Eur. J . Biochem. 62. 37-43 (1976)

Subcellular Distribution of Histone-Degrading Enzyme Activities from Rat Liver Peter C. HEINRICH, Gerhard RAYDT, Bernd PUSCHENDORF, and Mirna JUSIC Biochemisches Institut der Universitat, Freiburg (Received May 27/October 30, 1975)

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0- 5.0 was found for the lysosomal fraction, whereas 7.5- 8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.O mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. Proteinases associated with chromatin isolated from calf thymus [I - 41 or rat liver [5,6] have been described. The biological role of this enzyme is still unknown. During the preparation of this manuscript two controversial publications on this subject have appeared. Chong et al. [7] have described the purification of a neutral chromatin-associated proteinase from rat liver, whereas Destree et al. [8) have concluded from their studies that the neutral proteinase found in rat liver chromatin originates from cytoplasmic organelles. The results of our work strongly support the experiments of Destrke et al.

(Trasylolm) was a gift of Dr E. Truscheit, Bayer AG, Wuppertal-Elberfeld. All chemicals were of highest purity grade available. Animals

Male Wistar rats between 150 g and 200 g were used. Ehrlich ascites tumor cells were grown in NMRI mice (Zentralinstitut fur Versuchstierzucht, Hannover, Germany) and harvested as described by Grunicke et al. [9]. Chromatin Preparation

MATERIALS AND METHODS Protein kinase (beef heart), was purchased from Sigma (St Louis, U.S.A.), Triton RW 1339 and PhMeS0,F from Serva (Heidelberg, Germany). [y32P]ATP (16 Ci/mmol) and [4,5-3H]leucine (53 Ci/ mmol) were obtained from the Radiochemical Centre (Amersham, England). Kallikrein-trypsin inhibitor Abbreviations. PhMeSO,F, phenylmethanesulfonyl fluoride; succinatc-tetrazolium reductase, succinate-2-(p-iodophenyl)-3-@nitrophen yl)-5-phenyltetrazolium reductase. Enzyme. Acid phosphatase (EC 3.1.3.2).

Chromatin was isolated (A) from purified nuclei [lo] according to the procedure described by Spelsberg and Hnilica [ll], and (B) from a whole homogenate according to Bonner et al. [12], with the exception that the liver was not frozen. Preparation of Escherichia coli RNA Polymerase and Transcription of Chromatin

RNA polymerase was prepared according to Burgess [13]. The RNA-synthesizing system contained

Histone-Degrading Enzyme Activities from Rat Liver

38

in a total volume of 0.25 ml (in pmol): Tris/HCl (pH 7.9) 10; KCl40; 2-mercaptoethanol3; EDTA 2; Mg Clz 2; CTP, UTP, GTP, ATP 0.05 each; 10 pCi [5,6-3H]uridine 5'-triphosphate, ammonium salt (specific activity 20 Ci/mmol); 11.5 pg E. coli RNA polymerase (specific activity 310 units/mg) and 10 pg of DNA from calf thymus or chromatin. The mixture was incubated for 20 min at 37 "C. The reaction was stopped by the addition of 0.2 ml of bovine serum albumin (0.3%) and 10 ml of trichloroacetic acid ( 5 %). The precipitate was spun down by centrifugation at 4000 x g for 20 min, washed twice with 30 ml of 5 % trichloroacetic acid, and dissolved in 0.3 ml of 0.2 N NaOH. The samples were suspended in 10 ml of scintillation fluid (naphthalene 738 g, 2,5-diphenyloxazol 46 g, 2-(1-naphthyl)-5-phenyloxazol 0.46 g, xylene 3.5 I, dioxane 3.5 I, ethanol 2.1 1) and counted in a liquid-scintillation spectrophotometer after 12 h at 4 "C in the dark. Preparation of [3H]Leucine-Laheled Histones .from Ehrlich Ascites Tumor Cells 4- 5 days after transplantation of Ehrlich ascites tumor cells 150 pCi of [4,5-3H]leucine (53 Ci/mmol) was injected into each of 10 mice of 25- 30 g weight. The animals were killed after 60 min and the histones were prepared according to [141. Specific activities of 1.0- 1.9 x lo6 dis x min-' mg-' of total histone protein were obtained. Proteinuse Assay. The proteinase assay contained in a total volume of 90 PI 0.1 M Tris/HCl (between pH 7 and 9.5) or 0.1 M citrate buffer (between pH 2 and 6), 20 pg of total [4,5-3H]leucine-labeled histones (approx. 21 000 dis./min) and the proteinase to be assayed. After a 3-h incubation time at 37 "C 0.2 mg of bovine serum in 20 p1 was added, immediately followed by the addition of 20 p1 of trichloroacetic acid to give a final acid concentration of 20 After centrifugation at 12000 x g for 5 min 100 pl of clear supernatant in 10 ml of scintillator fluid containing 333 ml of Triton X-100, 666 ml toluene, 2.7g of 2,5-diphenyloxazole and 66 mg of 1,4-bis-2-(4-methyl5-phenoxazolyl)benzene/l, was used for scintillation counting.

x.

Preparation of Suhcellular Particles and Assays of' Respective Marker Enzymes

Nuclei were prepared according to Blobel and Potter [lo], mitochondria according to Loewenstein et al. [15]. Lysosomes have been isolated 3.5 days after injection of Triton RW 1339 into 200-g male Wistar rats [16]. Microsomes were prepared according to [I71 and cytosol was obtained by very gentle homogenization of liver tissue and high-speed centrifugation [18]. Glucose 6-phosphatase [19], acid phosphatase

1201 and succinate tetrazolium reductase [21] have been used as marker enzymes. Protein determinations were performed according to Lowry [22], with bovine serum albumin as standard.

RESULTS Two different methods for the preparation of chromatin have been used. Method A . Chromatin was prepared from purified nuclei by several washings with different salt solutions according to the procedure described by Spelsberg and Hnilica [ll]. Method B. Chromatin was prepared from liver tissue by homogenization in a Waring blender according to Bonner et al. [12]. The composition of the chromatin preparations is shown in Table 1. Chromatin obtained from isolated nuclei (chromatin A) has a lower template activity than the chromatin preparation obtained by sedimentation from a liver homogenate (chromatin B). Histone-degrading proteinases associated with chromatin have been described for chromatin from calf thymus [l -41 and from rat liver [5,6]. These proteolytic enzymes have been assayed by densitometric measurements of the stained histone bands after acrylamide gel electrophoresis [3,8]. In order to assay proteolytic activity more rapidly and with a higher sensitivity a radioactive assay was needed. Since it is not possible to obtain histones of high specific activity in adult rat liver, and since histones do not show significant differences in a wide spectrum of species [25], Ehrlich ascites tumor cells have been used for labelling and isolation of histones. The 3H-labeled total histones from Ehrlich ascites tumor cells showing the typical protein bands after acrylamide gel electrophoresis [26] served as proteinase

Tablc 1. Composition and teniplate ric/ivities of d(fleren/ elirormtin prep urations Values for histone and nonhistone proteins as well as RNA are expressed as mg based on 1 mg of DNA. DNA was determined according to [23], RNA according to [24]. Histones were extracted with 0.25 N H,SO,, nonhistone proteins with 0.1 N NaOH. The values of the template activities are related to 100% obtained with calf thymus DNA. The assay conditions have been described in Materials and Methods Preparation

DNA

Protein

RNA

-

~

-

histone

nonhistone

Chromatin A 1.0

0.98

0.51

0.09

ChromatinB 1.00

1.3

1.0

0.03

Template activity

2.4 27

P. C. Heinrich, G. Raydt. B. Puschendorf, and M. Jusii.

I

1

39 Table 2. Comparison of histone-degrading activity of variousfiac,tions during the preparation of chromatin from pur[fied nuclei I A j and j r o m whole liver homogenate ( B ) The proteinase assay conditions are described under Materials and Methods Prcparation

x Specific activity

Fraction

counts min-' mg protein-'

100

90

ao

70

60

A

homogenate (Potter-Elvehjem) 5.48 nuclei c 0.04 nuclear sap < 0.04 chromatin A c 0.04

B

homogenate (waring blender) 'nuclei' crude chromatin chromatin B

9.20 12.2 10.2 21.3

50

Intensity ( % )

Fig. 1. Correhlion qf tric/iloroacetic-acid-soluble radioactivity tiith the decrease in intensity of stuined total histones. The incubation mixture contained in a total volume of 180 PI: 0.1 M Tris/HCl, pH 8, 80 pg of total [4,5-3H]leucine-labeled histones and 0.03 mg of chromatin isolated according to [12]. After incubation for 0, 1, 2 and 3 h a t 37 "C an aliquot of 90 p1 was mixed with 20 p1 of bovine serum albumin (10 mg/ml) and precipitated with 20 p1 of trichloroacetic acid (110%). After centrifugation at 12OOOxg for 5 min 100 pl of supernatant was used for the determination of radioactivity. To the remaining 90 pl of incubation mixture 50 p1 of 10 M urea in 3 M acetic acid was added after 0, 1, 2 and 3 h of incubation at 37 "C. 100 pI of this mixture was used for acrykdmide gel electrophoresis [26],the gels were stained with amido black overnight and destained with 7 acetic acid. The amount of stain in each histone band was determined by scanning the gel with a Gilford gel scanner model 2520. The traces for all histone bands were cut out and weighed. The values given on the abscissa are percentages of untreated histones

substrate. It was checked that trichloroacetic-acidsoluble radioactivity measured in the proteinase assay correlated with the decrease in intensity of the stained histone bands (Fig. 1). The different methods for the preparation of chromatin have been compared in respect to histonedegrading enzyme activities, assayed at pH 7.5 (Table 2). The homogenate for the chromatin preparation according to Bonner et al. (B) exhibits already a 2-fold higher proteinase activity in the homogenate compared with the homogenate used for the preparation of nuclei (A). The histone-degrading enzyme activity was absent in the nuclear fraction (A), whereas proteinase activity was found in all steps during the chromatin preparation according to Bonner et al. [12]. Fig. 2 shows that the pH optimum of the proteinase associated with chromatin is around 7.5- 8.0. The fact that chromatin prepared from isolated nuclei was devoid of proteinase activity might be due to the loss of endogenous chromatin-associated pro-

o ! 1

. . .

2

3

4

.

5

I

I

6 7

I

8

I

.

I

I

9 1 O 1 1 1 2

PH

Fig. 2. CIfect oJ'pH on chromatin-associated activity. (0)Chromatin prepared according to [12]; (A) chromatin isolated from nuclei [ll]. Assay conditions are given in Materials and Methods. 0.03 mg of chromatin = 0.12 A260unit was used in the incubation mixtures of various pH values

teinase(s) during the preparation of nuclei. On the other hand, the proteinase activity found in the chromatin preparation (B) might well be a contamination due to extrachromosomal material generated by the vigorous homogenization of the liver cells. Therefore, it was of interest to study the susceptibility of 3H-labeled histones to various subcellular fractions. Nuclei, microsomes and cytosol did not show histonedegrading activities. Fig. 3 shows that the lysosomal fraction contained the highest specific proteinase activity over a pH range from 2- 9. The mitochondria exhibited two p H maxima of lower specific proteinase activities. The first pH maximum at pH 4-5 (not shown) disappeared when the mitochondria were treated with digitonin, a procedure which preferentially destroys lysosomes [15]. This indicates that the shoulder at pH 3 - 5 is due to lysosomal contamina-

Histone-Degrading Enzyme Activities from Rat Liver

40 , 9

Table 3. Marker enzyme activities of

the sub~ellulurfractionsisol(itcd

,from rut liver

The isolation methods for mitochondria and lysosomcs as well as the procedures for the respective cnzyme assays are mentioned in Materials and Methods. The percentage recoveries for the marker enzymes arc given in parentheses F

c .-

Fraction

E

Succinatetetrazoliumreductase

Acid phosphatase

Glucose6-phosphatasc

(x)

pmolh-' mg-l pmoI min-' ing-' ( %)

0.02 ( 4) 2.82 ( 11) 0.05 (100)

Mitochondria 8.1 ( 42) Lysosomes t 0 . 1 ( 0) Homogenatc 1.1 (100)

al

c m ._ al

-e

0.015 ( 2.5) 0.48 ( 0.6) 0.06 (100 )

a

Table 4. Binding studirs of mitochondrial or lysosomal proteinases to proteinase+ree chromatin Preparation 0 1

2 3 4 5 6 7 8 9 1 0 1 1

PH Fig. 3. p H depependencc. ofthe [ 3H]liwc~ne-labeled histone degradation hy subcellular fruczions. (0)Lysosornal ; (A) mitochondrial fraction after digitonin treatment. Assay conditions are given in Materials and Methods

Sucrose gradient centrifugation bcfore

after

total total proteinase protein activity added

prokin/ pellet

pH5

pH5 p H 8

pH8 mg

mg

tion. Nevertheless, the marker enzyme activities demonstrate a high degree of purity for both fractions (Table 3). Fig. 4 shows the degradation of [3H]leucine-labeled histones analyzed on acrylamide gels and stained with amido black. The histones were incubated at 37 "C and 0 "C for 3 h at pH 8.0 with chromatin obtained according to [12] (a,b), with mitochondria (c,d) or with lysosomes (e,f). (g) shows histones incubated at 37 "C for 3 h. The degradation patterns for the chromatin-associated and the mitochondrial proteinases are very similar compared to the lysosomes, which give rise to different bands already during incubation at 0 "C for 3 h (f). At pH 5.0 and at 0 "C the lysosomal proteinases are so active that an extensive degradation is found during the 3-h incubation period. However, the H1 histone seems to be resistent to the proteinase action (not shown). In order to exclude the possibility that histones isolated from Ehrlich ascites tumor cells might have a different proteinase susceptibility compared to histones from normal liver (because the rapidly dividing tumor cells exhibit a higher degree of phosphorylation [27]) these histones have been extensively phosphorylated by use of protein kinase. The degree of phosphorylation was estimated by measurement of [32P]phosphate incorporation. A ratio of 4.3 nmol [32P]phosphate/mgof total histone protein has been determined.

proteinasc activity/ pellet

Chromatin + sucrose

0.26

0

0

0.25

0

Chromatin + mitochondria

4.30

0 8000

0.32

0 4370

Chromatin lysosomes

0.70

88000 4100

0.27

+

1800

0

300

The time course of proteolysis of extensively phosphorylated histones and the untreated histone preparation was studied. A negligible difference has been found in the susceptibility of both substrates to lysosomal, mitochondrial or chromatin-associated proteinases at optimal pH conditions (not shown). The lysosomal fraction, which contains an active acid phosphatase activity did not liberate any [32P]phosphate from the phosphorylated histones. Therfore, the degree of histone phosphorylation does not affect the susceptibility to proteolytic degradation. In order to study the possible interaction of lysosoma1 or mitochondrial proteinases with proteinasefree chromatin, lysosomes or mitochondria disintegrated by freezing and thawing have been incubated at 4 "C for 2 h with proteinase-free chromatin and applied to a discontinuous sucrose gradient with a bottom layer of 1.7 M sucrose/lO mM Tris/HCl, pH 8.0, as used for the isolation of chromatin [12]. It can be seen in Table 4 that chromatin, which sediments through the sucrose, binds preferentially pro-

P. C. Heinrich, G. Raydt, B. Puschendorf, and M. Jusic

41

Fig.4. Drgradrrtion o f [ 3H]leucine-labeled histones,from Ehrlich ascires frirnor cells. The assay mixture contained in a total volume of 90 pI 0.1 M Tris/HCl. pH 8.0; 20 pg of total [4,5-3H]leucine-labeledhistones and 0.03 mg of chromatin isolated according to [I21 (a, b), or 0.09 mg of mitochondria, frozen and thawed twice (c,d). or 0.03 mg of lysosomes, frozen and thawed twice (e,f), or 5 pmol of sucrose (g). (a,c,e,g) were incubated for 3 h at 37 "C then 50 pl of 10 M urea in 3 M acetid acid was added and 100 pI of the mixture was used for electrophoresis [26]. Individual controls (b.d, f) wcre prepared by the addition of 50 pI 10 M urea in 3 M acetic acid prior to the addition of [3H]lcucine-labeled histones. These mixtures were kept at 0 'C for 3 h. To the control (g), which was incubated at 37 'C for 3 h, no proteinase had been added

c e ' j E ofproteinase inhihitors on proteolytic activities Table 5. t Data are expressed as percentage of controls=no inhibitors added. The proteinase assay conditions are given in Materials and Methods. The different proteinase inhibitors were added to chromatin, mitochondrial and lysosomal fractions respectively and incubated at 25 "C for 30 inin before proteolysis was started by addition of 3H-labeled histones. The pH of thc incubation mixtures was 8. The incubation mixture contained 0.03 mg of chromatin, 0.09 mg of mitochondria and 0.03 mg of lysosomes respectively. Final inhibitor concentrations are given in the table Fraction

PhMeS0,F (1 mM)

NaHSO, (20mM)

Trdsylol (1 mg/ml)

",., i0

Chromatin B

35

124

Mitochondria1 fraction

61

127

90

Lysosomal fraction

72

140

24

91

teinase activities from the mitochondrial fraction. Only about 2% of the lysosomal proteinases could be detected and no acid phosphatase or succinatetetrazolium reductase activities have been found in the chromatin pellet. No pellet was obtained when the mitochondrial or lysosomal fractions were subjected to the same centrifugation in the absence of chromatin. However, when larger amounts of mitochondria (50- 100 mg) were centrifuged proteinase activity was found in the pellet. The specific activity of the proteinase which sedimented as a membrane-bound enzyme through the 1.7 M sucrose layer increased ten-fold. Table 5 shows the effect of various proteinase inhibitors. Sulfite, which has been used by several authors to inhibit proteinase activity, stimulated

proteinase activities in all cases. It turned out that PhMeS0,F was a potent inhibitor of the chromatinassociated and the mitochondrial proteinase. It also inhibited the lysosomal proteinases assayed at pH 5 to 70% of its original activity. On the other hand, Trasylol was pratically without effect on thechromatinassociated and the mitochondrial proteinases, but it was strongly inhibitory to the lysosomal proteinases.

DISCUSSION It has been shown (Table 1) that higher template activities were obtained for chromatin prepared from liver tissue homogenized in a Waring blender compared to chromatin obtained from purified nuclei. There are several explanations for the difference observed in template activities. One possibility could be the shearing of the chromatin 128,291, since the two chromatin preparations were exposed to different shearing forces. On the other hand, proteolytic degradation of chromosomal proteins had to be considered. The present study was directed only to proteolytic effects. The fact that chromatin prepared from purified nuclei did not contain proteinase activity led to a critical examination of the origin of the proteinase found associated with chromatin, which had been isolated from liver tissue by rigorous homogenization [12]. The latter isolation technique results in a high degree of damage of organelles, particularly cell nuclei, mitochondria, and lysosomes. It also involves the exposure of the deoxyribonucleoprotein to ionic conditions which are empirically chosen and may not correspond to those within the nucleus. Therefore,

Histone-Degrading Enzyme Activities from Rat Liver

42

significant quantities of exogenous proteins may bind to chromatin, including proteinases from different organelles. Chong et al. [7] have described an activation of the chromatin-associated proteinase by NaC1. In our experiments with proteinase-free chromatin from rat liver nuclei NaCl concentrations up to 1.0 M did not result in a stimulation of measurable proteinase activity. Therefore, the lack of proteinase activity in chromatin from purified nuclei cannot be due to suboptimal assay conditions in this respect. The use of radioactively labeled total histones from Ehrlich ascites tumor cells instead of total liver histones as a substrate in the proteinase assay is justified, since the histones of both species do not differ in their banding pattern in acrylamide electrophoresis. It is known, however, that histones of rapidly proliferating cells exhibit a higher degree of phosphorylation than histones from normal liver cells [27]. In order to study the effect of histone phosphorylation on proteinase susceptibility, total histones from Ehrlich ascites tumor cells should have been treated with a histone-specific phosphatase to remove covalently bound phosphate. Because such a phosphatase was not available the total histones from Ehrlich ascites cells were extensively phosphorylated by means of protein kinase and [y3’P]ATP. No significant differences in respect to proteinase susceptibility between modified and native histones could be demonstrated. Among the subcellular fractions assayed for histone-degrading activities only lysosomes and mitochondria had proteinase activity (Fig. 3). The lysosomes have been prepared after Triton RW 1339 injection and should be free of mitochondrial contamination. The mitochondria, on the other hand, should not contain lysosomal contamination after digitonin treatment. Indeed purc subcellular fractions can be assumed from the marker enzyme activities measured (Table 3). After incubation of proteinase-free chromatin with mitochondrial or lysosomal fractions and subsequent sucrose gradient centrifugation it became evident that a mitochondrial proteinase with a wide pH optimum around 7.5 binds to the proteinase-free chromatin, whereas no neutral lysosomal proteinases are bound under these experimental conditions. Neutral mitochondrial proteinases from rat liver have been described by Alberti and Bartley [30]. Although we have not purified this mitochondrial neutral proteinase it seems likely that Chong et al. are dealing with the same enzyme, associated with chromatin. They suggest a physiological role in the turnover of histones for this proteinase. It has been shown, however, that histones have an extremely low turnover and there is hardly a need for a chromatin-associated proteinase for histones [31]. The results of our studies on the chromatin-associated

proteinase are in favour of the findings of Destree [8], although these authors have attributed the proteinase associated with chromatin to a lysosomal contamination. They did not study the pH dependence of their lysosomal proteinases in respect to histone degradation. Furthermore, mitochondrial contamination in the lysosomal preparation has not been excluded in these experiments. As a result of our studies it is suggested that chromatin should be isolated from pure nuclei in order to avoid the binding of proteinases from damaged mitochondria.

el af.

The authors thank Dr Ch. Barth for several samples of liver cytosol and Dr H. Betz for advice in the preparation of lysosomes. We also wish to thank Professor D r H. Grunicke for criticism and helpful discussions. The technical assistance of Mrs S. Seiferl is gratefully acknowledged. The authors also thank Professor Dr H. Holzer for his interest in and support of this work.

REFERENCES 1. Furlan, M. & Jericijo, M. (1967) Biochim. Biopliys. Acia, 147, 135 - 144. 2. Furlan, M., Jericijo, M. & Suhar, A. (1968) Biochim. Biophp. Acta, 167, 154- 160. 3. Bartley, J . & Chalkley, R. (1970) J. B i d . Chem. 245, 42864292. 4. Kurecki, T. & Toczko, K. (1974) Actu Biochim. Pal. 21, 225233. 5. Garrels, J . I.. Elgin, S. C . R. & Bonncr, J. (1972) Biochem. Biophys. Res. Commun. 46, 545 - 551. 6. Chae, Ch. & Carter, D. B. (1974) Biochim Biophys. Res. Commun. 57,740- 746. 7. Chong, M . T., Garrard. W . T. & Bonner, J. (1974) Biochemistry, 13, 5128-5134. 8. Destree, 0. H., D’Adelhart-Toorop, H. A. & Charles, R. (1975) Biochim. Biophys. Actu, 378, 450-458. 9. Grunicke, H., Liersch, M., Hinz, M., Puschendorf, B., Richter, E. Sr Holzer, H. (1966) Bioc,hirn.Biopli.vx. Actu. 121,228 - 240. 10. Blobel, 0. & Pottcr, R. (1966) Science (Wash. D.C.j 154, 1662- 1665. 11. Spelsberg, T. C. & Hnilica, L. S. (1971) Biochim. Bioph)~. Acru. 228,202-211. 12. Bonner, J., Chalkley, G. R., Dahmus, M., Famhrough. D.. Fujimura, F., Huang, R. C. C., Hubcrinan. J., Jensen, R., Marusliige, K., Ohlenbusch, H., Olivera, B. & Widholm, J. (1968) Methods Enzymol. 12, 3 - 65. 13 Burgess, R. R. (1969) J . B i d . Chem. 244, 6160-6167. 14. Puschendorf, B., Wolf, H. & Grunicke, H. (1971) Biochem. Phurmucol. 20, 3039- 3050. 15 Loewenstein, J., Scholtc, H. R . & Wit-Peetcrs, E. M . (1970) Biochim. Biriphys. Acta, 223, 432 -436. 16 Trouet, A. (1974) Methods Enzymol. 3 1 A , 323. 17 Grossi, L. G. & Moldave, K., (1960) .I. B i d . Chum. 235. 2370- 2374. 18 Pctte, D. (1969) in Praktischr Enzymologie (Schmidt, F. W., ed.) p. 35, Verlag Halhuber, Bern, Switzerland. 19 De Duve, C. (1955) Biochem. J . 60, 604-617. 20 Wattiaux, R. & de Duvc, C. (1956) Biocliem. J . 63, 606-608. 21 Pennington, J. (1961) Biochem. J . 80,649-654. 22 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 23 Ceriotti, G. (1952) J . Bid. Chem. 198, 297-303.

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P. C. Heinrich, G. Raydt, B. Puschendorf, and M. Jusii. 24. Fleck, A . & Munro, H. N. (1962) Bioc,him. Biophys. Aclu. 55, 571 - 583. 25. Wilhelm, J. A,, Spelsberg, T. C. & Hnilica, L. S . (1971) Sub-cell Biochetn. 1, 39-65. 26. Panyim, S. & Chalkley, R. (1969) Arch. Biochcm. Biophys. 130, 337- 346. 27. Balhorn, R., Chalklcy, R. & Granner, D. (1972) Biochemistry, 11, 1094-1098.

28. de Pomerai. D. J., Chesterton, C. J. & Buttcrworth. H. W. (1974) Eur, J. Biocliem. 46, 461 -471. 29. Tan, C. H. & Miyagi, M. (1970) J . Mol. Biol. 50, 641 -653. 30. Alberti, K. G. M. M. & Bartley, W. (1965) Bioclwn. J . 95, 641 - 656. 31. Hnilica, L. S . , McClure, M. E. & Spelsberg, T.C. (1971) in Histones and Nucleohistones (D. M. P. Phillips, ed.) pp. 199204, Plenum Press, London, New York.

P. C. Heinrich and M. JusiC, Biochemiscbes Institut der Albert-Ludwigs-Universitlt Freiburg. D-7800 Freiburg i. Br., Hermann-Herder-StraDe 7, Federal Republic of Germany G. Raydt, Institut f i r Physiologische Chemic und Physikalische Biochemie der Ludwig-Maximilians-UniversitLtMunchen, D-8000 Munchen 2, Goethestralje 33, Federal Republic of Germany B. Puschendorf, Medizinisch-Chemisches Institut der Leopold-Franzens-Universitat Innsbruck, Miillerstralje 44. A-6020 Innsbruck, Austria