- Email: [email protected]
[15] Immunochemical characteristics of chromosomal proteins
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
191
choice of assay conditions, the purity and physical state of the DNA, and the technique chosen to monitor protein-DNA binding are all important variables t h a t must be considered when attempting to study the interaction of phosphorylated nonhistone proteins with DNA.
[15] I m m u n o c h e m i e a l C h a r a c t e r i s t i c s Chromosomal Proteins
of
B y F. CHYTIL
This chapter describes procedures for production of antibodies against histones and nonhistone chromosomal proteins, as well as against the nonfractionated chromatin. The conditions for testing the antigenicity of the chromosomal proteins by the method of quantitative complement fixation will be given also. This method appears to be a method of choice as it requires relatively small amounts of the chromosomal material which become very often a limiting factor in the determination of the properties of these proteins. The reader should consult first the introduction in general immunochemical techniques published in this series I as well as the detailed description of the method of quantitative complement fixation published earlierY Immunogens
Histones
To facilitate the production of antibodies against these basic proteins it is advisable either to bind them covalently to serum albumin 3 or to complex them with phosphorylated serum albumin 3 or to form a complex with R N A or DNA, 4 though free histones apparently could be used also2 Coupling the Histones with H u m a n Serum A l b u m i n ( H S A ) . ~ HSA, 100 mg, and 40 mg of whole histone are dissolved in 3 ml of H~0. Three milliliters of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p1See this series, Vol. 11 [91]. 2See this series, Vol. 11 [92]. SA. L. Sandberg, M. Liss, and B. D. Stollar. J. Immunol. 98, 1182 (1967). 4B. D. Stollar and M. Ward, J. Biol. Chem. 245, 1261 (1970). 5p. Rumke and M. Sluyser, Biochem. J. 1Ol, lc (1966).
192
THE CELL NUCLEUS AND CHROMATIN PROTEINS
[15]
toluenesulfonate 6 in H..O (75 mg/ml) are added, and the solution is stirred for 30 minutes at room temperature and then dialyzed for 24 hours against distilled H~O at 4 ° (25 mg of HSA, 10 mg of histone fraction, and 75 mg of the above carbodiimide can be used). Conjugates of HSA and the carbodiimide without histones can be also prepared. Coupling o] Histones with Phosphorylated Bovine Serum Albumin (BSA) PHOSPHORYLATED BSA. 7,8 BSA, 1.5 g is dissolved in 55 ml of 5% Na,.,HPO4 containing one drop of a phenolphthalein solution and placed in an ice-salt bath. Three milliliters of POC13 dissolved in 25 ml of carbon tetrachloride are added dropwise to the stirring BSA solution. Simultaneously, 1 M N a O H is added dropwise to maintain a p H of approximately 9 during the 4-hour reaction period. The reaction mixture is then poured into a dialysis bag and dialyzed overnight in the cold room against three changes of H~O. By twisting the dialysis bag at a level well above the carbon tetrachloride-water interphase, the aqueous phase could be separated. Alternatively, the carbon tetrachloride layer could be removed by centrifugation. Aliquots of the reaction mixture (at pH 8 to 9) are delivered into small tubes and stored frozen. The phosphorylated BSA can be isolated by acidification of the aqueous layer in the cold with 1 N hydrochloric acid to achieve maximum precipitation. The phosphorylated BSA is separated by centrifugation. COUPLING7 Phosphorylated BSA, 250 tLg in 50 td of water, is added to 2 ml of 0.15 sodium chloride solution containing 250 t~g of histone. The resulting solution is used for immunization. Histone Complexes with Nucleic Acids. 4,~ Equal amounts of yeast RNA and histones in 0.14 NaC1, 10 m M sodium phosphate buffer, pH 6.8, are mixed. In another procedure 600 ~g of each histone fraction is mixed with 200 t~g of RNA, or 2 mg of whole histone in 2 ml is added to 0.6 mg of DNA in 0.75 ml. Nonhistone Proteins
It is the author's experience that acidic proteins still complexed with DNA or free are good immunogens. 1°,1~ e T. L. Goodfriend, L. Levine, and G. Fasman, Science 144, 1344 (1964). 7H. Van Vunakis, J. Kaplan, H. Lehrer, and L. Levine, Immunochemistry 3, 393 (1966). 8 M. Heidelberger, B. Davis, and H. P. Treffers, J. Amer. Chem. Soc. 63, 498 (1941). g M. Bustin and B. D. Stollar, J. Biol. Chem. 247, 5716 (1972). ~oF. Chytil and T. C. Spelsberg, Nature (London) New Biol. 233, 215 (1971). ~1F. Chytil, unpublished observations.
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
193
Chromatin The preparation can be used directly for immunization.11
Immunization Procedure Rabbits: New Zealand White rabbits weighing between 3 and 4 kg are usually used. Complete Freund adjuvant is mixed with the solution of immunogen (1:1 or 1:1.5 v/v) in a syringe or homogenized in a small Teflon pestle glass homogenizer. Usually the immunization schedule used in this laboratory involves a toe pad injection of as much of the immunogen as possible on the first and eighth days, the rest being administered intramuscularly in multiple sites. About 10 days after administration of the complete Freund adjuvant, inflammation of the toe pads is observed. Then the injection in the toes has to be avoided, and the immunogen is administered intramuscularly only. Immunization Doses. The table shows the immunization doses for different chromosomal components. Intravenous Booster. Often an intravenous iniection into the marginal vein is given (Freund adjuvant has to be omitted) a week before bleeding. Bleeding. The animals are usually bled a week after the intravenous injection from the ear vein, and the sera are prepared as described earlier. 1 Antisera. Usually the sera can be used for testing without purification. However, sometimes, especially when a low dilution of the serum has to be employed, a high fixation of the complement in the absence of an antigen is observed in the complement fixation assay. In order to lower this property ("anticomplementarity") the globulin fraction can be purifled either by ammonium sulfate precipitation 12 or by ion exchange chromatography (DEAE-cellulose).12 In the author's laboratory the following procedure was found to remove substantially the "anticomplementarity." The sera are diluted with 9 volumes of 0.14 M sodium chloride and centrifuged at 105,000 g at 4 ° for 60 minutes. The serum is then carefully siphoned off, sterilized by Millipore filtration, and stored at --20 ° . Antigens. Varying amounts of antigens are used by diluting with the diluent used for the complement fixation assay. The antigens are stored frozen at --20 °. The histones and chromatins do not exert any "anticomplementarity." On the other hand, the nonhistone protein-DNA complexes may show limited binding of complement in the absence of anti12H. H. Fudenberg, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 306. Academic Press, New York, 1967.
194
THE
CELL
NUCLEUS
AND
CHROMATIN
[15]
PROTEINS
¢; o
o
o ::k o ~
0
0
0
g
o ~
o
Z
0
Z ['-. ~r:
Z ¢o © oo ~
..-4
© o o
v
o
v
Z ca
0 Z ;2 r~ W o
o
o
o
o
z
.
.~
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
195
body, which can be subtracted from that observed in the presence of antibody-antigen complexes. Repeated thawing and freezing of the antigens might induce a rise in "anticomplementarity." It is therefore advisable to store the antigens in small aliquots. Low "anticomplementarity" of nonhistone protein-DNA complexes is observed when the histones are removed by high urea, high salt treatment. TM On the other hand, when histones are extracted with acid the "anticomplementarity" of these preparations is frequently very high. Testing the Antigenicity The method of quantitative microcomplement fixation described earlier ~ is employed with small modification which consists in using onefifth of the incubation volume. Briefly, to disposable glass tubes 13 X 100 mm the following components are added: 0.6 ml of the diluent, 0.2 ml of diluted antibody, 0.2 ml of the antigen, and 0.2 ml of diluted complement to make the total volume 1.2 ml. After an overnight incubation at 4 °, 0.2 ml of the sensitized sheep red blood cells are added and the hemolysis is stopped usually after 45-60 minutes. The dilution of the antisera was found to range between 1/200 and 1/3200. The maximum complement fixation by chromosomal protein-antibody complexes varies from 0.5 to 2.0 t~g per assay tube. This amount is substantially higher than that found for another antigen. 2 The titer of the complement which has to be determined for each shipment varies in this laboratory between 1/125 and 1/175. Anti-histone Antibody. Figure 1 shows typical curves obtained when complement fixation assay was employed to test the antigenicity of antihistone antibody in the presence of different preparations of F1 histone. These results show the extent of reproducibility of this method. This laboratory has similar experience when different batches of nonhistone protein-DNA complexes were tested against homologous antibody. Anti-nonhistone Protein Antibody. Nonhistone proteins are apparently good immunogens. Complement-fixing antibody can be obtained when the rabbits are immunized with these proteins still attached to DNA 1° or with the free form. 11 Histones, double- or single-stranded DNA, do not interact with these antibodies. The antibodies react also, but to a smaller extent, with the chromatin preparation from the same organ 1° and show tissue specificity, which is demonstrated in Fig. 2. They react also with free nonhistone proteins. ~ Anti-chromatin Antibody. Antibody against the whole chromatin can be induced rather easily.~1 The results from this laboratory show in Fig. 3 i, F. Chytil and T. C. Spelsberg, unpublished observations.
196
THE
CELL
NUCLEUS
AND
CHROMATIN
PROTEINS
[15]
IO0 A
75
Z
_o
~ 5c X
7~ 25
0
1,0
2.0
ANTIGEN (~ug)
Fie. 1. C' fixation of antiserum to whole calf thymus F1 histone with varying preparations of whole calf thymus F1 histone. O, An old chromatographically purified preparation frozen and thawed several times; @, a fresh chromatographically purified preparation frozen and thawed once; X, a preparation before purification by column chromatography. Serum dilution was 1:1000. From M. Bustin and B. D. Stollar, J. Biol. Chem. 247, 5716 (1972). IOC
Z 0
Z) 25
/
•
o.o25 o.os o.,
o'.s
i
ANTIGEN Lug )
FIG. 2. C' fixation of antiserum to nonhistone protein-DNA complexes from chick oviduct by varying quantities of nonhistone protein-DNA complexes from chick oviduct, @; heart, X ; and liver, II. Serum dilution was 1:400. From F. Chytil and T. C. Spelsberg, Nalure (London) New Biol. 233, 215 (1971). that the immunization with chromatin leads to antibody reacting also with free histones. Moreover, these antibodies fix complement in the presence of nonhistone p r o t e i n - D N A complexes and do not react with DNA. Testing the Nuclear Origin of the I m m u n o g e n I n order to obtain additional evidence as to whether the antichromosomal protein antibody is directed against the nuclear material, and not
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
197
I00
75
Z 0
so
X LL
i)
25
.os
,,
.s
i
ANTIGEN (#g)
Fro. 3. C' fixation of antiserum to chromatin from chick oviduct by varying quantities of chromatin, O, and whole histone, ×. Serum dilution was 1:400. From unpublished data of F. Chytil and T. C. Spelsberg.
FIG. 4. Intracellular localization of antiliver nonhistone protein-DNA antibodies in liver slices by peroxidase bridge analysis described in this series (Vol. 37 [9]). LeJt: Adult rat liver slices incubated with control serum. Right: With anti-liver nonhistone protein antibody. From unpublished experiments of F. Chytil and P. K. Nakane. against some cytoplasmic components that could have been adsorbed to the nuclei or chromatin during the isolation, it is advisable to localize the antibody in the cell histochemically. Figure 4 shows the intracellular localization of anti-nonhistone protein antibody by the peroxidase bridge technique. 14 The immunogen was a nonhistone p r o t e i n - D N A complex isolated from adult rat liver by the method described earlier. 1° I t is evident 1~See this series Vol. 37 [9].
198
TI~IE CELL NUCLEUS AND CHROMATIN PROTEINS
that the antibody reacts with the nuclear material. fluorescent-labeled antisera can be used for this purpose. 15
[16]
Alternatively,
1~L. S. Desai, L. Pothier, G. E. Foley, and R. A. Adams, Exp. Cell Res. 70, 468 (1971).
[16] C h r o m a t i n P r o t e i n K i n a s e s 1 B y VALERIEM. KISH and LEWIS J. KLEINSMITH
The phosphorylation of nonhistone chromatin proteins has been suggested to play a key role in the regulation of gene activity in higher organisms 2-~2 (also see Kleinsmith and Kish, this volume [14]). Currently, however, there is little information concerning the enzyme (s) involved in the phosphorylation of these proteins. The nonhistone chromatin phosphoprotein fraction is known to contain an endogenous protein kinase activity which catalyzes the phosphorylation of these proteins in the absence of added exogenous substrate2 ,13 This chapter is concerned with methods for fractionation and assay of these chromatin-associated protein kinases, and will show that chromatin contains a broad spectrum of different types of protein kinases with differing substrate specificities and sensitivity to control by cyclic AMP. The general experimental approach employed involves purification of phosphorylated nonhistone 1Studies on this subject in our laboratory have been supported by grants from the National Science Foundation (GB-8123 and GB-23921). V.M.K. held a predoctoral fellowship from U.S. Public Health Service Training Grant 5-T01-GM72-15. L. J. Kleinsmith, V. G. Allfrey, and A. E. Mirsky, Proc. Nat. Acad. Sci. U.S. 55, 1182 (1966). T. A. Langan, in "Regulation of Nucleic Acid and Protein Biosynthesis" (V. V. Koningsberger and L. Bosch, eds.), p. 233. Elsevier, Amsterdam, 1967. 4E. L. Gershey and L. J. Kleinsmith, Biochim. Biophys. Acta 194, 519 (1969). R. W. Turkington and M. Riddle, J. Biol. Chem. 244, 6040 (1969). K. Ahmed and H. Ishida, Mol. Pharmacol. 7, 323 (1971). 7R. D. Platz, V. M. Kish, and L. J. Kleinsmith, FEBS Lett. 12, 38 (1970). s C. S. Teng, C. T. Teng, and V. G. Allfrey, J. Biol. Chem. 246, 3597 (1971). M. Kamiyama, B. Dastugue, and J. Kruh, Biochem. Biophys. Res. Commun. 44, 1345 (1971). 1oN. C. Kostraba and T. Y. Wang, Biochim. Biophys. Acta 262, 169 (1972). 11p. B. Kaplowitz, R. D. Platz, and L. J. Kleinsmith, Biochim. Biophys. Acla 229, 739 (1971). i~ L. J. KIeinsmith and V. G. Allfrey, Biochim. Biophys. Acta 175, 136 (1969). 13L. J. Kleinsmith and V. G. Allfrey, Biochim. Biophys. Acta 175, 123 (1969).
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
191
choice of assay conditions, the purity and physical state of the DNA, and the technique chosen to monitor protein-DNA binding are all important variables t h a t must be considered when attempting to study the interaction of phosphorylated nonhistone proteins with DNA.
[15] I m m u n o c h e m i e a l C h a r a c t e r i s t i c s Chromosomal Proteins
of
B y F. CHYTIL
This chapter describes procedures for production of antibodies against histones and nonhistone chromosomal proteins, as well as against the nonfractionated chromatin. The conditions for testing the antigenicity of the chromosomal proteins by the method of quantitative complement fixation will be given also. This method appears to be a method of choice as it requires relatively small amounts of the chromosomal material which become very often a limiting factor in the determination of the properties of these proteins. The reader should consult first the introduction in general immunochemical techniques published in this series I as well as the detailed description of the method of quantitative complement fixation published earlierY Immunogens
Histones
To facilitate the production of antibodies against these basic proteins it is advisable either to bind them covalently to serum albumin 3 or to complex them with phosphorylated serum albumin 3 or to form a complex with R N A or DNA, 4 though free histones apparently could be used also2 Coupling the Histones with H u m a n Serum A l b u m i n ( H S A ) . ~ HSA, 100 mg, and 40 mg of whole histone are dissolved in 3 ml of H~0. Three milliliters of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p1See this series, Vol. 11 [91]. 2See this series, Vol. 11 [92]. SA. L. Sandberg, M. Liss, and B. D. Stollar. J. Immunol. 98, 1182 (1967). 4B. D. Stollar and M. Ward, J. Biol. Chem. 245, 1261 (1970). 5p. Rumke and M. Sluyser, Biochem. J. 1Ol, lc (1966).
192
THE CELL NUCLEUS AND CHROMATIN PROTEINS
[15]
toluenesulfonate 6 in H..O (75 mg/ml) are added, and the solution is stirred for 30 minutes at room temperature and then dialyzed for 24 hours against distilled H~O at 4 ° (25 mg of HSA, 10 mg of histone fraction, and 75 mg of the above carbodiimide can be used). Conjugates of HSA and the carbodiimide without histones can be also prepared. Coupling o] Histones with Phosphorylated Bovine Serum Albumin (BSA) PHOSPHORYLATED BSA. 7,8 BSA, 1.5 g is dissolved in 55 ml of 5% Na,.,HPO4 containing one drop of a phenolphthalein solution and placed in an ice-salt bath. Three milliliters of POC13 dissolved in 25 ml of carbon tetrachloride are added dropwise to the stirring BSA solution. Simultaneously, 1 M N a O H is added dropwise to maintain a p H of approximately 9 during the 4-hour reaction period. The reaction mixture is then poured into a dialysis bag and dialyzed overnight in the cold room against three changes of H~O. By twisting the dialysis bag at a level well above the carbon tetrachloride-water interphase, the aqueous phase could be separated. Alternatively, the carbon tetrachloride layer could be removed by centrifugation. Aliquots of the reaction mixture (at pH 8 to 9) are delivered into small tubes and stored frozen. The phosphorylated BSA can be isolated by acidification of the aqueous layer in the cold with 1 N hydrochloric acid to achieve maximum precipitation. The phosphorylated BSA is separated by centrifugation. COUPLING7 Phosphorylated BSA, 250 tLg in 50 td of water, is added to 2 ml of 0.15 sodium chloride solution containing 250 t~g of histone. The resulting solution is used for immunization. Histone Complexes with Nucleic Acids. 4,~ Equal amounts of yeast RNA and histones in 0.14 NaC1, 10 m M sodium phosphate buffer, pH 6.8, are mixed. In another procedure 600 ~g of each histone fraction is mixed with 200 t~g of RNA, or 2 mg of whole histone in 2 ml is added to 0.6 mg of DNA in 0.75 ml. Nonhistone Proteins
It is the author's experience that acidic proteins still complexed with DNA or free are good immunogens. 1°,1~ e T. L. Goodfriend, L. Levine, and G. Fasman, Science 144, 1344 (1964). 7H. Van Vunakis, J. Kaplan, H. Lehrer, and L. Levine, Immunochemistry 3, 393 (1966). 8 M. Heidelberger, B. Davis, and H. P. Treffers, J. Amer. Chem. Soc. 63, 498 (1941). g M. Bustin and B. D. Stollar, J. Biol. Chem. 247, 5716 (1972). ~oF. Chytil and T. C. Spelsberg, Nature (London) New Biol. 233, 215 (1971). ~1F. Chytil, unpublished observations.
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
193
Chromatin The preparation can be used directly for immunization.11
Immunization Procedure Rabbits: New Zealand White rabbits weighing between 3 and 4 kg are usually used. Complete Freund adjuvant is mixed with the solution of immunogen (1:1 or 1:1.5 v/v) in a syringe or homogenized in a small Teflon pestle glass homogenizer. Usually the immunization schedule used in this laboratory involves a toe pad injection of as much of the immunogen as possible on the first and eighth days, the rest being administered intramuscularly in multiple sites. About 10 days after administration of the complete Freund adjuvant, inflammation of the toe pads is observed. Then the injection in the toes has to be avoided, and the immunogen is administered intramuscularly only. Immunization Doses. The table shows the immunization doses for different chromosomal components. Intravenous Booster. Often an intravenous iniection into the marginal vein is given (Freund adjuvant has to be omitted) a week before bleeding. Bleeding. The animals are usually bled a week after the intravenous injection from the ear vein, and the sera are prepared as described earlier. 1 Antisera. Usually the sera can be used for testing without purification. However, sometimes, especially when a low dilution of the serum has to be employed, a high fixation of the complement in the absence of an antigen is observed in the complement fixation assay. In order to lower this property ("anticomplementarity") the globulin fraction can be purifled either by ammonium sulfate precipitation 12 or by ion exchange chromatography (DEAE-cellulose).12 In the author's laboratory the following procedure was found to remove substantially the "anticomplementarity." The sera are diluted with 9 volumes of 0.14 M sodium chloride and centrifuged at 105,000 g at 4 ° for 60 minutes. The serum is then carefully siphoned off, sterilized by Millipore filtration, and stored at --20 ° . Antigens. Varying amounts of antigens are used by diluting with the diluent used for the complement fixation assay. The antigens are stored frozen at --20 °. The histones and chromatins do not exert any "anticomplementarity." On the other hand, the nonhistone protein-DNA complexes may show limited binding of complement in the absence of anti12H. H. Fudenberg, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 306. Academic Press, New York, 1967.
194
THE
CELL
NUCLEUS
AND
CHROMATIN
[15]
PROTEINS
¢; o
o
o ::k o ~
0
0
0
g
o ~
o
Z
0
Z ['-. ~r:
Z ¢o © oo ~
..-4
© o o
v
o
v
Z ca
0 Z ;2 r~ W o
o
o
o
o
z
.
.~
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
195
body, which can be subtracted from that observed in the presence of antibody-antigen complexes. Repeated thawing and freezing of the antigens might induce a rise in "anticomplementarity." It is therefore advisable to store the antigens in small aliquots. Low "anticomplementarity" of nonhistone protein-DNA complexes is observed when the histones are removed by high urea, high salt treatment. TM On the other hand, when histones are extracted with acid the "anticomplementarity" of these preparations is frequently very high. Testing the Antigenicity The method of quantitative microcomplement fixation described earlier ~ is employed with small modification which consists in using onefifth of the incubation volume. Briefly, to disposable glass tubes 13 X 100 mm the following components are added: 0.6 ml of the diluent, 0.2 ml of diluted antibody, 0.2 ml of the antigen, and 0.2 ml of diluted complement to make the total volume 1.2 ml. After an overnight incubation at 4 °, 0.2 ml of the sensitized sheep red blood cells are added and the hemolysis is stopped usually after 45-60 minutes. The dilution of the antisera was found to range between 1/200 and 1/3200. The maximum complement fixation by chromosomal protein-antibody complexes varies from 0.5 to 2.0 t~g per assay tube. This amount is substantially higher than that found for another antigen. 2 The titer of the complement which has to be determined for each shipment varies in this laboratory between 1/125 and 1/175. Anti-histone Antibody. Figure 1 shows typical curves obtained when complement fixation assay was employed to test the antigenicity of antihistone antibody in the presence of different preparations of F1 histone. These results show the extent of reproducibility of this method. This laboratory has similar experience when different batches of nonhistone protein-DNA complexes were tested against homologous antibody. Anti-nonhistone Protein Antibody. Nonhistone proteins are apparently good immunogens. Complement-fixing antibody can be obtained when the rabbits are immunized with these proteins still attached to DNA 1° or with the free form. 11 Histones, double- or single-stranded DNA, do not interact with these antibodies. The antibodies react also, but to a smaller extent, with the chromatin preparation from the same organ 1° and show tissue specificity, which is demonstrated in Fig. 2. They react also with free nonhistone proteins. ~ Anti-chromatin Antibody. Antibody against the whole chromatin can be induced rather easily.~1 The results from this laboratory show in Fig. 3 i, F. Chytil and T. C. Spelsberg, unpublished observations.
196
THE
CELL
NUCLEUS
AND
CHROMATIN
PROTEINS
[15]
IO0 A
75
Z
_o
~ 5c X
7~ 25
0
1,0
2.0
ANTIGEN (~ug)
Fie. 1. C' fixation of antiserum to whole calf thymus F1 histone with varying preparations of whole calf thymus F1 histone. O, An old chromatographically purified preparation frozen and thawed several times; @, a fresh chromatographically purified preparation frozen and thawed once; X, a preparation before purification by column chromatography. Serum dilution was 1:1000. From M. Bustin and B. D. Stollar, J. Biol. Chem. 247, 5716 (1972). IOC
Z 0
Z) 25
/
•
o.o25 o.os o.,
o'.s
i
ANTIGEN Lug )
FIG. 2. C' fixation of antiserum to nonhistone protein-DNA complexes from chick oviduct by varying quantities of nonhistone protein-DNA complexes from chick oviduct, @; heart, X ; and liver, II. Serum dilution was 1:400. From F. Chytil and T. C. Spelsberg, Nalure (London) New Biol. 233, 215 (1971). that the immunization with chromatin leads to antibody reacting also with free histones. Moreover, these antibodies fix complement in the presence of nonhistone p r o t e i n - D N A complexes and do not react with DNA. Testing the Nuclear Origin of the I m m u n o g e n I n order to obtain additional evidence as to whether the antichromosomal protein antibody is directed against the nuclear material, and not
[15]
CHROMOSOMAL PROTEINS--IMMUNOCHEMISTRY
197
I00
75
Z 0
so
X LL
i)
25
.os
,,
.s
i
ANTIGEN (#g)
Fro. 3. C' fixation of antiserum to chromatin from chick oviduct by varying quantities of chromatin, O, and whole histone, ×. Serum dilution was 1:400. From unpublished data of F. Chytil and T. C. Spelsberg.
FIG. 4. Intracellular localization of antiliver nonhistone protein-DNA antibodies in liver slices by peroxidase bridge analysis described in this series (Vol. 37 [9]). LeJt: Adult rat liver slices incubated with control serum. Right: With anti-liver nonhistone protein antibody. From unpublished experiments of F. Chytil and P. K. Nakane. against some cytoplasmic components that could have been adsorbed to the nuclei or chromatin during the isolation, it is advisable to localize the antibody in the cell histochemically. Figure 4 shows the intracellular localization of anti-nonhistone protein antibody by the peroxidase bridge technique. 14 The immunogen was a nonhistone p r o t e i n - D N A complex isolated from adult rat liver by the method described earlier. 1° I t is evident 1~See this series Vol. 37 [9].
198
TI~IE CELL NUCLEUS AND CHROMATIN PROTEINS
that the antibody reacts with the nuclear material. fluorescent-labeled antisera can be used for this purpose. 15
[16]
Alternatively,
1~L. S. Desai, L. Pothier, G. E. Foley, and R. A. Adams, Exp. Cell Res. 70, 468 (1971).
[16] C h r o m a t i n P r o t e i n K i n a s e s 1 B y VALERIEM. KISH and LEWIS J. KLEINSMITH
The phosphorylation of nonhistone chromatin proteins has been suggested to play a key role in the regulation of gene activity in higher organisms 2-~2 (also see Kleinsmith and Kish, this volume [14]). Currently, however, there is little information concerning the enzyme (s) involved in the phosphorylation of these proteins. The nonhistone chromatin phosphoprotein fraction is known to contain an endogenous protein kinase activity which catalyzes the phosphorylation of these proteins in the absence of added exogenous substrate2 ,13 This chapter is concerned with methods for fractionation and assay of these chromatin-associated protein kinases, and will show that chromatin contains a broad spectrum of different types of protein kinases with differing substrate specificities and sensitivity to control by cyclic AMP. The general experimental approach employed involves purification of phosphorylated nonhistone 1Studies on this subject in our laboratory have been supported by grants from the National Science Foundation (GB-8123 and GB-23921). V.M.K. held a predoctoral fellowship from U.S. Public Health Service Training Grant 5-T01-GM72-15. L. J. Kleinsmith, V. G. Allfrey, and A. E. Mirsky, Proc. Nat. Acad. Sci. U.S. 55, 1182 (1966). T. A. Langan, in "Regulation of Nucleic Acid and Protein Biosynthesis" (V. V. Koningsberger and L. Bosch, eds.), p. 233. Elsevier, Amsterdam, 1967. 4E. L. Gershey and L. J. Kleinsmith, Biochim. Biophys. Acta 194, 519 (1969). R. W. Turkington and M. Riddle, J. Biol. Chem. 244, 6040 (1969). K. Ahmed and H. Ishida, Mol. Pharmacol. 7, 323 (1971). 7R. D. Platz, V. M. Kish, and L. J. Kleinsmith, FEBS Lett. 12, 38 (1970). s C. S. Teng, C. T. Teng, and V. G. Allfrey, J. Biol. Chem. 246, 3597 (1971). M. Kamiyama, B. Dastugue, and J. Kruh, Biochem. Biophys. Res. Commun. 44, 1345 (1971). 1oN. C. Kostraba and T. Y. Wang, Biochim. Biophys. Acta 262, 169 (1972). 11p. B. Kaplowitz, R. D. Platz, and L. J. Kleinsmith, Biochim. Biophys. Acla 229, 739 (1971). i~ L. J. KIeinsmith and V. G. Allfrey, Biochim. Biophys. Acta 175, 136 (1969). 13L. J. Kleinsmith and V. G. Allfrey, Biochim. Biophys. Acta 175, 123 (1969).