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Similarities in the cytoplasmic proteins of different organs and species examined by SDS gel electrophoresis
Copyright All rights
0 1972 by Academic Press, Inc. in any form reserved
of reproduction
Experimental Cell Research 75 (1972) 73-78
SIMILARITIES
IN THE
ORGANS
AND
CYTOPLASMIC SPECIES
PROTEINS
EXAMINED
OF DIFFERENT
BY SDS GEL
ELECTROPHORESIS D. E. COMINGS Department
of Medical
Genetics,
City
of Hope
and LOIS C. TACK National
Medical
Center,
Duarte,
Calif.
91010,
USA
SUMMARY Cytoplasmic proteins from different organs of the rat, mouse, quail, frog, and fish were compared by SDS gel electrophoresis. Since this technique separates proteins on the basis of molecular weight rather than charge, the evolutionary changes due simply to amino acid substitutions were eliminated. The brain proteins of these five widely divergent species were virtually identical, and the proteins from the liver and kidneys also showed many cross species similarities. This suggests that by the time fish had evolved the biochemical sophistication necessary to run the various organs had been developed and become relatively fixed. When proteins of different organs within the same species were compared there were again many similarities. These were most marked between the liver and kidney, and between the spleen and fibroblasts. There are probably a large number of proteins which all vertebrate cells share in common.
How different are the cytoplasmic proteins of organs from widely divergent species?It is clear that because of base substitutions a given protein may vary greatly in amino acid composition and rate of electrophoretic migration. Thus when many proteins are examined, and when the marked morphological and behavioral differences between such divergent species as man and fish are considered, one might expect the differences to be great. However, as far as the running of the metabolic machinery of a cell is concerned the amino acid substitutions in the proteins should make little difference. Thus, to examine whether the same general types and amounts of proteins are present in the cytoplasm of organs of divergent speciesthe use of SDS gel electrophoresis, which eliminates charge differences, is ideal. A second and related question is, how different are the cytoplasmic proteins of
different organs within a given species?It has been repeatedly implied that differentiation in higher organisms leads to marked organ specificity for many enzymes and proteins, and that many genesare ‘turned on’ in one organ and ‘turned off’ in another. While to a certain extent this is undoubtedly true, a case could also be made for the opposite conclusion that the cytoplasmic proteins of different organs should show more similarities than differences. Thus, if we assumethat there are somewherebetween two and four thousand ‘household’ genes which must be ‘on’ in the cells of all organs just to run the many metabolic pathways, and that there are probably no more than a hundred unique ‘luxury’ proteins in various differentiated cells, this leaves a 4% difference compared to a 97% similarity between different cells. These two questions have been examined Exptl
Cell Res 75 (1972)
74 D. E. Comings & Lois C. Tack by SDS gel electrophoresis of the cytoplasmic proteins of the brain, liver, kidney, and in some cases spleen and testis, of the rat, mouse, quail, frog, and fish.
mammals than the frog. The kidney proteins did not electrophorese consistently well in all species but it could be seen that the patterns were similar between the mouse, rat, quail, and frog, and the few bands that were seen in the fish tended to match the other MATERIALS AND METHODS species. Brain, liver, kidney, spleen and testis tissues from Figs 4-7 allow comparisons between the the mouse (Mm musculus), rat (Raftus norvegicus), cytoplasmic proteins of different organs withJapanese quail (Coturnix coturnix Japonica), frog (Rana pipiens) and fish (rainbow trout, Salmo iriin a species. In the mouse (fig. 4) the first deus Gibbons) were minced and washed in 0.01 M five bands in the brain, kidney, liver and phosphate buffer, pH 6.0 containing lo+ M NaSO,. The low pH and sodium bisulfite [5] were incorpospleenmatch almost exactly and a number of rated to inhibit proteolysis. The tissues were homothe lower molecular weight bands match but genized gently until about 50 % of the cells were ruptured with no rupture of nuclear membranes. This show variations in amount. In the rat (fig. 5) was spun at 10 000 g for 10 min and the pellet disthe kidney and liver matched well, as did carded. The sunernatant was further centrifuged at 100 000 g for 1 h and the pellet discarded. An ahquot the spleen and testis, and several bands could of the supernatant was brought to 1% SDS (sodium be followed through all the organs. In the dodecyl sulphate), then dialysed overnight at room temnerature against 0.1 % SDS, 0.1 % /3-mercaptoquail (fig. 6) the kidney and liver again ethanol, 0.01 M sodium phosphate, pH 7’.1. The promatched well, although there were three bands tein concentration was determined by TCA turbidity using albumin standards. This involved dilution of present in the liver (arrows) that were faint 20 to 150 pg of protein to 2.0 ml with distilled water. or absent in the kidney. Although the three 1.O ml of 50 % TCA was added and quickly vortexed. The samples were left at room temperature for 50 large middle bands in the brain proteins were min and read at 400 nm. 75 pg of protein was added quantitatively increased they seemedto also to each gel. SDS gel electrophoresis was carried out by the technique of Maize1 [4, 61 in 10 % acrylamide be represented in the other organs. In the gels. The details are described elsewhere [l]. The following standards were utilized for estimation of frog (fig. 7) the lower molecular weight molecular weight: albumin (A-65 000); ovalbumin proteins (from T to 0) are very similar while (O-48 000), chymotrypsinogen A (T-26 000) and cytosome differences were present in those of chrome c (C-12 400). The molecular weights represent the reduced form 171. higher molecular weight. Again there were quantitative differences but qualitative similarities between the brain proteins and those RESULTS of the kidney and liver. The fish (not shown) The three organs which consistently gave the were similar to the frog, but the poor quality best patterns and thus allowed comparison of the kidney bands precluded good combetween all the speciestested, were the brain, parisons. liver, and kidney. The cytoplasmic proteins To further examine the similarities between of these organs for the five speciesare shown organs, the cytoplasmic proteins of Chinese in figs l-3. The comparison of the brain hamster kidney, liver, spleen and V79 tissue proteins illustrates the remarkable similarity culture cells were examined (fig. 8). The in the pattern of these proteins throughout pattern of the liver and kidney proteins were evolution. In the liver proteins the mouse and virtually identical; there were many simirat are essentially identical, and some bands larities between the proteins of the spleen can be followed throughout all five species. and tissue cultured cells, and many bands The fish showed the greatest variation and the could be followed through all four sources quail appeared to be more different from the of protein. Exptl Cell Res 75 (1972)
Similarities
in cytoplasmic proteins of different organs and species
75
Fig. I. SDS gel electrophoresis of brain cytoplasmic proteins of the mouse (M), rat (R), Japanese quail (Q), frog (Fr) and fish (Fi). A, albumin; 0, ovalbumin; T, chymotrypsin. Fig. 2. SDS gel electrophoresis of liver cytoplasmic proteins. A, 0, T, as in fig. 1; C, cytochrome c. Fig. 3. SDS gel electrophoresis of kidney cytoplasmic proteins. Fig. 4. SDS gel electrophoresis of brain (B), kidney (K), liver (L), and spleen (S) cytoplasmic proteins of the mouse.
Exptl
Ceil Res 75 (1972)
76 D. E. Comings & Lois C. Tack
T ,/c
*
6
K
L
7 Fig. Fig.
liver Fig. Fig.
The
5. SDS gel electrophoresis of brain, kidney, liver spleen and testis (T), cytoplasmic proteins of the rat. 6. SDS gel electrophoresis of the brain, kidney and liver cytoplasmic proteins of the Japanese quail. The proteins possess three bands (arrows) not seen in the brain and kidney. 7. SDS gel electrophoresis of brain, kidney and liver cytoplasmic proteins of the frog. 8. SDS gel electrophoresis of the kidney, liver, spleen and tissue culture (V79 cells) of the Chinese hamster. histones are unfractionated calf thymus histones.
Exptl
Cell Res 75 (1972)
Similarities
in cytoplasmic proteins of different organs and species
DISCUSSION The remarkable similarities in the pattern of cytoplasmic proteins from such widely divergent species suggest that the biochemical equipment needed for rat organs to function is basically the same as that for the fish. This implies that by the time fish appeared, during the Silurian period 320 to 350 million years ago, a level of biochemical sophistication necessary for the basic functioning of vertebrate organs had already developed and become more or less fixed. Subsequent evolution, especially of the brain, presumably involved changes in form, organ size, and cellto-cell interaction rather than basic biochemical and structural functions. This, of course, must be qualified to the extent that only some classes of cytoplasmic proteins are present in sufficient quantity to be visualized by electrophoresis (see below). The between organ comparisons within a given species also showed many similarities. The kidney and liver consistently showed similar patterns and there were many similarities between the Chinese hamster tissue culture cells and the spleen. With the exception of a quantitative increase in a set of three large, middle molecular weight bands, the brain proteins also showed many bands that were common to the other organs. There are obviously wide variations in the amounts of different cellular proteins. Those that can be observed by electrophoresis would have to be individually or collectively present in high concentrations. These would presumably include structural proteins (such as microtubular and membrane proteins), special products (such as hemoglobin in erythrocytes, and gamma globulin in immunocytes), and certain enzymes present in high concentration (such as carbonic anhydrase and catalase [3] in erythrocytes). A second class, composed of metabolic enzymes, and
77
probably present in amounts that would be insufficient for them to be detected by electrophoresis and protein staining alone. In addition, there is presumably a third class composed of regulatory proteins that would be present at still lower concentrations. Although proteins in the latter two categories might not be individually discernible, a group of them with similar molecular weights, would be. A couple of features of this study should be emphasized. First, a concerted effort was made to examine primarily those gene products which are most likely to be different in different cells. Thus the 10 000 g and 100 000 g pellets were discarded. These would contain, among other things, ribosomes, mitochondria, and membranes; structures whose proteins would be very similar in different organs. Secondly, while SDS gel electrophoresis had the advantage of removing charge differences and separating on the basis of molecular weight, it had the disadvantage of dividing proteins into their subunits. It is unlikely, however, that this materially affected the results. Significant differences in cytoplasmic proteins would be apparent whether by electrophoresis of whole proteins or subunits. These electrophoretic similarities that transcend both species and organ lines, provide some qualified support for the proposal that there is a basic set of structural and enzymatic proteins that are common to all vertebrate cells. They also provide a base line for similar comparisons with proteins from other intracellular sources. For example, Elgin & Bonner [2] have shown that the non-histone chromosomal proteins of different organs and different species show many similarities by SDS gel electrophoresis. The present results indicate that this cannot be taken as a characteristic unique to the non-histones. Transspecies and trans-organ similarity is also a characteristic of many cellular proteins. Exptl
Cell Res 75 (1972)
18 D. E. Comings h Lois C. Tack This work was supported 15886and HD 03637.
by NIH
Grants
GM
REFERENCES 1. Comings, D E & Tack, L C. Submitted for publication. 2. Elgin, S C R & Bonner, 3, Biochemistry 9 (1970) 4440. 3. Haut, A, Tudhope, G R, Cartwright, G E & Wintrobe, M M, J clin invest 41 (1962) 579.
Exptl
Cell Res 75 (1972)
4. Maizel, J V, Fundamental techniques in virology (ed K Habel & N P Salzman) p. 334. Academic Press, New York (1969). 5. Panyim, S, Jensen, R H & Chalkley, R, Biochim biophys acta 160 (1968) 252. 6. Shapiro, A L, Vinuela, E & Maizel, J V, Biochem biophys res commun 28 (1967) 815. 7. Weber, K & Osborn, M, J biol them 244 (1969) 4406. Received March 22, 1971 Revised version received May 8, 1972
0 1972 by Academic Press, Inc. in any form reserved
of reproduction
Experimental Cell Research 75 (1972) 73-78
SIMILARITIES
IN THE
ORGANS
AND
CYTOPLASMIC SPECIES
PROTEINS
EXAMINED
OF DIFFERENT
BY SDS GEL
ELECTROPHORESIS D. E. COMINGS Department
of Medical
Genetics,
City
of Hope
and LOIS C. TACK National
Medical
Center,
Duarte,
Calif.
91010,
USA
SUMMARY Cytoplasmic proteins from different organs of the rat, mouse, quail, frog, and fish were compared by SDS gel electrophoresis. Since this technique separates proteins on the basis of molecular weight rather than charge, the evolutionary changes due simply to amino acid substitutions were eliminated. The brain proteins of these five widely divergent species were virtually identical, and the proteins from the liver and kidneys also showed many cross species similarities. This suggests that by the time fish had evolved the biochemical sophistication necessary to run the various organs had been developed and become relatively fixed. When proteins of different organs within the same species were compared there were again many similarities. These were most marked between the liver and kidney, and between the spleen and fibroblasts. There are probably a large number of proteins which all vertebrate cells share in common.
How different are the cytoplasmic proteins of organs from widely divergent species?It is clear that because of base substitutions a given protein may vary greatly in amino acid composition and rate of electrophoretic migration. Thus when many proteins are examined, and when the marked morphological and behavioral differences between such divergent species as man and fish are considered, one might expect the differences to be great. However, as far as the running of the metabolic machinery of a cell is concerned the amino acid substitutions in the proteins should make little difference. Thus, to examine whether the same general types and amounts of proteins are present in the cytoplasm of organs of divergent speciesthe use of SDS gel electrophoresis, which eliminates charge differences, is ideal. A second and related question is, how different are the cytoplasmic proteins of
different organs within a given species?It has been repeatedly implied that differentiation in higher organisms leads to marked organ specificity for many enzymes and proteins, and that many genesare ‘turned on’ in one organ and ‘turned off’ in another. While to a certain extent this is undoubtedly true, a case could also be made for the opposite conclusion that the cytoplasmic proteins of different organs should show more similarities than differences. Thus, if we assumethat there are somewherebetween two and four thousand ‘household’ genes which must be ‘on’ in the cells of all organs just to run the many metabolic pathways, and that there are probably no more than a hundred unique ‘luxury’ proteins in various differentiated cells, this leaves a 4% difference compared to a 97% similarity between different cells. These two questions have been examined Exptl
Cell Res 75 (1972)
74 D. E. Comings & Lois C. Tack by SDS gel electrophoresis of the cytoplasmic proteins of the brain, liver, kidney, and in some cases spleen and testis, of the rat, mouse, quail, frog, and fish.
mammals than the frog. The kidney proteins did not electrophorese consistently well in all species but it could be seen that the patterns were similar between the mouse, rat, quail, and frog, and the few bands that were seen in the fish tended to match the other MATERIALS AND METHODS species. Brain, liver, kidney, spleen and testis tissues from Figs 4-7 allow comparisons between the the mouse (Mm musculus), rat (Raftus norvegicus), cytoplasmic proteins of different organs withJapanese quail (Coturnix coturnix Japonica), frog (Rana pipiens) and fish (rainbow trout, Salmo iriin a species. In the mouse (fig. 4) the first deus Gibbons) were minced and washed in 0.01 M five bands in the brain, kidney, liver and phosphate buffer, pH 6.0 containing lo+ M NaSO,. The low pH and sodium bisulfite [5] were incorpospleenmatch almost exactly and a number of rated to inhibit proteolysis. The tissues were homothe lower molecular weight bands match but genized gently until about 50 % of the cells were ruptured with no rupture of nuclear membranes. This show variations in amount. In the rat (fig. 5) was spun at 10 000 g for 10 min and the pellet disthe kidney and liver matched well, as did carded. The sunernatant was further centrifuged at 100 000 g for 1 h and the pellet discarded. An ahquot the spleen and testis, and several bands could of the supernatant was brought to 1% SDS (sodium be followed through all the organs. In the dodecyl sulphate), then dialysed overnight at room temnerature against 0.1 % SDS, 0.1 % /3-mercaptoquail (fig. 6) the kidney and liver again ethanol, 0.01 M sodium phosphate, pH 7’.1. The promatched well, although there were three bands tein concentration was determined by TCA turbidity using albumin standards. This involved dilution of present in the liver (arrows) that were faint 20 to 150 pg of protein to 2.0 ml with distilled water. or absent in the kidney. Although the three 1.O ml of 50 % TCA was added and quickly vortexed. The samples were left at room temperature for 50 large middle bands in the brain proteins were min and read at 400 nm. 75 pg of protein was added quantitatively increased they seemedto also to each gel. SDS gel electrophoresis was carried out by the technique of Maize1 [4, 61 in 10 % acrylamide be represented in the other organs. In the gels. The details are described elsewhere [l]. The following standards were utilized for estimation of frog (fig. 7) the lower molecular weight molecular weight: albumin (A-65 000); ovalbumin proteins (from T to 0) are very similar while (O-48 000), chymotrypsinogen A (T-26 000) and cytosome differences were present in those of chrome c (C-12 400). The molecular weights represent the reduced form 171. higher molecular weight. Again there were quantitative differences but qualitative similarities between the brain proteins and those RESULTS of the kidney and liver. The fish (not shown) The three organs which consistently gave the were similar to the frog, but the poor quality best patterns and thus allowed comparison of the kidney bands precluded good combetween all the speciestested, were the brain, parisons. liver, and kidney. The cytoplasmic proteins To further examine the similarities between of these organs for the five speciesare shown organs, the cytoplasmic proteins of Chinese in figs l-3. The comparison of the brain hamster kidney, liver, spleen and V79 tissue proteins illustrates the remarkable similarity culture cells were examined (fig. 8). The in the pattern of these proteins throughout pattern of the liver and kidney proteins were evolution. In the liver proteins the mouse and virtually identical; there were many simirat are essentially identical, and some bands larities between the proteins of the spleen can be followed throughout all five species. and tissue cultured cells, and many bands The fish showed the greatest variation and the could be followed through all four sources quail appeared to be more different from the of protein. Exptl Cell Res 75 (1972)
Similarities
in cytoplasmic proteins of different organs and species
75
Fig. I. SDS gel electrophoresis of brain cytoplasmic proteins of the mouse (M), rat (R), Japanese quail (Q), frog (Fr) and fish (Fi). A, albumin; 0, ovalbumin; T, chymotrypsin. Fig. 2. SDS gel electrophoresis of liver cytoplasmic proteins. A, 0, T, as in fig. 1; C, cytochrome c. Fig. 3. SDS gel electrophoresis of kidney cytoplasmic proteins. Fig. 4. SDS gel electrophoresis of brain (B), kidney (K), liver (L), and spleen (S) cytoplasmic proteins of the mouse.
Exptl
Ceil Res 75 (1972)
76 D. E. Comings & Lois C. Tack
T ,/c
*
6
K
L
7 Fig. Fig.
liver Fig. Fig.
The
5. SDS gel electrophoresis of brain, kidney, liver spleen and testis (T), cytoplasmic proteins of the rat. 6. SDS gel electrophoresis of the brain, kidney and liver cytoplasmic proteins of the Japanese quail. The proteins possess three bands (arrows) not seen in the brain and kidney. 7. SDS gel electrophoresis of brain, kidney and liver cytoplasmic proteins of the frog. 8. SDS gel electrophoresis of the kidney, liver, spleen and tissue culture (V79 cells) of the Chinese hamster. histones are unfractionated calf thymus histones.
Exptl
Cell Res 75 (1972)
Similarities
in cytoplasmic proteins of different organs and species
DISCUSSION The remarkable similarities in the pattern of cytoplasmic proteins from such widely divergent species suggest that the biochemical equipment needed for rat organs to function is basically the same as that for the fish. This implies that by the time fish appeared, during the Silurian period 320 to 350 million years ago, a level of biochemical sophistication necessary for the basic functioning of vertebrate organs had already developed and become more or less fixed. Subsequent evolution, especially of the brain, presumably involved changes in form, organ size, and cellto-cell interaction rather than basic biochemical and structural functions. This, of course, must be qualified to the extent that only some classes of cytoplasmic proteins are present in sufficient quantity to be visualized by electrophoresis (see below). The between organ comparisons within a given species also showed many similarities. The kidney and liver consistently showed similar patterns and there were many similarities between the Chinese hamster tissue culture cells and the spleen. With the exception of a quantitative increase in a set of three large, middle molecular weight bands, the brain proteins also showed many bands that were common to the other organs. There are obviously wide variations in the amounts of different cellular proteins. Those that can be observed by electrophoresis would have to be individually or collectively present in high concentrations. These would presumably include structural proteins (such as microtubular and membrane proteins), special products (such as hemoglobin in erythrocytes, and gamma globulin in immunocytes), and certain enzymes present in high concentration (such as carbonic anhydrase and catalase [3] in erythrocytes). A second class, composed of metabolic enzymes, and
77
probably present in amounts that would be insufficient for them to be detected by electrophoresis and protein staining alone. In addition, there is presumably a third class composed of regulatory proteins that would be present at still lower concentrations. Although proteins in the latter two categories might not be individually discernible, a group of them with similar molecular weights, would be. A couple of features of this study should be emphasized. First, a concerted effort was made to examine primarily those gene products which are most likely to be different in different cells. Thus the 10 000 g and 100 000 g pellets were discarded. These would contain, among other things, ribosomes, mitochondria, and membranes; structures whose proteins would be very similar in different organs. Secondly, while SDS gel electrophoresis had the advantage of removing charge differences and separating on the basis of molecular weight, it had the disadvantage of dividing proteins into their subunits. It is unlikely, however, that this materially affected the results. Significant differences in cytoplasmic proteins would be apparent whether by electrophoresis of whole proteins or subunits. These electrophoretic similarities that transcend both species and organ lines, provide some qualified support for the proposal that there is a basic set of structural and enzymatic proteins that are common to all vertebrate cells. They also provide a base line for similar comparisons with proteins from other intracellular sources. For example, Elgin & Bonner [2] have shown that the non-histone chromosomal proteins of different organs and different species show many similarities by SDS gel electrophoresis. The present results indicate that this cannot be taken as a characteristic unique to the non-histones. Transspecies and trans-organ similarity is also a characteristic of many cellular proteins. Exptl
Cell Res 75 (1972)
18 D. E. Comings h Lois C. Tack This work was supported 15886and HD 03637.
by NIH
Grants
GM
REFERENCES 1. Comings, D E & Tack, L C. Submitted for publication. 2. Elgin, S C R & Bonner, 3, Biochemistry 9 (1970) 4440. 3. Haut, A, Tudhope, G R, Cartwright, G E & Wintrobe, M M, J clin invest 41 (1962) 579.
Exptl
Cell Res 75 (1972)
4. Maizel, J V, Fundamental techniques in virology (ed K Habel & N P Salzman) p. 334. Academic Press, New York (1969). 5. Panyim, S, Jensen, R H & Chalkley, R, Biochim biophys acta 160 (1968) 252. 6. Shapiro, A L, Vinuela, E & Maizel, J V, Biochem biophys res commun 28 (1967) 815. 7. Weber, K & Osborn, M, J biol them 244 (1969) 4406. Received March 22, 1971 Revised version received May 8, 1972