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Chapter 19. Histone Hydrolase
Chapter 19 Histone Hydrolase WOON KI PAIK
AND
SANGDUK KIM
Fels Research Institute and Department of Biochemistry, Temple University School ojiuedicine, Philadelphia, Pennsylvania
I. Introduction Histones are synthesized in the cytoplasm and are transported into the nucleus, where they exist in conjunction with DNA at an approximate ratio of unity (I). The role that histones play in controlling genetic expression is not yet clear. The cell apparently maintains a vital regulatory process in controlling the amount of histone available in order to limit its binding to DNA in a constant ratio, thus it would seem that any excess histones must be disposed of in some way. One of the mechanisms to maintain the homeostasis is the presence of proteolytic enzymes which are highly specific toward histones. Neutral histone hydrolase has been observed in various cellular fractions; chromatin ( 2 4 , microsomes (9,or cytosol (6,7).
11. Assay Method A.
Principle
Histones are hydrolyzed by the enzyme and the material, which is then rendered soluble in 5% trichloroacetic acid, is determined by allowing it to react with ninhydrin. Since the optimum pH for histone hydrolase activity varies with the tissue, one must first determine this parameter. The following assay condition has been used for the enzyme from rat kidney.
B.
Reagents
Histone suspension (3 mg per milliliter of water; histone type 11-A of Sigma Chemical Co. is routinely used) 19 1
192
WOON K1 PAIK AND SANGDUK KIM
Tris-HC1 buffer (PH 9.0),0.5 M Trichloroacetic acid, 10% NaOH, 1 N Sodium citrate buffer 0.2 M, @H 5.0) Ninhydrin, 4%, in ethyl Cellosolve (ethylene glycol monoethyl ether; Fisher Chemical Co.) SnCl, suspension in water, 50 mg/ml; prepare immediately before use
C. Procedure The tissue was homogenized in 0.25 M sucrose-6 mM CaCl, or in cold water to yield a 20% homogenate with an electrically driven Teflon-glass homogenizer. The homogenate was passed through adouble layer of cheesecloth. One-tenth or 0.2 ml of tissue homogenate or a fractionated enzyme preparation, 0.2 ml of histone suspension, 0.1 ml of Tris-HC1 buffer in a total volume of 0.5 ml were incubated at 37OC for 20 minutes. Thereaction was terminated by the addition of 0.5 ml of loo/, trichloroacetic acid solution. For a control, the enzyme suspension was added after the reaction was stopped by trichloroacetic acid. The mixture was transferred into a Sorvall centrifuge tube and was centrifuged at 39,000 g for 10minutes. A portion of the clear supernatant (usually 0.1 x 0.2 ml) was transferred to a Coleman spectrophotometer cuvette (19 x 105 mm), and apredetermined amount of NaOH solution was added to bring the pH of the solution to about 5 . The volume was adjusted to 1.0 ml with water, and the ninhydrin color was developed as follows. Thirty milliliters of sodium citrate buffer and an equal volume of ninhydrin solution was first mixed thoroughly, and 1 ml of SnCl, solution was added. The solution was completely mixed. One milliliter of this ninhydrin solution was added into the above Coleman cuvette and the cuvette was capped with a rubber stopper with a capillary tube. The cuvette was heated in a boiling water bath for 5 minutes, and was then cooled for 3 minutes. Five milliliters of water was added into the cuvette, and absorbancy at 580 nm was read in a Coleman spectrophotometer. Duplicate determinations at two enzyme concentrations have been determined and the value was corrected for the control.
D. Definition of Specific Activity The enzyme activity is expressed as specific activity, which corresponds to A,,,/20 minutes per milligram of enzyme protein. One unit of A,,, corresponds to 0.24 pmole of leucine. The enzyme activity represents an increase of ninhydrin color in 5% trichloroacetic acid-soluble fraction after
19.
HISTONE HYDROLASE
193
allowing the histone to react with the enzyme preparation. Enzyme protein concentration was determined by the method of Lowry et al. (8).
111. Properties A. Specificity The enzyme is highly specific toward histones. Table I lists specificity of histone hydrolase obtained from various sources. With calf thymus chromatin-associated histone hydrolase, the susceptibility of various histones to hydrolysis is greatly dependent on whether the histones are conjugated with DNA or not (3). In the nucleohistone complex, the lysine-rich (Fl) and arginine-rich (F3) histones are degraded, and the rest of the histones are resistant. When freed, only lysine-rich histone becomes resistant to the hydrolytic activity, and the rest of the histones are rapidly degraded.
B. OptimumpH Optimum pH for histone hydrolase varies depending on the sources. However, the enzyme activities listed in Table I indicate that the enzyme in general may be characterized as a neutral protease.
C. Subcellular Distribution The neutral histone hydrolase has been found in chromatin isolated from both calf thymus (2,3) and rat liver nuclei (4), in the microsomal fraction of rat kidney (9,in the cytosol fraction of tadpole liver (6) and regenerating limb of the adult newt Diemicfylusviridescens (7).
D. Inhibitors Chromatin-bound enzyme is completely inhibited by sodium bisulfite at concentration of 0.05 M (4). Serine-specific protease inhibitors, such as phenylmethanesulfonyl fluoride and diisopropylfluorophosphate, and the alkylating reagent, carbobenzoxyphenylalanine chloromethylketone inhibit the enzyme reversibly at 1 mMconcentration (9). Rat liver chromatin-bound protease is still active in the presence of 2 NNaCl-5 Murea @H 6 4 9 , which has been most often used for dissociation and reconstitution of chromatin (9).
194
WOON KI PAIK AND SANGDUK KIM
SPECInClTY Organisms and organs: Subcellular localization: Optimum pH: Substrate Histone (crude) F1 histone F2 histone F3 histone Polylysine Rotamine Casein Hemoglobin (denatured) Ribonuclease Lysozyme Polyarginine Albumin Globulin Polyleucine Polyglutamic acid Reference no.
TABLE I HISTONE HYDROLASE
OF
Rat liver Chromatin 8.2
IW
320 9 18 0 5
2
Rat kidney Tadpole liver Newt limb Microsome cytosol cytosol 8-9 6-8 6-7
100 157 128 44 43
I29
100 180
100 24
227 72 290 150
17 74 4
44 32 6 0 0
0 0 (4)
(5)
a Percentage activity.
E. Enzyme Purification Furlan et al. (10)purified histone hydrolase from calf thymus nuclei 220fold with 64% yield. Approximately 700-fold purification was also achieved from rat liver chromatin (11). It has a molecular weight of 200,000.
IV. Biological Significance Biological significance of histone hydrolase is not clear at present. Since nuclear histones are turning over at an extremely slow rate (IZ),it does not seem probable that histone hydrolase removes histones as a means of gene activation. However, the cytosolic histone hydrolase of regenerating limb of the adult newt D. viridescens increased about 2-fold during the wound healing period (7). A similar increase in hydrolase activity was observed in liver of tadpoles during thyroxine-induced metamorphosis (13). These two observations suggest that histone hydrolase might play an important role in maintaining the balance between histone synthesis and the amount of histone influx to the nucleus.
19.
HISTONE HYDROLASE
195
It should be noted here that recent evidence indicates that so-called chromatin-bound histone hydrolase might represent contamination with a cytosolic enzyme (14,15).
REFERENCES 1. Hnilica, L. S., “The Structure and Biological Function of Histones,” p. 57. CRC Prcss,
Cleveland, Ohio (1972). 2. Furlan, M., and Jericijo, M.,Biochim. Biophys. Actu 147, 135 (1967). 3. Bartley, J., and ChalkIey, R., J. BioI. Chem. 245, 4286 (1970). 4. Garrels, J . I., Elgin, S. C. R., and Bonner, J., Biochem. Biophys. Res. Comrmcn. 46,545 (1 972). 5. Paik, W. K., and Lee, H. W., Biodrem. Biophys. Res. Commun. 38,333 (1970). 6. Paik, W . K., and Lee, H. W., Experlenriu27,630 (1971). 7. Procaccini, D. J., Procaccini, R. L.,and Pease, J. B., Oncologv29, 265 (1974). 8. Lowry, 0. H., Rosebrough, M. J., Farr, A. L., and Randall, R. J.,J. Biol. Chem. 193, 265 (1951). 9. Carter, D. B., and Chae, C. B., Biochemistry 15, 180 (1976). 10. Furlan, M., Jericijo, M., and Suhar, A., Bioclrim, Biophys. Ado 167, 154 (1%8). 11. Chong, M. T., Garrard, W. T., and Bonner, J., Biochemistry 13,5128 (1974). 12. Hnilica, L. S., “The Structure and Biological Function of Histones,” p. 64.CRC Press, Cleveland, Ohio (1972). 13. Paik, W. K.,and Kim,S., unpublished results. 14. Raydt, G., and Heinrich, P. S., Hoppe-Seyler’s Z.Physiol. Chem. 356,267 (1975). 15. Destree, 0. H. J., D’Adelhart-Toorop, H. A., and Charles, R., Biochim. Biophys. Ado 378,450 (1975).
AND
SANGDUK KIM
Fels Research Institute and Department of Biochemistry, Temple University School ojiuedicine, Philadelphia, Pennsylvania
I. Introduction Histones are synthesized in the cytoplasm and are transported into the nucleus, where they exist in conjunction with DNA at an approximate ratio of unity (I). The role that histones play in controlling genetic expression is not yet clear. The cell apparently maintains a vital regulatory process in controlling the amount of histone available in order to limit its binding to DNA in a constant ratio, thus it would seem that any excess histones must be disposed of in some way. One of the mechanisms to maintain the homeostasis is the presence of proteolytic enzymes which are highly specific toward histones. Neutral histone hydrolase has been observed in various cellular fractions; chromatin ( 2 4 , microsomes (9,or cytosol (6,7).
11. Assay Method A.
Principle
Histones are hydrolyzed by the enzyme and the material, which is then rendered soluble in 5% trichloroacetic acid, is determined by allowing it to react with ninhydrin. Since the optimum pH for histone hydrolase activity varies with the tissue, one must first determine this parameter. The following assay condition has been used for the enzyme from rat kidney.
B.
Reagents
Histone suspension (3 mg per milliliter of water; histone type 11-A of Sigma Chemical Co. is routinely used) 19 1
192
WOON K1 PAIK AND SANGDUK KIM
Tris-HC1 buffer (PH 9.0),0.5 M Trichloroacetic acid, 10% NaOH, 1 N Sodium citrate buffer 0.2 M, @H 5.0) Ninhydrin, 4%, in ethyl Cellosolve (ethylene glycol monoethyl ether; Fisher Chemical Co.) SnCl, suspension in water, 50 mg/ml; prepare immediately before use
C. Procedure The tissue was homogenized in 0.25 M sucrose-6 mM CaCl, or in cold water to yield a 20% homogenate with an electrically driven Teflon-glass homogenizer. The homogenate was passed through adouble layer of cheesecloth. One-tenth or 0.2 ml of tissue homogenate or a fractionated enzyme preparation, 0.2 ml of histone suspension, 0.1 ml of Tris-HC1 buffer in a total volume of 0.5 ml were incubated at 37OC for 20 minutes. Thereaction was terminated by the addition of 0.5 ml of loo/, trichloroacetic acid solution. For a control, the enzyme suspension was added after the reaction was stopped by trichloroacetic acid. The mixture was transferred into a Sorvall centrifuge tube and was centrifuged at 39,000 g for 10minutes. A portion of the clear supernatant (usually 0.1 x 0.2 ml) was transferred to a Coleman spectrophotometer cuvette (19 x 105 mm), and apredetermined amount of NaOH solution was added to bring the pH of the solution to about 5 . The volume was adjusted to 1.0 ml with water, and the ninhydrin color was developed as follows. Thirty milliliters of sodium citrate buffer and an equal volume of ninhydrin solution was first mixed thoroughly, and 1 ml of SnCl, solution was added. The solution was completely mixed. One milliliter of this ninhydrin solution was added into the above Coleman cuvette and the cuvette was capped with a rubber stopper with a capillary tube. The cuvette was heated in a boiling water bath for 5 minutes, and was then cooled for 3 minutes. Five milliliters of water was added into the cuvette, and absorbancy at 580 nm was read in a Coleman spectrophotometer. Duplicate determinations at two enzyme concentrations have been determined and the value was corrected for the control.
D. Definition of Specific Activity The enzyme activity is expressed as specific activity, which corresponds to A,,,/20 minutes per milligram of enzyme protein. One unit of A,,, corresponds to 0.24 pmole of leucine. The enzyme activity represents an increase of ninhydrin color in 5% trichloroacetic acid-soluble fraction after
19.
HISTONE HYDROLASE
193
allowing the histone to react with the enzyme preparation. Enzyme protein concentration was determined by the method of Lowry et al. (8).
111. Properties A. Specificity The enzyme is highly specific toward histones. Table I lists specificity of histone hydrolase obtained from various sources. With calf thymus chromatin-associated histone hydrolase, the susceptibility of various histones to hydrolysis is greatly dependent on whether the histones are conjugated with DNA or not (3). In the nucleohistone complex, the lysine-rich (Fl) and arginine-rich (F3) histones are degraded, and the rest of the histones are resistant. When freed, only lysine-rich histone becomes resistant to the hydrolytic activity, and the rest of the histones are rapidly degraded.
B. OptimumpH Optimum pH for histone hydrolase varies depending on the sources. However, the enzyme activities listed in Table I indicate that the enzyme in general may be characterized as a neutral protease.
C. Subcellular Distribution The neutral histone hydrolase has been found in chromatin isolated from both calf thymus (2,3) and rat liver nuclei (4), in the microsomal fraction of rat kidney (9,in the cytosol fraction of tadpole liver (6) and regenerating limb of the adult newt Diemicfylusviridescens (7).
D. Inhibitors Chromatin-bound enzyme is completely inhibited by sodium bisulfite at concentration of 0.05 M (4). Serine-specific protease inhibitors, such as phenylmethanesulfonyl fluoride and diisopropylfluorophosphate, and the alkylating reagent, carbobenzoxyphenylalanine chloromethylketone inhibit the enzyme reversibly at 1 mMconcentration (9). Rat liver chromatin-bound protease is still active in the presence of 2 NNaCl-5 Murea @H 6 4 9 , which has been most often used for dissociation and reconstitution of chromatin (9).
194
WOON KI PAIK AND SANGDUK KIM
SPECInClTY Organisms and organs: Subcellular localization: Optimum pH: Substrate Histone (crude) F1 histone F2 histone F3 histone Polylysine Rotamine Casein Hemoglobin (denatured) Ribonuclease Lysozyme Polyarginine Albumin Globulin Polyleucine Polyglutamic acid Reference no.
TABLE I HISTONE HYDROLASE
OF
Rat liver Chromatin 8.2
IW
320 9 18 0 5
2
Rat kidney Tadpole liver Newt limb Microsome cytosol cytosol 8-9 6-8 6-7
100 157 128 44 43
I29
100 180
100 24
227 72 290 150
17 74 4
44 32 6 0 0
0 0 (4)
(5)
a Percentage activity.
E. Enzyme Purification Furlan et al. (10)purified histone hydrolase from calf thymus nuclei 220fold with 64% yield. Approximately 700-fold purification was also achieved from rat liver chromatin (11). It has a molecular weight of 200,000.
IV. Biological Significance Biological significance of histone hydrolase is not clear at present. Since nuclear histones are turning over at an extremely slow rate (IZ),it does not seem probable that histone hydrolase removes histones as a means of gene activation. However, the cytosolic histone hydrolase of regenerating limb of the adult newt D. viridescens increased about 2-fold during the wound healing period (7). A similar increase in hydrolase activity was observed in liver of tadpoles during thyroxine-induced metamorphosis (13). These two observations suggest that histone hydrolase might play an important role in maintaining the balance between histone synthesis and the amount of histone influx to the nucleus.
19.
HISTONE HYDROLASE
195
It should be noted here that recent evidence indicates that so-called chromatin-bound histone hydrolase might represent contamination with a cytosolic enzyme (14,15).
REFERENCES 1. Hnilica, L. S., “The Structure and Biological Function of Histones,” p. 57. CRC Prcss,
Cleveland, Ohio (1972). 2. Furlan, M., and Jericijo, M.,Biochim. Biophys. Actu 147, 135 (1967). 3. Bartley, J., and ChalkIey, R., J. BioI. Chem. 245, 4286 (1970). 4. Garrels, J . I., Elgin, S. C. R., and Bonner, J., Biochem. Biophys. Res. Comrmcn. 46,545 (1 972). 5. Paik, W. K., and Lee, H. W., Biodrem. Biophys. Res. Commun. 38,333 (1970). 6. Paik, W . K., and Lee, H. W., Experlenriu27,630 (1971). 7. Procaccini, D. J., Procaccini, R. L.,and Pease, J. B., Oncologv29, 265 (1974). 8. Lowry, 0. H., Rosebrough, M. J., Farr, A. L., and Randall, R. J.,J. Biol. Chem. 193, 265 (1951). 9. Carter, D. B., and Chae, C. B., Biochemistry 15, 180 (1976). 10. Furlan, M., Jericijo, M., and Suhar, A., Bioclrim, Biophys. Ado 167, 154 (1%8). 11. Chong, M. T., Garrard, W. T., and Bonner, J., Biochemistry 13,5128 (1974). 12. Hnilica, L. S., “The Structure and Biological Function of Histones,” p. 64.CRC Press, Cleveland, Ohio (1972). 13. Paik, W. K.,and Kim,S., unpublished results. 14. Raydt, G., and Heinrich, P. S., Hoppe-Seyler’s Z.Physiol. Chem. 356,267 (1975). 15. Destree, 0. H. J., D’Adelhart-Toorop, H. A., and Charles, R., Biochim. Biophys. Ado 378,450 (1975).