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A lipoprotein of rat liver nuclei
102
A LIPOPROTEIN T.Y. Departmente
of Biochemistry
WANG,
OF RAT LIVER D. T. MAYER
and Agricultural
and L. E. THOMAS
Chemistry,
Received January
NUCLE11v2
University
of Missouri,
Columbia,
Miss.
28, 1952
CELLULAR proteins insoluble
in water or salt solutions but soluble in alkaline solutions have been reported several times. Bensley and Hoerr (5) obtained preparations from rabbit and guinea pig liver which they named “ellipsin”. The material was precipitated by the addition of acetic acid to the alkaline solution and was reported to contain 30 per cent “fatty substances”. Luck (10) obtained an alkali-soluble protein fraction from dog liver. This material, by the addition of acid. These designated “globulin II”, was precipitated preparations probably contained proteins from the cytoplasm and nucleus. (We have found such proteins in both cytoplasmic and nuclear fractions of rat liver.) Mayer (11) reported the presence of an alkali-soluble protein fraction in the nuclei of calf thymus, isolated by a modified Behrens technique. It was precipitated at pH 5.8 to 6.2 by acetic acid. Boshijan (6), Mayer and Gulick (12), Stedman and Stedman (16, 17, IS), Jeener (8), and Zbarski and Debov (23) have described similar preparations. Two of the present authors (21) described a protein fraction extracted from boar spermatozoa with alkali and precipitated at about pH 6 with acetic acid. A preliminary report (22) was also made indicating that this material could not be obtained from the tail-midpiece fraction of boar spermatozoa, but only from the heads and that similar proteins had been obtained from rat liver nuclei and “isolated chromosomes” of calf thymus. In the latter case it was found (9) that the “residual prepared by the method of Mirsky and Ris, consist almost chromosomes”, entirely of this protein fraction. Evidence has now been obtained indicating that the proteins insoluble in neutral salt solutions, but soluble in alkaline solutions, obtained from the nuclei of rat liver, calf liver, calf thymus, ox spleen and chicken erythrocytes and from the heads of bull sperm all have tightly bound lipid, removable 1 Missouri Agricultural Experiment Station Journal Series No. 1289. s This work was supported (in part) by a grant from the National Cancer Institute, National Institutes of Health, Public Health Service.
of the
A lipoprotein
of rat liver nuclei
by hot alcohol but not by ether. The present paper reports some properties the alkali-soluble lipoprotein of rat liver nuclei. EXPERIMENTAL
METHODS
AND
of
RESULTS
Rat liver nuclei were isolated by the method of Dounce (7), modified slightly, or by Behrens’ method (a), also modified somewhat. l In either procedure, the liver was frozen as soon as possible after the animals were killed and all operations used in isolating nuclei and extracting proteins (except the lyophilizing in the Behrens method) were carried out at 0”--4” C. The nuclei were extracted exhaustively with 1 M sodium chloride solution to remove the nucleohistone, using, in each extraction, approximately 100 ml of solution for each 5 ml of packed nuclei. Ordinarily, three extractions were sufficient for the nuclei prepared by the procedure of Dounce. The nuclei obtained by the Behrens method required more extraction due to the formation of a more viscous solution at the outset. The first centrifugation was carried out at 4 500 r.p.m. for about three hours with shorter periods for subsequent centrifugations. After exhaustive extraction with sodium chloride some of the nuclei retained their original shape and size and the nuclear membrane of those nuclei appeared to be intact. The nuclear contents appeared to have been mostly removed, and the “ghost” nuclei were clumped. Many of the nuclei were broken into fragments. The nuclei were then stirred three times with 0.1 N sodium hydroxide solution (about 100 ml of solution for each 5 ml of packed nuclei) for 30 minute periods, the mixture being centrifuged each time at 4 500 r.p.m. until a clear solution was obtained. From 3 to 5 hours were required. The pH of the extract was approximately 12.5. Extraction can be carried out at a lower pH, but the lipoprotein goes into solution much more slowly under those conditions, its solubility apparently being quite low over a wide pH range. It was therefore deemed better to extract for a shorter period at the higher pH, as described. Acetic acid was added to the extract slowly and a precipitate, having the characteristics of a lipoprotein, was obtained at pH 5.7-6.0. It was collected by centrifugation at about 3 000 r.p.m. for 10 minutes. The precipitate was dissolved in 0.1 N sodium hydroxide and reprecipitated with acetic acid three times. The alkaline solution was centrifuged each time to remove any insoluble material before the reprecipitation. The material prepared in this way is referred to below as purified lipoprotein. Ultraviolet light absorption by an alkaline solution of the precipitated lipoprotein fraction showed no indication of the presence of nucleic acid, 1 In the Behrens procedure we used a mixture of benzene and carbon tetrachloride of low specific gravity to suspend the lyophilized tissue for grinding, and mixtures of these two liquids of increasing specific gravity for the differential centrifugation. A detailed description is not given here because the proportions of the two liquids used must necessarily be varied somewhat each time the procedure is used to obtain a good separation of the nuclei.
T. Y. Wang, D. T. Mayer and L,. E. Thomas
Fig. 1. Electrophoretic Patterns of the Lipoprotein: (a) ascending pattern in 0.1 M sodium citrate, pH 7.5, after 3630 seconds, field strength 18.0 volts/cm, (b) same, descending pattern, (c) ascending pattern in 0.1 M sodium acetate buffer, pH 7.8, after 2280 seconds, field strength 18.6 volts/cm, (d) same, descending pattern. TABLE
I
Analysis of the Protein Part of the Lipoprotein Percentage Nitrogen . . . . . . . . . . . . . . 14.5 Phosphorus . . . . . ?. . . . . . 0.07 Arginine . . . . . . . . . . . . . . 5.7 Tyrosine . . . . . . . . . . . . . . . 3.2 Tryptophane . . . . . . . . . . . 2.9
Method Micro-Kjeldahl Allen (1) Thomas, Ingalls and Luck (20) Thomas (19) Spies and Chambers (15)
but the supernatant solution after the first precipitation showed a small absorption peak at 256 mp. A small residue remained after the extraction of the lipoprotein, but no “ghost” nuclei could be seen in this residue. It contained only small fragments. The lipoprotein was found to constitute roughly 50 per cent of the dry weight of the nuclei isolated by the Dounce method. with hot alcohol in a Soxhlet The nature of the lipoprotein. Extraction apparatus removed lipid from the purified lipoprotein. The lipid constituted about 10 per cent of the total complex and gave positive tests for phospholipid and cholesterol. After the lipoprotein is purified as described above, no appreciable amount of lipid was removed during several more repre-
A lipoprotein of rat liver nuclei
105
cipitations. Ether did not extract an appreciable quantity of the lipid from the moist precipitate of the lipoprotein nor from its alkaline solution. Table I shows the results of analysis of the protein part of the lipoprotein (after removal of the lipid portion). Two-dimensional filter paper chromotography of an acid hydrolysate of the lipoprotein gave a pattern very similar amino acids were to that of many other tissue proteins. The following demonstrated to be present: alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, hydroxyproline, leucine (and isoleucine), lysine, methionine, proline, serine, threonine, tyrosine, valine. Electrophoresis was carried.out on the purified lipoprotein, using a PerkinThe results obtained indicate that only Elmer electrophoresis apparatus. one component is present in the region of pH 7.5. Preliminary inorganic analysis indicates that the purified lipoprotein complex contains approximately 0.8 per cent calcium and no sodium or potassium. DISCUSSION
It would be of interest to know what morphological part or parts of the nucleus contain the lipoprotein. The answer to this question is uncertain on the basis of evidence available at present. The lipoprotein fraction is essentially the same substance that Stedman and Stedman (16) called “chromosomin.” The “residual chromosomes” of Mirsky and Ris (14), as prepared in our laboratory, were made up almost entirely of this same material (9). Nevertheless, it seems that the possibility of the nuclear membrane being made up of a lipoprotein complex should, be given serious consideration. Only meager evidence has been obtained for that view, viz., the “ghost” appearance of many nuclei after extraction of the nucleohistone (referred to above) and the lack of any “ghost nuclei” after the extraction of the lipoprotein. We have not made careful microscopic observations to test this point, however. These observations were made incidentally during the progress of the chemical work and they are not new. Miescher (13) made some similar observations long ago. Bensley and Hoerr (5) and Bensley (3, 4) also reported somewhat similar findings. SUMMARY
A lipoprotein, soluble in alkali and insoluble in water and neutral salt solutions, has been prepared from rat liver nuclei isolated by the Dounce and Behrens methods.
106
T. Y. Wang, D. T. Mayer and L. E. Thomas
Hot alcohol extracted the lipid from the lipoprotein, but ether did not extract an appreciable quantity of lipid. Repeatedly dissolving the lipoprotein in alkali and reprecipitating with acid did not remove an appreciable amount of lipid. Tests on the lipid portion indicated the presence of phospholipid and cholesterol. The amino acid content of the protein part of the complex was found by chromatography to be similar to that of many tissue proteins. It was found by chemical analysis to contain: nitrogen - 14.5 per cent, phosphorus - 0.07 per cent, arginine - 5.7 per cent, tyrosine - 3.2 per cent, and tryptophane - 2.9 per cent. Electrophoretic patterns indicate that the lipoprotein complex contains only one component inothe region of pH 7.5. The possibility of the nuclear membrane being composed of a lipoprotein complex is discussed briefly. We are indebted to Dr. Eugene Roberts of the Cancer Research Division, Washington University Medical School, St. Louis, Missouri, for carrying out the chromatography and to Dr. Charles W. Turner of the Dairy Husbandry Department, University of Missouri, who generously allowed us to use the Tiselius apparatus. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
ALLEN, R. J. L., Biochem. .I., 34, 858 (1940). BEHRENS, M., Z. Physiol. Chem., 209, 59 (1932). BENSLEY, R. R., Anat. Rec., 72, 351 (1938). Biol. Symposia, 10, 323 (1943). BENSLEY, R. R., and HOERR, N. L., Anat. Rec., 60, 251 (1934). BOSHIJAN, G. M., Trudy Lab. Zzucheniyu Belka, Akad. Nauk. S.S.S.R., No. DOUNCE, A. L., J. Biol. Chem., 147, 685 (1943). JEENER, J., Corn@. rend. Sot. beige Biol., 140, 1103 (1946). KIRKHAM, W. R., unpublished data. LUCK, J. M., J. Biol. Chem., 115, 491 (1936). MAYER, D. T., Thesis, Univerisity of Missouri, 1938. MAYER, D. T., and GULICK, A., J. Biol. Chem., 146, 433 (1942). MIESCHER, F., Die Histochemischen und Physiologischen Arbeiten. Leipzig, Part 2, p. 15-16, 1897. MIRSKY, A. E., and RIS, H., J. Gen. Physiol., 31, 7 (1947). SPIES, J. R., and CHAMBERS, D. C., Anal. Chem., 21, 1249 (1949). STEDMAN, E., and STEDMAN, E., Nature, 152, 267 (1943). Cold Spring Harbor Symposia @ant. Biol., 12, 224 (1947). ~ Symposia Sot. Expptl. Biol., 1, 232 (1947). THOMAS, L. E., Arch. Biochem., 5, 175 (1944). THOMAS. L. E.. INGALLS. J. K.. and LUCK. J. M., J. Biol. Chem., 129, 263 THOMAS; L. E.; and MA&ER, c. T., Science, 110,‘393 (1949). WANG, T., KIRKHAM, W. R., DALLAM, R. D., MAYER, D. T., and THOMAS, 165, 974 (1950.) ZBARSKI, I. B., and DEBOV, S. S., Dokladi Akad. Nauk. S.S.S.R., 62, 795
1, 49 (1940).
F. C. W. Fogel.
(1939). L.
E.,
(1948).
Nature,
A LIPOPROTEIN T.Y. Departmente
of Biochemistry
WANG,
OF RAT LIVER D. T. MAYER
and Agricultural
and L. E. THOMAS
Chemistry,
Received January
NUCLE11v2
University
of Missouri,
Columbia,
Miss.
28, 1952
CELLULAR proteins insoluble
in water or salt solutions but soluble in alkaline solutions have been reported several times. Bensley and Hoerr (5) obtained preparations from rabbit and guinea pig liver which they named “ellipsin”. The material was precipitated by the addition of acetic acid to the alkaline solution and was reported to contain 30 per cent “fatty substances”. Luck (10) obtained an alkali-soluble protein fraction from dog liver. This material, by the addition of acid. These designated “globulin II”, was precipitated preparations probably contained proteins from the cytoplasm and nucleus. (We have found such proteins in both cytoplasmic and nuclear fractions of rat liver.) Mayer (11) reported the presence of an alkali-soluble protein fraction in the nuclei of calf thymus, isolated by a modified Behrens technique. It was precipitated at pH 5.8 to 6.2 by acetic acid. Boshijan (6), Mayer and Gulick (12), Stedman and Stedman (16, 17, IS), Jeener (8), and Zbarski and Debov (23) have described similar preparations. Two of the present authors (21) described a protein fraction extracted from boar spermatozoa with alkali and precipitated at about pH 6 with acetic acid. A preliminary report (22) was also made indicating that this material could not be obtained from the tail-midpiece fraction of boar spermatozoa, but only from the heads and that similar proteins had been obtained from rat liver nuclei and “isolated chromosomes” of calf thymus. In the latter case it was found (9) that the “residual prepared by the method of Mirsky and Ris, consist almost chromosomes”, entirely of this protein fraction. Evidence has now been obtained indicating that the proteins insoluble in neutral salt solutions, but soluble in alkaline solutions, obtained from the nuclei of rat liver, calf liver, calf thymus, ox spleen and chicken erythrocytes and from the heads of bull sperm all have tightly bound lipid, removable 1 Missouri Agricultural Experiment Station Journal Series No. 1289. s This work was supported (in part) by a grant from the National Cancer Institute, National Institutes of Health, Public Health Service.
of the
A lipoprotein
of rat liver nuclei
by hot alcohol but not by ether. The present paper reports some properties the alkali-soluble lipoprotein of rat liver nuclei. EXPERIMENTAL
METHODS
AND
of
RESULTS
Rat liver nuclei were isolated by the method of Dounce (7), modified slightly, or by Behrens’ method (a), also modified somewhat. l In either procedure, the liver was frozen as soon as possible after the animals were killed and all operations used in isolating nuclei and extracting proteins (except the lyophilizing in the Behrens method) were carried out at 0”--4” C. The nuclei were extracted exhaustively with 1 M sodium chloride solution to remove the nucleohistone, using, in each extraction, approximately 100 ml of solution for each 5 ml of packed nuclei. Ordinarily, three extractions were sufficient for the nuclei prepared by the procedure of Dounce. The nuclei obtained by the Behrens method required more extraction due to the formation of a more viscous solution at the outset. The first centrifugation was carried out at 4 500 r.p.m. for about three hours with shorter periods for subsequent centrifugations. After exhaustive extraction with sodium chloride some of the nuclei retained their original shape and size and the nuclear membrane of those nuclei appeared to be intact. The nuclear contents appeared to have been mostly removed, and the “ghost” nuclei were clumped. Many of the nuclei were broken into fragments. The nuclei were then stirred three times with 0.1 N sodium hydroxide solution (about 100 ml of solution for each 5 ml of packed nuclei) for 30 minute periods, the mixture being centrifuged each time at 4 500 r.p.m. until a clear solution was obtained. From 3 to 5 hours were required. The pH of the extract was approximately 12.5. Extraction can be carried out at a lower pH, but the lipoprotein goes into solution much more slowly under those conditions, its solubility apparently being quite low over a wide pH range. It was therefore deemed better to extract for a shorter period at the higher pH, as described. Acetic acid was added to the extract slowly and a precipitate, having the characteristics of a lipoprotein, was obtained at pH 5.7-6.0. It was collected by centrifugation at about 3 000 r.p.m. for 10 minutes. The precipitate was dissolved in 0.1 N sodium hydroxide and reprecipitated with acetic acid three times. The alkaline solution was centrifuged each time to remove any insoluble material before the reprecipitation. The material prepared in this way is referred to below as purified lipoprotein. Ultraviolet light absorption by an alkaline solution of the precipitated lipoprotein fraction showed no indication of the presence of nucleic acid, 1 In the Behrens procedure we used a mixture of benzene and carbon tetrachloride of low specific gravity to suspend the lyophilized tissue for grinding, and mixtures of these two liquids of increasing specific gravity for the differential centrifugation. A detailed description is not given here because the proportions of the two liquids used must necessarily be varied somewhat each time the procedure is used to obtain a good separation of the nuclei.
T. Y. Wang, D. T. Mayer and L,. E. Thomas
Fig. 1. Electrophoretic Patterns of the Lipoprotein: (a) ascending pattern in 0.1 M sodium citrate, pH 7.5, after 3630 seconds, field strength 18.0 volts/cm, (b) same, descending pattern, (c) ascending pattern in 0.1 M sodium acetate buffer, pH 7.8, after 2280 seconds, field strength 18.6 volts/cm, (d) same, descending pattern. TABLE
I
Analysis of the Protein Part of the Lipoprotein Percentage Nitrogen . . . . . . . . . . . . . . 14.5 Phosphorus . . . . . ?. . . . . . 0.07 Arginine . . . . . . . . . . . . . . 5.7 Tyrosine . . . . . . . . . . . . . . . 3.2 Tryptophane . . . . . . . . . . . 2.9
Method Micro-Kjeldahl Allen (1) Thomas, Ingalls and Luck (20) Thomas (19) Spies and Chambers (15)
but the supernatant solution after the first precipitation showed a small absorption peak at 256 mp. A small residue remained after the extraction of the lipoprotein, but no “ghost” nuclei could be seen in this residue. It contained only small fragments. The lipoprotein was found to constitute roughly 50 per cent of the dry weight of the nuclei isolated by the Dounce method. with hot alcohol in a Soxhlet The nature of the lipoprotein. Extraction apparatus removed lipid from the purified lipoprotein. The lipid constituted about 10 per cent of the total complex and gave positive tests for phospholipid and cholesterol. After the lipoprotein is purified as described above, no appreciable amount of lipid was removed during several more repre-
A lipoprotein of rat liver nuclei
105
cipitations. Ether did not extract an appreciable quantity of the lipid from the moist precipitate of the lipoprotein nor from its alkaline solution. Table I shows the results of analysis of the protein part of the lipoprotein (after removal of the lipid portion). Two-dimensional filter paper chromotography of an acid hydrolysate of the lipoprotein gave a pattern very similar amino acids were to that of many other tissue proteins. The following demonstrated to be present: alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, hydroxyproline, leucine (and isoleucine), lysine, methionine, proline, serine, threonine, tyrosine, valine. Electrophoresis was carried.out on the purified lipoprotein, using a PerkinThe results obtained indicate that only Elmer electrophoresis apparatus. one component is present in the region of pH 7.5. Preliminary inorganic analysis indicates that the purified lipoprotein complex contains approximately 0.8 per cent calcium and no sodium or potassium. DISCUSSION
It would be of interest to know what morphological part or parts of the nucleus contain the lipoprotein. The answer to this question is uncertain on the basis of evidence available at present. The lipoprotein fraction is essentially the same substance that Stedman and Stedman (16) called “chromosomin.” The “residual chromosomes” of Mirsky and Ris (14), as prepared in our laboratory, were made up almost entirely of this same material (9). Nevertheless, it seems that the possibility of the nuclear membrane being made up of a lipoprotein complex should, be given serious consideration. Only meager evidence has been obtained for that view, viz., the “ghost” appearance of many nuclei after extraction of the nucleohistone (referred to above) and the lack of any “ghost nuclei” after the extraction of the lipoprotein. We have not made careful microscopic observations to test this point, however. These observations were made incidentally during the progress of the chemical work and they are not new. Miescher (13) made some similar observations long ago. Bensley and Hoerr (5) and Bensley (3, 4) also reported somewhat similar findings. SUMMARY
A lipoprotein, soluble in alkali and insoluble in water and neutral salt solutions, has been prepared from rat liver nuclei isolated by the Dounce and Behrens methods.
106
T. Y. Wang, D. T. Mayer and L. E. Thomas
Hot alcohol extracted the lipid from the lipoprotein, but ether did not extract an appreciable quantity of lipid. Repeatedly dissolving the lipoprotein in alkali and reprecipitating with acid did not remove an appreciable amount of lipid. Tests on the lipid portion indicated the presence of phospholipid and cholesterol. The amino acid content of the protein part of the complex was found by chromatography to be similar to that of many tissue proteins. It was found by chemical analysis to contain: nitrogen - 14.5 per cent, phosphorus - 0.07 per cent, arginine - 5.7 per cent, tyrosine - 3.2 per cent, and tryptophane - 2.9 per cent. Electrophoretic patterns indicate that the lipoprotein complex contains only one component inothe region of pH 7.5. The possibility of the nuclear membrane being composed of a lipoprotein complex is discussed briefly. We are indebted to Dr. Eugene Roberts of the Cancer Research Division, Washington University Medical School, St. Louis, Missouri, for carrying out the chromatography and to Dr. Charles W. Turner of the Dairy Husbandry Department, University of Missouri, who generously allowed us to use the Tiselius apparatus. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
ALLEN, R. J. L., Biochem. .I., 34, 858 (1940). BEHRENS, M., Z. Physiol. Chem., 209, 59 (1932). BENSLEY, R. R., Anat. Rec., 72, 351 (1938). Biol. Symposia, 10, 323 (1943). BENSLEY, R. R., and HOERR, N. L., Anat. Rec., 60, 251 (1934). BOSHIJAN, G. M., Trudy Lab. Zzucheniyu Belka, Akad. Nauk. S.S.S.R., No. DOUNCE, A. L., J. Biol. Chem., 147, 685 (1943). JEENER, J., Corn@. rend. Sot. beige Biol., 140, 1103 (1946). KIRKHAM, W. R., unpublished data. LUCK, J. M., J. Biol. Chem., 115, 491 (1936). MAYER, D. T., Thesis, Univerisity of Missouri, 1938. MAYER, D. T., and GULICK, A., J. Biol. Chem., 146, 433 (1942). MIESCHER, F., Die Histochemischen und Physiologischen Arbeiten. Leipzig, Part 2, p. 15-16, 1897. MIRSKY, A. E., and RIS, H., J. Gen. Physiol., 31, 7 (1947). SPIES, J. R., and CHAMBERS, D. C., Anal. Chem., 21, 1249 (1949). STEDMAN, E., and STEDMAN, E., Nature, 152, 267 (1943). Cold Spring Harbor Symposia @ant. Biol., 12, 224 (1947). ~ Symposia Sot. Expptl. Biol., 1, 232 (1947). THOMAS, L. E., Arch. Biochem., 5, 175 (1944). THOMAS. L. E.. INGALLS. J. K.. and LUCK. J. M., J. Biol. Chem., 129, 263 THOMAS; L. E.; and MA&ER, c. T., Science, 110,‘393 (1949). WANG, T., KIRKHAM, W. R., DALLAM, R. D., MAYER, D. T., and THOMAS, 165, 974 (1950.) ZBARSKI, I. B., and DEBOV, S. S., Dokladi Akad. Nauk. S.S.S.R., 62, 795
1, 49 (1940).
F. C. W. Fogel.
(1939). L.
E.,
(1948).
Nature,