 54 000) is not documented convincingly enough to identify them as two CP subunits of somewhat different molecular weights. These apparent molecular weight values are at least twice less than the molecular weight of circulating CP (130 000). On the other hand, they approach rather closely the molecular weight of a CP half-molecule (60 000-65 000), although being apparently smaller. This difference might well be due to the failure of heterologous cell-free system to perform posttranslational modifications, such as glycosylation and copper incorporation into CP chains. The majority of molecular weight estimations mentioned above were made in studies on human CP preparations. However, previous investigations from our laboratory [4] showed that there is no significant difference between human and rat CPs with respect to their molecular sizes. Therefore, the data obtained do not contradict the suggestion concerning the existence of two subunits in the CP molecule [5]. The data reported demonstrate the isolation of translatable CP mRNA which appeared to be functionally enriched more than 40 times and highly purified according to electrophoresis under conditions of mild denaturation. The calculated value of the apparent molecular weight of CP mRNA is as high as 1.0X 1 0 6 , i.e. its molecule is long enough to code for a putative CP subunit with a molecular weight of 60 000-65 000. If these calculations are correct, the CP mRNA seems to contain, besides the coding sequence, extra-sequences constituting about 25-30% of its total molecular length. One of these non-coding sequences is poly(A) whose existence was demonstrated in our experiments described above. The isolation of highly enriched CP mRNA preparations functionally active in cell-free translation experiments seems to be a prerequisite for the study of inherited defects of CP synthesis in patients with hepatolenticular degeneration.
240
1000 qO0
B
800 700 ~00 500 qOD 5~
200 IO0
900
800 700
A
600 5O0 400
I00 0
m
Fig. 3. Electrophoretic analysis of the products of cell-free translation of CP-mRNA in the submitochondrial system. The preparation of the system and incubation conditions are described elsewhere [19]. The newly formed CP polypeptides were isolated by indirect immunoprecipitation. Eleetrophoresis was performed in 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate [20]. The radioactivity of gel slices (1.5 mm thick) was measured using 'Unisolv' mixture in a 'Nuclear Chicago', Mark II scintillation counter. Abscissa: Shce number. Ordinate: 14 C radioactivity, cpm. A. Immunoprecipitated product of the translation of the non-fractionated RNA from CP polyribosomes. B. lmmunoprecipitated product of the translation of poly(A)-containing RNA from CP polysomes. Serum albumin (mol.wt 68 000), catalase (mol.wt 60 000), ovalbumin (mol.wt 43 000) and cytochrome c (mol.wt 12 400) were used as markers in the calculations of the molecular weight of CP polypeptides. ACKNOWLEDGEMENT The w o r k was s u p p o r t e d b y the grant f r o m the World Health Organization ( A g r e e m e n t No. G3/181/38).
241
REFERENCES 1. Neifakh, S.A., Monakhov, N.K., Shaposhnikov, A.M., and Zubzhitski, Yu. N., Experienna 25,337-344 (1969). 2. Shaposhnikov, A.M., Zubzhitski, Yu. N., and Shulman, V.S., Experientia 25,424-426 (1969). 3. Mukasa, H., Hajiyama, S., Sugiyama, K., Itoh, M., Nosoh, G., and Sato, T., Biochim. Biophys. Acta 168, 132-141 (!968). 4. Vassiletz, I.M., Shavlovski, M.M., and Mukha, G.V., Biokhimiya (Russ.) 35, 1139-1146 (1970). 5. Vassiletz, I.M., Kushner, V.P., Moshkov, K.A., and Neifakh, S.A., Doklady Akademii Nauk S.S.S.R. (Russ.) 208,729-732 (1973). 6. Shokeir, M.H.K., Clin. Biochem. 6, 9-14 (1973). 7. Ryden L.,Eur. J. Biochem. 26,380-386 (1972). 8. Neifakh, S.A., Vassiletz, I.M., and Shavlovski, M.M., Biochem. Genet. 6,231-238 (1972). 9. Gaitskhoki, V.S., Kisselev, O.I., Moshkov, K.A., Puchkova, L.V., Shavlovski, M.M., Shulman, V.S., Vakharlovski, V.G., and Neifakh, S.A., Biochem. Genet. 13, 533-550 (1975). 10. Broman, L., Acta Soc. Med. Upsal 69, Suppl. 7 (1964). 11. Palmiter, R., Biochemistry 13, 3606-3615 (1974). 12. Schechter, I.,Biochemistry 13, 1875-1885 (1974). 13. Boyer, S., Smith, K.D., and Noyes, A.,Ann. N. Y. Acad. Sci. 241,204-222 (1974). 14. Rhoads, R.E.,J. Biol. Chem. 250, 8088-8097 (1975). 15. Lindberg, U. and Persson, T.,Eur. J. Biochem. 31,246-254 (1972). 16. Scherberg, S. and Refetoff, S., Nature New BioL 242, 142-144 (1973). 17. Greenberg, J. and Perry, R.,J. Mol. Biol. 72, 91-98 (1972). 18. Loening, U.E.,Biochem. J. 113, 131-138 (1969). 19. Kisselev, O.I., Gaitskhoki, V.S., and Neifakh, S.A., Mol. Cell. Biochem., in press. 20. Rustum, Y.M., Massaro, E.J., and Bamard, E.A.,Biochemistry 10, 3509-3516 (1971).
242
S.A. NEIFAKH0V.S. GAITSKHOKI,N.A. KLIMOV,L.V. PUCHKOVA. M.M. SHAVLOVSKI,and A.L. SCHWARTZMAN Laboratory of Biochemical Genetics of the Institute of Experimental Medicine, Leningrad, U.S.S.R.
(Received 29 November, 1976) Abstract. Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1• 106 daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cellfree translation had molecular weights in the range 4.5-5.4X 104 daltons as estimated by gelelectrophoresis under denaturating conditions. These values approach the weight of the halfmolecule of native ceruloplasmin.
I. INTRODUCTION Ceruloplasmin (CP)* is a copper-containing glycoprotein of the o~2-globulin fraction of human and other mammalian plasma proteins [1-4]. CP molecular structure is not completely understood. It is known that the molecular weight of human CP is about 130 000 and the protein consists of either two equal size subunits [5, 6] or even a single polypeptide chain [7]. CP synthesis is a liver-specific process [ 1, 2]. The study of the synthesis of this protein and the mechanisms controlling different stages of CP gene expression is of interest in view of the fact that hepatolenticular degeneration, a human hereditary disease, is known to be a result of the deficiency of CP synthesis. This defect is apparently based on the mutation in either a CP structural gene or some other genes involved in the control of the CP synthesis [1,8, 9]. The isolation and cell-free translation of CP messenger RNA seems to be a valuable approach to these unsolved problems. We report here our data on the isolation of partially purified preparations of CP mRNA from rat liver.
* Unusual abbreviations: CP - ceruloplasmin,TCA - trichloroaceticacid. 235 Molecular Biology Reports 3 (1977) 235-242. All Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
II. MATERIALS ANDMETHODS Rabbits were immunized with homogeneous CP preparations isolated from rat serum as described earlier [ 10]. The immunoglobulin fraction from the sera of immunized animals was monospecific to CP in agar gel immunodiffusion tests. Commercially available preparations of anti-rabbit donkey immunoglobulin served as the 'second' antibodies. Total polysome preparations were isolated from rat liver. The tissue was homogenized in 0.25 M sucrose containing 25 mM potassium chloride, 5 mM magnesium chloride, 0.05 m trisHC1, pH 7.6 and 50/ag rn1-1 heparin. The postmitochondrial supematant was lysed with 2% Triton X- 100. Polysomes were precipitated with 100 mM magnesium chloride [ 11] and then pelleted through 2 M sucrose (39 000 rpm, 18 hr, Spinco rotor Ti 42). CP polysomes were obtained from the total polysome preparations by means of indirect immunoprecipitation [ 12, 13]. 50 A26 o-units m1-1 polysomes were incubated with 100/~g ml-~ rabbit anti-CP immunoglobulin for 1 hr at 37 ~ and for 6 - 8 hr at 4 ~ Then the 'second' antibodies (200/ag mY 1) were added and the mixture was incubated again at 37 ~ for one hour and at 4 ~ for 6 hr. The immunoprecipitate formed during the incubation was collected by centrifugation at 6000 rpm for 40 min. Total high molecular weight polysomal RNA was isolated with 4 M urea-4 M lithium chloride according to Rhoads [14] and then desalted by 3 - 4 cycles of ethanol precipitation. Poly(A)-containing RNA was isolated using poly(U)-Sepharose columns according to Lindberg and Persson [ 15 ]. RNA preparations were iodinated with potassium [12 s I]-iodide as reported earlier [ 16]. Poly(U) hybridization with subsequent hydroxyapatite fractionation [17] was used for the analytical estimation of the proportions of poly(A)-containing RNA in the preparations. Electrophoresis of RNA was performed in 2.7% polyacrylamide gels containing 1% sodium dodecyl sulfate in the buffer system described by Loening [ 18]. The methylene blue-stained gels were traced at 580 nm in the densitometric device adapted to a Hitachi-356 spectrophotometer. Total polysomal RNA [14] served as a source of molecular weight markers (28 S and 18 S ribosomal RNAs). The cell-free submitochondrial system described earlier [ 19 ] was used for the translation of the RNA preparations. Mitochondria were isolated from the livers of fasting rats. The newly formed CP polypeptides were isolated with the aid of indirect immunoprecipitation. The molecular weight distribution of the immunoprecipitable radioactive material was analyzed by gel electrophoresis under denaturing conditions [20] using 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. The molecular weight markers ('Serva') were run in parallel tubes.
Reagents: Poly(U)-Sepharose (Pharmacia, Sweden), heparin, poly(U), ATP, GTP, phosphoenol pyruvate, pyruvate kinase (Sigma, U.S.A.), 14C.amino acid mixture (sp. act. 45 mCi/matom C, Amersham, England), K 12 s I (carrier-free, Isotop, U.S.S.R.), donkey anti-rabbit immunoglobulins (N. F. Gamaleya Institute for Epidemiology and Microbiology, Moscow, U.S.S.R.). III. RESULTS AND DISCUSSION The data of a series of experiments on the quantitation of immunoprecipitated CP polysomes and the determination of the poly(U)-retained poly(A)-containing RNA in the precipitated material are summarized in the Table I. It is seen that CP-synthesizing polysomes contain about 236
TABLE I: Yield of CP-synthesizing polysomes and CP mRNA from the total polysomal preparations of rat liver No. Expt.
Proportion of CP polysomes,% A26o a
RNA b"
1
2.1
0.7 c
2 3 4 5
2.6 3.2 3.3 1.6
0.75 0.72 0.66 0.56
Proportion of poly(A)-containing RNA in CP polysomes, % d 0.7 0.6 0.3 -
A260 of immunoprecipitate A260 of total polysomes b RNA from CP polysomes
x 100.
x 100. RNA from total polysomes The amount of RNA in CP polysomeswas measured after deproteinization.The RNA content in the total polysomeswas calculatedassumingthat I A260 unit of polysomalmaterial corresponds to 50/dg of polysomalRNA. c The differencebetween values in these columnsseems to be due to significantcontribution of immunoglobulins to the measured absorption of the irnmunoprecipitateat 260 rim. d poly(U)-Sepharoseretained RNA x 100. total RNA
0.5-0.7% of the total polysomal RNA from the rat liver. These data are consistent with the results of our previous determinations on the proportion of CP polysomes in liver biopsy specimens from patients with non-hereditary diseases [9]. The proportion of poly(A)-containing RNA was 0.3-0.7% of the total high molecular weight polysomal RNA recovered from the immunoprecipitate. After in vitro [12 s I]-labelling the poly(A)-containing RNA from CP polysomes annealed almost quantitatively with poly(U) (Figure 1). Thus, these RNA preparations were not contaminated with measurable amounts of ribosomal RNA and other poly(A)-free RNAs. Electrophoretic analysis showed that poly(A)-containing RNA from immunoprecipitated polysomes migrated as a homogeneous major component between 28 S and 18 S ribosomal RNA markers (Figure 2A). The radioactivity peak of [12 s I]-RNA followed closely the peak of the material stained with methylene blue (Figure 2B). The densitometric recording revealed also a minor, somewhat lighter, component. It could be suggested therefore that CP mRNA preparations isolated from immunoprecipitated polysomes are represented by two mRNA species of similar though not identical molecular weights. Minor contamination with other (nonCP) mRNAs cannot be excluded. The asymmetric distribution of [12 s I]-RNA in the gels with a shoulder of the same mobility as the minor peak in the stained gels is also suggestive for the presence of a minor component differing in electrophoretic mobility from the major RNA fraction. The apparent molecular weight of the major poly(A)-containing RNA from CP polysomes is as high as 1.0• 106 daltons assuming an inverse relationship between the rates of RNA migration and the logarithms of their molecular weights. 237
0,5 M
o,25 u I0
I0
20
3O
40
Fig. 1. Poly(U) hybridization of poly(A)-containing RNA from immunoprecipitated polyribosomes. [125i].labelle d RNA (30 000 cpm) was incubated with 100 #g m1-1 poly(U) in 1 M sodium chloride - 10 mM sodium phosphate buffer, pH 6.8 - 0.1% sodium dodecyl sulfate for 1 hr at 45 ~ The incubated samples were diluted 10 times with 0.2 M sodium chloride - 0.01 M phosphate buffer, pH 6.8 - 0.1% sodium dodecyl sulfate and chromatographed on hydroxyapatite columns IX 1.5 cm at 45 ~ [17]. Abscissa - number of eluate fractions (1.5 ml each). Ordinate - [ 1 2 5 I] radioactivity, cpm• 10-3 . The arrows indicate the change in the molarity of the eluting phosphate buffer, e - - e - $ RNA incubated without poly(U), o-o--o RNA annealed with poly(U). Cell-free translation of various RNA preparations from rat liver polysomes in the submitochondrial system was performed in order to identify CP-mRNA functionally. The results of these studies (Table II) demonstrated that non-fractionated RNA from precipitated polysomes is three times enriched (in the terms of specific template activity) in CP mRNA content as compared to the total polysomal RNA preparation from rat liver. Further enrichment (more than tenfold) was achieved when poly(A)-containing RNA was isolated from CP polysomes. On the other hand, poly(A)-free RNA from these polysomes was characterized by very low specific template activity in the cell-free CP synthesis. The electrophoretic analysis of radioactive newly formed polypeptides recovered from the immunoprecipitates (Figure 3) showed a rather broad distribution in sodium dodecyl sulfate-containing gels. Two unresolved peaks were observed at 238
28S
ISS
02
t0
o,,
,
iO
I
I
20
30
B
~0
Fig. 2. Polyacrylamide gel electrophoresis of poly(A)-containing RNA from immunoprecipitated polysomes. Electrophoresis was performed in 2.7% polyacrylamide gel containing 1% sodium dodecyl sulfate. A. Photographs of the gels stained with methylene blue. 1. Total RNA from polysomes (100 #g tube-1). 2. Poly(A)-containing RNA from immunoprecipitated polysomes (15 #g tube-1). B. Densitometric tracing of the gel A-I and the distribution of [125i].labelle d poly(A)-containing RNA from CP polysomes. Abscissa: Slice number. Ordinate: Asso (right, - -- ) and radioactivity of 1 2 5 I-RNA, cpm• 10-2 (left, 0__0_@). The arrows indicate the position of markers (28 S and 18 S ribosomal RNAs from rat liver cytoplasm).
positions corresponding to the apparent molecular weights of 54 000 and 45 000, respectively. The electrophoretic distribution of CP immunoreactive material from the samples containing the total RNA of CP polysomes (Figure 3A) and its poly(A)-containing fraction (Figure 3B) was similar. The molecular size heterogeneity of the radioactive polypeptides could be a result 239
TABLE II: Synthesis of immunoreactive CP in the submitochondrial system programmed with various preparations of polysomal RNA from rat liver C.amin o acid incorporation, cpm/mg RNA 14
Enrichment factor
RNA preparation
Total polysomal RNA Total RNA from CP polysomes Poly(A).containing RNA from CP polysomes Poly(A)-free RNA from CP polysomes
TCA-insoluble material
immunoprecipitate
350 000 280 000 1 400 000
2 500 8 250 100 000
250 000
4 000
x 1.0 x 3.3 x 40.0 x
1.6
of contribution of polysome-bound uncompleted CP chains because the immunoprecipitation of CP polypeptides was performed directly from incubation mixtures without removal of polysomes. The existence of two electrophoretic components (mol. wts 45 000 and 54 000) is not documented convincingly enough to identify them as two CP subunits of somewhat different molecular weights. These apparent molecular weight values are at least twice less than the molecular weight of circulating CP (130 000). On the other hand, they approach rather closely the molecular weight of a CP half-molecule (60 000-65 000), although being apparently smaller. This difference might well be due to the failure of heterologous cell-free system to perform posttranslational modifications, such as glycosylation and copper incorporation into CP chains. The majority of molecular weight estimations mentioned above were made in studies on human CP preparations. However, previous investigations from our laboratory [4] showed that there is no significant difference between human and rat CPs with respect to their molecular sizes. Therefore, the data obtained do not contradict the suggestion concerning the existence of two subunits in the CP molecule [5]. The data reported demonstrate the isolation of translatable CP mRNA which appeared to be functionally enriched more than 40 times and highly purified according to electrophoresis under conditions of mild denaturation. The calculated value of the apparent molecular weight of CP mRNA is as high as 1.0X 1 0 6 , i.e. its molecule is long enough to code for a putative CP subunit with a molecular weight of 60 000-65 000. If these calculations are correct, the CP mRNA seems to contain, besides the coding sequence, extra-sequences constituting about 25-30% of its total molecular length. One of these non-coding sequences is poly(A) whose existence was demonstrated in our experiments described above. The isolation of highly enriched CP mRNA preparations functionally active in cell-free translation experiments seems to be a prerequisite for the study of inherited defects of CP synthesis in patients with hepatolenticular degeneration.
240
1000 qO0
B
800 700 ~00 500 qOD 5~
200 IO0
900
800 700
A
600 5O0 400
I00 0
m
Fig. 3. Electrophoretic analysis of the products of cell-free translation of CP-mRNA in the submitochondrial system. The preparation of the system and incubation conditions are described elsewhere [19]. The newly formed CP polypeptides were isolated by indirect immunoprecipitation. Eleetrophoresis was performed in 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate [20]. The radioactivity of gel slices (1.5 mm thick) was measured using 'Unisolv' mixture in a 'Nuclear Chicago', Mark II scintillation counter. Abscissa: Shce number. Ordinate: 14 C radioactivity, cpm. A. Immunoprecipitated product of the translation of the non-fractionated RNA from CP polyribosomes. B. lmmunoprecipitated product of the translation of poly(A)-containing RNA from CP polysomes. Serum albumin (mol.wt 68 000), catalase (mol.wt 60 000), ovalbumin (mol.wt 43 000) and cytochrome c (mol.wt 12 400) were used as markers in the calculations of the molecular weight of CP polypeptides. ACKNOWLEDGEMENT The w o r k was s u p p o r t e d b y the grant f r o m the World Health Organization ( A g r e e m e n t No. G3/181/38).
241
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