Purification of an inhibitor of pancreatic deoxyribonuclease from calf spleen

BIOCHIMICA ET BIOPHYSICA ACTA

237

BBA 4194

P U R I F I C A T I O N O F AN I N H I B I T O R PANCREATIC DEOXYRIBONUCLEASE

OF

FROM CALF SPLEEN

UNO L I N D B E R G

Department of Medical Chemislrt,, Uppsala University, Uppsala (Sweden) (Received May 3rd, 1963)

SUMMARY

An inhibitor of pancreatic deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5) was partially purified from calf spleen. The inhibitor had the general properties of a protein. It was thermolabile with a stability optimum around pH 7.6 and showed a typical "protein spectrum" between 25o and 35o m/~. It was stabilized by SH compounds such as mercaptoethanol and glutathione. The purified inhibitor was inactivated by digestion with low concentrations of trypsin (EC 3.4.4.4). Preliminary studies on the mechanism of inhibition indicated that the inhibitor acts by being bound to deoxyribonuclease. A spectrophotometric method for the assay of the inhibitor is described.

INTRODUCTION

Different types of inhibitors have been described for various DNAases. Thus two deoxyribonucleases of bacterial origin were reported to be inhibited by RNA 1-3. Protein inhibitors have also been described for a DNAase from yeast4, 5 and for some animal DNAases 6-n. Deoxyribonuclease (deoxyribonucleate oligonucleotido-hydrolase, EC3.I.4.5) from pancreas was crystallized by KDNITZ in 1948 (see refs. 12 and 13). The enzyme is an endonuclease, which splits DNA to oligonucleotides of varying size. In 195o LASKOWSKI et alY reported that pancreatic DNAase was inhibited by extracts from most animal tissues. Thereafter such observations were made by several authors though the inhibitor substance was in no case purified. MATERIALS AND METHODS

Glass-distilled water was used exclusively. Crystalline pancreatic DNAase was purchased from the Worthington Biochemical Laboratories. Before use, it was dried over NaOH in a vacuum desiccator. A stock solution containing I mg/ml was made in 0.05 M Tris buffer (pH 7.5) and stored frozen at --16 ° in o.3-ml portions. Each portion was then used for one day's experiments. New stock solutions were prepared monthly. Crystalline trypsin (EC 3.4.4.4) and soy-bean trypsin inhibitor were also obtained Biochim. Biophys. Aeta, 82 (1964) 237-248

23 S

u. LINDBERG

from the Worthington Biochemical Laboratories. Stock solutions were prepared as described above. Highly polymerized DNA, type I, was obtained from Sigma Comp. St. Louis, Mo. (U.S.A.). Solutions of DNA (substrate solutions) were prepared as follows: DNA (24 rag) was dissolved in distilled water at room temperature (21°) by vigorous stirring for at least 5 h. Then 2.4 mmoles of MgSO 4, 1.o5 mmoles of CaC12 and 6o mmoles of Tris buffer (pH 7.5) were added and the mixture diluted to 6oo ml. The solution was filtered and stored at o-4 °. Usually the substrate was used within a week. DEAE-Sephadex A5o medium was washed with o.5 M HC1, followed by water, then treated with o.5 M NaOH and again with water, as suggested by the supplier (Pharmacia, Uppsala (Sweden)). A colunm of suitable size was then packed in o.22 M phosphate buffer (pH 7.6) and washed with a large volume (about 15o ml per g of dry substance) of this buffer to convert it into the phosphate form. Just before an experiment the column was again washed with the same buffer now also containing o.oo5 M mercaptoethanol. A fresh column was made for each experiment. Used ion exchanger was washed with 0.3 M Na:~P04 (at least 3 1 for 25 g of DEAE-Sephadex) to remove remaining proteins, and then rinsed with water. Aged (3 years) C7 alumina hydroxide gel, prepared according to WILLST.'a~TTER14 was available in this laboratory. The gel suspension contained 20 mg of dry substance per ml. Protein was determined by a turbidometric method described by BUCHER1~ with serum albumin as standard. A recording Zeiss spectrophotometer (RPQ 20 A) with its temperature adjusted to 2I ° was used for some of the measurements described in this paper. RESULTS

Assay of pancreatic DNAase: DNAase activity was measured by a rapid spectrophotometric method adapted from the method originally described by Kunitz. It was based upon the hyperchromicity, which could be observed at 26o m/~, when DNA was depolymerized. The substrate solution, described above, contained the different electrolytes in optimal concentrations. Before each assay replicate tubes, containing 3 nT1 of the substrate, were set up and warmed to 21 ° (usually room temperature). A typical assay was then performed as follows: 5-3o/~1 (o.25-1.5/~g) of the enzyme solution (stock solution diluted 2o-fold with 0.05 M Tris buffer (pH 7-5)) was pipetted into a separate tube. This volume was considered negligible compared to the volume of the substrate. Then the content of one substrate tube was added and complete mixture of the components was ensured by rapid rotation of the tube by a Vortex Jr. Mixer. The mixing time required was about I5 sec. The reaction mixture was then poured into a quartz cuvette and the increase in absorbancy at 26o m/z was read at I5-sec intervals for 2 rain in a Zeiss spectrophotometer PMQ II. The readings were made against substrate solution (without enzyme) as blank. The maximum firal increase in absorbancy was about 3o % over the absorbancy of the substrate solution. Except from an initial lag period the increase was linear with time during the first part of the reaction (Fig. ia). The rate for the reaction calculated from the li,:ear slope of the time curve was directly proportional to the amount of enzyme (Fig. Ib). The reaction rate was expressed as change in absorbancy Biochim. I3ioph3,x. ,4cta, 82 (1964) 237 24,3

DEOXYRIBONUCLEASE INHIBITOR FROM CALF SPLEEN

239

p e r min : one unit of D N A a s e a c t i v i t y was defined as the a m o u n t of e n z y m e which u n d e r s t a n d a r d conditions caused an increase of o . o o I / m i n in t h e a b s o r b a n c y at 260 m F. Effect of divalent ions on DNAase reaction I n order to be able to m e a s u r e D N A a s e a c t i v i t y u n d e r o p t i m a l conditions, t h e effect of d i v a l e n t ions was s t u d i e d with the present a s s a y m e t h o d . As can be seen in Fig. 2 the initial reaction r a t e w i t h a given

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Fig. Ia. Time course of DNAase action. V a r y i n g amounts of DNAase (indicated in the figure in ml) were incubated w i t h s u b s t r a t e u n d e r s t a n d a r d conditions and the increase in abs o r b a n c y at 260 m p was registered with a rec o r d i n g Zeiss s p e c t r o p h o t o m e t e r ( R P Q 20 A).

Fig. lb. Dependence of reaction rate (calculated from Fig. Ia) on enzyme concentration, o.oi ml of D N A a s e = 0. 5/*g.

._~06

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O.01

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Concentra~on(M) Fig 2. Effect of divalent ions on D N A a s e activity. T h e substrate mixture contained : 4 °/~g of D N A , 3oo/~moles of Tris buffer (pH 7.5) and v a r y i n g a m o u n t s of the divalent ions u n d e r investigation in a total volume of 3 ml. The s u b s t r a t e was t h e n added to the a m o u n t of D N A a s e given below and the increase in a b s o r b a n c y followed as usual. O - - O , v a r y i n g Mg 2+ concentration, 50/xg of D N A a s e ; x - X, v a r y i n g Mn ~+ concentration, o.75/2g of D N A a s e , O - - O , v a r y i n g Ca ~+ concent r a t i o n at a Mg ~+ c o n c e n t r a t i o n of o.005 M, 0.75 tig of DNAase.

Biochim. Biophys. Acta, 82 (1964) 237-248

24o

U. LINI)BERG

a m o u n t of enzvmes was 5o times higher in t h e presence of o p t i m a l concentrations of Mn ~+ as c o m p a r e d to Mg 2+. Ca 2+ as such h a d v e r y little s t i m u l a t o r y effect on the reaction, b u t in the presence of Mg 2+ a further strong s t i m u l a t i o n b y Ca °-+ was observed a n d an o p t i m a l c o m b i n a t i o n of Mg 2+ a n d Ca 2+ gave as efficient a s t i m u l a t i o n as Mn 2. alone. These effects were v e r y simular to those described b y WIBERG 16, who used a t i t r i m e t r i c m e t h o d for m e a s u r i n g D N A a s e a c t i v i t y . 7

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Fig. 3a. Inhibition of D N A a s e activity by chrom a t o g r a p h e d inhibitor. The u p p e r curve gives the reaction rate of unhibited D N A a s e (l yg), while the lower curves show the effect of increasing a m o u n t s of inhibitor. S t a n d a r d inhibitor assay procedure was employed.

0

2

4

spleen inhibitor(~g) Fig. 3b. Inhibitor action as a function of inhibitor concentration. The values were calculated from the data of Fig. 3a.

Assay of DNAase inhibitor: F o r the d e t e r m i n a t i o n of inhibitor a c t i v i t y ~ t*g of D N A a s e was first m i x e d with 5-20 t~l of the active i n h i b i t o r fraction. T h e n 3 ml of s u b s t r a t e solution were added, a n d after m i x i n g w i t h t h e V o r t e x m i x e r the increase in a b s o r b a n c y at 260 mtz was followed as described above. The difference in a c t i v i t y thus o b t a i n e d between the change in a b s o r b a n c y at 260 m/, in the absence and presence of i n h i b i t o r was t a k e n as a measure of the i n h i b i t o r a c t i v i t y . One unit of i n h i b i t o r a c t i v i t y was defined as t h e a m o u n t of i n h i b i t o r causing a decrease of o.ooi a b s o r b a n c v unit in the reaction r a t e of I / z g of DNAase. A linear relation between the a m o u n t of i n h i b i t o r a d d e d a n d decrease in reaction rate was o b t a i n e d a l r e a d y in t h e crude e x t r a c t . Fig. 3a shows t i m e curves of I / z g of D N A a s e with a n d w i t h o u t purified spleen i n h i b i t o r a d d e d a n d Fig. 3 b the decrease in change in a b s o r b a n c y p e r min as a function of the a m o u n t of i n h i b i t o r added. The degree of inhibition was p r o p o r t i o n a l to the a m o u n t of i n h i b i t o r a d d e d up to 7 ° % inhibition. DNAase inhibitor activity in different organs: Crude e x t r a c t s of different organs were m a d e as described below for spleen inhibitor. A c t i v i t y a n d protein were determ i n e d in the extracts. Table I shows t h a t i n h i b i t o r a c t i v i t y is present in several calf organs a n d t h a t the spleen e x t r a c t c o n t a i n e d the highest a c t i v i t y . Purification of DNAase inhibitor All o p e r a t i o n s were carried out at 0 - 4 °. M e r c a p t o e t h a n o l was found to stabilize the i n h i b i t o r a n d was a d d e d d u r i n g all procedures. All centrifugations were carried out in an I n t e r n a t i o n a l high-speed refrigerated centrifuge, model H R - I . Biochim. Biophys. Acta, 82 ([964) z37 248

DEOXYRIBONUCLEASE INHIBITOR FROM CALF SPLEEN

241

Preparation of crude extract: Spleens from young calves were taken directly at slaughter and cooled on ice during the transport to the laboratory, where they were either used immediately or frozen and stored at --16 ° . The spleens could be stored frozen up to 3 weeks without any observable loss of activity. The distal parts of the spleen were cut off and the parenchyme scraped out with a spoon. 200 g of parenchyme were then treated in a Waring Blendor for 3 rain with 5 vol. of o.oi M mercaptoethanol. The homogenate was then centrifuged for 15 min at 9000 rev./min. TABLE INHIBITOR

ACTIVITY

IN

I

DIFFERENT

ORGANS

OF T H E

CALF

Organ

Inhibitor (units/ml~

Protein (mg/ml)

Specific a:tivity

Spleen Thymus Testes Brain Liver

16 ooo 8000 360o 2200 2600

13 9 8 8 ~9

I2OO 890 45o 280 14o

The cloudy supernatant solution, here referred to as the crude extract, had a p H of about 7.o and could not be kept frozen without considerable loss of activity. Precipitation of nucleic acid with MnCl 2 (see ref. I7): This step was introduced to remove the major part of the nucleic acids which otherwise interfered with the following purification steps. To the crude extract was added slowly o.I M MnC12 to give a final concentration of 0.oo4 M. During the addition the solution was stirred vigorously. The heavy precipitate was removed by centrifugation. The resulting supernatant solution, which was used for the further purification of the inhibitor contained only about 5o % of the activity of the crude extract. Ammonium sulfate fractionation: The supernatant solution from the MnCI~ step was diluted with o.05 M Tris buffer (pH 7-5) to give a protein concentration of 9 mg/ml. The diluted solution (7o0 ml) was then brought to 30 % saturation with ammonium sulfate b y slow addition of 3o0 ml of a saturated, neutralized solution of ammonium sulfate. The addition took about 45 min. After another 15 min of stirring the precipitate was centrifuged off and discarded. The supernatant solution was brought to 5o % saturation with saturated ammonium sulfate (400 ml added during I h) and stirred for another hour. The second precipitate contained more than 9 ° To of the activity from the previous step. This precipitate was removed b y centrifugation and dissolved in the least possible amount of 0.05 M Tris buffer (pH 7.5) and then dialyzed against the same buffer containing o.o4 M mercaptoethanol. The concentration of mercaptoethanol in the earlier steps was o.oi M. The non-dialyzed ammonium sulfate fraction could be stored frozen for several months without appreciable loss of activity. Adsorption with CV alumina hydroxide gel: The dialyzed ammonium sulfate fraction was diluted with 0.o5 M Tris buffer (pH 7.5) to a protein concentration of 15 mg/ml. The inhibitor was then adsorbed to Cy alumina. For this purpose 0.04 ml of the C~, alumina solution was added for each absorbancy unit at 280 mt~. The suspension was stirred during 3 min and centrifuged. The gel was first washed twice with 40 ml of o.005 M phosphate buffer (pH 7.6) containing o.0o5 M mercaptoBiochim. Biophys. Acta, 82 (~964) 237-248

242

U. LINDBERG

ethanol, and the inhibitor was then eluted with two 4o-ml portions of o.o25 M phosphate buffer (pH 7.6) containing o.005 M mercaptoethanol. Chromatography on DEAE-Sephadex: The eluate from the C7 step (230 mg of proteins) was adsorbed to a column of DEAE-Sephadex (diameter 4 cm ; length 7 cm) which had been equilibrated with o.22 M phosphate buffer (pH 7.6)-0.o05 M mercaptoethanol. Two completely inactive peaks were eluted with this buffer (Fig. 4)-

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Fig. 4. C h r o m a t o g r a p h i c purification of the spleen inhibitor on D E A E - S e p h a d e x . Elution was s t a r t e d w i t h o.22 M p h o s p h a t e buffer (pH 7.6). The buffer was changed at the arrow to o.3o M (pH 7.6). The elution buffer was p u m p e d onto the column at a rate of 25 ml/h and fractions of a b o u t 2 ml were collected. The solid line represents the a b s o r b a n c y of the fractions at 28o ml, and the b r o k e n line inhibitor concentration in units/ml.

When the second peak had been almost completely eluted the molarity of the elution buffer was changed to 0.3 M and the inhibitor eluted as one further peak. The buffers were fed to the column with a micropump at a rate of 25 ml/h. The fraction size was 5 ml. The absorbancy of all the fractions was determined at 260, 280 and 31o m/, and each third fraction was tested for inhibitor activity. The active fractions were pooled and concentrated by ultrafiltration in a collodium bag. The ehromatographed inhibitor was used for the following experiments unless otherwise stated. A summary of the purification procedure is given in Table II.

General properties of the inhibitor Stability: The inhibitor was sensitive to both elevated temperature and acid and alkaline pH values. This was demonstrated by the following experiment: Equal volumes of crude spleen extract and a buffer solution with known p H (0.2 M acetate buffer for pH 4-6, 0.2 M phosphate buffer for pH 6-7.5, o.2 M Tris for pH 7.5-9) were mixed and incubated at the desired temperature. In the case shown in Fig. 5 Biochim. Bioph3's. Acla, 8'- (t9¢~4) 237 2.t8

243

DEOXYRIBONUCLEASE INHIBITOR FROM CALF SPLEEN TABLE II SUMMARY OF THI~ PURIFICATION PROCEDURE Fraction

Units/ml ( x to*)

Total units ( x zo*)

Pzotein {mg/ml)

Crude e x t r a c t

Total protein Specific activity (rag) (units/tag)

Ywld (°o)

i84

92 ooo

2i

io 500

880

[oo

After MnC1 z t r e a t m e n t

96

49 920

i2

6300

800

54

After a m m o n i u m sulfate-fractionation

735

46 3oo

23

[45o

32oo

5o

After dialysis

48o

36 5 oo

[9

I43o

26oo

4°

After CT a l u m i n a hydroxide gel adsorption

35 °

25 600

3

227

i i 300

2S

After D E A E Sephadex c h r o m a t o g r a p h y

960

12 ooo

2. 7

34

35 ooo

[3

the incubation temperature was 44 °. A series of mixtures with different pH values was kept at o ° as control. The inhibitor activity was then determined at two different time intervals. The inhibitor had its stability optimum in the crude extract between pH 7.5 and 8.o. The purified inhibitor was similarl}, completely inactivated by incubation for 5 rain at 44 ° (pH 7.6). The inhibitor was precipitated around pH 5 and little activity could be recovered from the precipitate. Ultraviolet absorbtion: The purified inhibitor had an absorption spectrum such as can be expected from a protein mixture devoid of nucleic acids (Fig. 6).

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Fig. 5. Effect of p H and temperature on the stability of inhibitor activity in the crude extract. T h e inhibitor was incubated at the desired p H and temperature for the times indicated. The inhibitor activity was then determined b y the standard assay procedure.

Fig. 6. Ultraviolet-absorption s p e c t r u m of the spleen inhibitor after D E A E - S e p h a d e x chromatography.

Biochim.

Biophys.

,4cta. 82 (1904) 237 248

244

u. LINDBEI~G

The ratio between the absorbancies at 26o and 28o m/~ was o.56. Effect of t~,psi~, on the inhibitor: In order to ascertain the protein nature of the inhibitor, its inactivation by a proteolytic enzyme was investigated. In the experiment shown in Fig. 7 4o0 ~g of the DNAase inhibitor (600 units) were incubated at 21 ° with different amounts of trypsin (6.25, 12.5 and 25/*g) in a total volume of o.15 ml.

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Fig. 7. I n a c t i v a t i o n of inhibitor b y different a m o u n t s of t r y p s i n (see text).

4

Fig. 8. Time course of inhibitor inactivation b y trypsin.

After half a minute the samples were placed in an ice-water bath and 5o/~g (o.o5 ml) of soy-bean trypsin inhibitor was added to stop the trypsin action. The mixture was then assayed for remaining DNAase inhibitor. For this purpose 5/xl of the incubation mixture were added to i ~g of DNAase and DNAase activity was measured in the usual way. In Fig. 8 the amount of trypsin (6.25 ~g) was kept constant while the incubation time was varied (o.5, 1.5, 3 and 5 rain). The same amount of DNAase inhibitor and trypsin inhibitor was used and after incubation the mixtures were assayed for remaining DNAase inhibitor as before. Both experiments show clearly that trypsin did effectively inactivate the DNAase inhibitor. Control experiments showed that although uninhibited trypsin rapidly inactivated DNAase, the amount of soy-bean trypsin inhibitor added was sufficient to abolish trypsin activity completely. The chosen amounts of trypsin inhibitor did in no way by itself influence the inhibition of DNAase by DNAase inhibitor. To exclude the possibility that the DNAase inhibitor itself was a proteolytic enzyme the following experiment was done: I ~g of DNAase was mixed with 19/zg of the inhibitor.This amount of DNAase inhibitor gave 5o % inhibition under standard assay conditions. The time for contact between the DNAase and the inhibitor before the addition of substrate was then varied. Maximal inhibition was reached at the earliest time at which it could be measured (4 sec) and no further increase could be observed.

Preliminary experiments on the mechanism of inhibition Two possible ways in which the inhibitor might act can be visualized: (a) the inhibitor might bind the substrate and there prevent the attack of the enzyme; (b) or it could bind the enzyme and thereby inactivate it. Biochim. Biophys..4cla, 82 (t964) 237 24~

DEOXYRIBONUCLEASE INHIBITOR FROM CALF SPLEEN

245

In an attempt to show which of these two possibilities was more likely, the dependence of the inhibition on substrate concentration and on enzyme concentration was studied. Inhibition at increasing substrate concentration: In his studies on the kinetics of the DNAase reaction KUNITZ13 showed that the optimal Mg2+ concentration depended on the concentration of DNA. This effect was further investigated by WIBERG 16, who showed a direct correlation between optimal binding of Mg2+ ions to DNA and optimal DNAase activity. Such a correlation was not found for Ca 2+. When the effect of the concentration of DNA on the inhibition of DNAase by the spleen inhibitor was now studied, the Mg 2+ concentration was varied together with the DNA concentration, while the Ca 2+ concentration was kept constant. The upper curve in Fig. 9 shows that with our DNA preparation and with the assay conditions used, DNAase activity in the absence of inhibitor was optimal at concentrations of DNA around 60/zg/ml. The same optimum was observed when two different amounts of the spleen inhibitor were added (two lower curves), but lower values for Vmax were obtained. Inhibition at increasing enzyme concentration: As demonstrated earlier in this paper the initial reaction rate of the DNAase reaction was directly proportional to the amounts of DNAase added. This is shown in Curve i of Fig. IO. Adding increasing amounts of the spleen inhibitor gave the results demonstrated by Curves 2-4. These

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D N A (~g/rnl ) Fig. 9. Effect of D N A c o n c e n t r a t i o n on i n h i b itor a c t i v i t y . T h e u p p e r c u r v e shows t h e dep e n d e n c e of D N A a s e a c t i v i t y (i /~g of enzyme) on D N A c o n c e n t r a t i o n . T h e t w o lower c u r v e s were o b t a i n e d w i t h 1.25 a n d 1.88 F g of i n h i b i t o r a d d e d . The c o n c e n t r a t i o n of Mg ~+ was v a r i e d in p a r a l l e l w i t h t h e D N A c o n c e n t r a t i o n . A rec o r d i n g Zeiss s p e c t r o p h o t o m e t e r was used.

DNAase(Fg) Fig. I o. Effect of e n z y m e c o n c e n t r a t i o n on i n h i b itor activity. Curve i was obtained w i t h o u t a n d C urve s 2 - 4 w i t h s pl e e n i n h i b i t o r a d d e d (1.88, 2.5 a n d 3.12 F g r e s p e c t i v e l y ) . A r e c o r d i n g Zeiss s p e c t r o p h o t o m e t e r w a s used i n t h e s t a n d a r d a s s a y p r o c e d u r e of i n h i b i t o r .

curves show that in the presence of the inhibitor the reaction rate did not increase linearly with increasing amount of enzyme. Instead an initial lag was observed. After this lag a straight-line relationship was again observed. Biochim. Biophys. Mcta, 82 (i964) 237-248

24(9

U. LINDBERG DISCUSSION

Three main groups of nucleodepolymerases can be distinguished: (a) ribonucleases (e.g. RNAase, EC 2.7.7.16), (b) deoxyribonucleases (e.g. DNAase, EC 3.1.4.5) and (c) a group of miscellaneous exonucleases (e.g. phosphodiesterase, EC 3.1.4.1 ). The first two groups are specific for RNA and DNA respectively, while the last group attacks phosphodiester linkages between nucleotides without regard to the nature of the sugar moiety. The biological function of these nucleolytic enzymes is still unclear and the picture becomes still more complicated when one considers that several of the nucleases exist in the cell in a strongly inhibited state. Naturally occurring inhibitors of both RNAases and DNAases have been described and an RNAase inhibitor extensively purified from rat-liver was shown to be a protein 18,19. Two types of DNAase inhibitors have been described. (I) Soluble RNA from E. coli inhibited an endonuclease from the same bacterium, and streptodornase (DNAase, EC 3.1.4.5) from Streptococcus Lancefield group A was specifically inhibited by RNA from the same bacterium. This RNA was inactive against streptodornase from Lancefields groups B or C. (2) DNAase inhibitors from yeast and animal cells were shown to have properties of proteins. In several cases the inhibitor was intimately associated in vivo with the corresponding DNAase indicating a possible flmctional relationship between DNAase and the inhibitor. None of the protein inhibitors were studied extensively. Model systems may be provided by the naturally occurring inhibitors of trypsin and chymotrypsin (EC 3.4.4.5). Several of these inhibitors have been obtained in crystalline form. The\inhibit proteolytic activity by being bound to the enzymes, but the binding is easily reversed by exposure of the complexes to pH extremes. This indicates that secondary rather than primary forces keep the complexes together. Other evidence indicates that the inhibitors are bound at or near the active site of the enzyme and that amino groups are involved in the linkage 2°-~2. The present work was started in order to obtain more information about a DNAase inhibitor from animal tissues. It is believed that an understanding of the mechanism of action of this inhibitor may contribute to our general knowledge of the action of nucleolytic enzymes. Furthermore several investigators 2a,24 have implicated the participation of DNAases in the regulation of growth and cell multiplication. Since DNAase inhibitors seem to be present in many different types of cells, their presence should be incorporated into all such theories. Finally the availability of a purified preparation of the inhibitor is believed to be of practical importance as a tool in many types of experiments in vitro when an inhibition of DNAase is aspired. Before a purification of the inhibitor could be attempted it was necessary to work out a rapid and relatively exact assay method for the inhibitor. The spectrophotometric method introduced by KUNITZla for the assay of DNAase appeared to be suitable also for the assay of the inhibitor. Bivalent ions are needed for full activity of DNAase. WlBER6 tG, using a pH-stat method, showed that Mn ~+ alone gives full activity but that in the absence of Mn 2+ both Mge+ and Ca 2+ are required. FEINSTEIN~5 reported that addition of boiled ratliver extract further stimulated DNAase activity in the presence of optimal amounts of Mg2+ and Ca 2+ ions. This author used a viscosimetrie assay method. The influence Biochim. Bioph3,s..4eta. ,$2 (lq64) 237-z48

DEOXYRIBONUCLEASE INHIBITOR FROM CALF SPLEEN

247

of divalent ions on DNAase activity, as measured in this paper by a spectrophotometric method agreed very well with the findings of WlBERGt6. It was not possible, however, to demonstrate an activating effect of heated rat-liver extract. The spectrophotometric assay could then be directly applied to the measurement of the DNAase inhibitor since a linear relationship was established between the amount of inhibitor added and the decrease in DNAase activity observed. When Mg2+ and Ca 2+ were used at optimal concentrations the assay was extremely sensitive and it was possible to measure inhibitor activities in crude extracts without interference from ultraviolet-absorbing contaminating material. DNAase is unstable in solution and it was not possible to compare inhibitor activities, obtained with one and the same DNAase solution, over longer periods than two weeks. Preliminary experiments indicate that inactive DNAase also can bind the inhibitor. This would mean that it might be suitable to define inhibitor activity on the basis of the inhibition of a certain amount of DNAase irrespective of the specific activity of the DNAase solution. In the present experiments this was not done, however, instead care was taken to use one and the same fresh solution of DNAase whenever it was necessary to obtain comparable results. Our best inhibitor preparations were only about 5o-fold purified over the activity of the crude extract and we intend to try further purification. It was nevertheless possible to arrive at certain tentative conclusions with respect to the nature of the inhibitor and the mechanism of its action. The general properties of the inhibitor suggest that it was a protein. This idea was further supported by the fact that it could be purified by conventional protein purification procedures, and by the rapidity with which it was inactivated by exposure to low concentrations of trypsin. Our preliminary experiments indicated that the inhibitor acted by being bound to the enzyme. The inhibition was studied at increasing substrate and enzyme concentrations respectively. In the first type of experiments the inhibition was not released by an excess of substrate. Thus the same optimal DNA concentration was found

{

0.5 DNAase ( p g )

1

Fig. I I. S t o i c h i o m e t r i c r e l a t i o n b e t w e e n D N A a s e a n d t h e spleen i n h i b i t o r . The a m o u n t of i n h i b i t o r was p l o t t e d a g a i n s t t h e a m o u n t of D N A a s e o b t a i n e d b y e x t r a p o l a t i o n t o t h e a b s c i s s a of t h e l i n e a r part~ of Curves 2 4 in Fig. io.

Biochim. Bioph3,s..4cta, 82 (1964) 237 248

248

U. LINDBERG

both with and without the inhibitor with decreasing values for Vm,~ with increasing amounts of the inhibitor. These experiments make it unlikely that the inhibitor was bound to DNA. When increasing amounts of enzyme were added at a fixed substrate concentration at three different concentrations of the inhibitor, the curves obtained could be easily explained by the assumption that a given amount of inhibitor inactivated a proportional amount of the enzyme. This is illustrated in Fig. zI. The amounts of DNAase plotted in this figure were obtained by extrapolation to the abscissa of the linear parts of Curves 2-4 in Fig. IO. In this way a straight line is obtained which passes through the origin. From this curve one can also calculate that ~ mg of DNAase was inactivated by 3 mg of the partially purified inhibitor preparation. These experiments strongly support the conclusion that the inhibitor inactivated DNAase by direct binding to the enzyme. ACKNOWLEDGEMENT

This investigation was supported by a grant from the United States Public Health Service (CA 06144) to Dr. P. REICHARD. REFERENCES 1 A. W. BERNHEIMER AND N. t4. RUFFIER, J. Exptl. Med., 83 (1946) 972 A. -W. BERNHEIMER, Biochem. J., 53 (1953) 53:~ I. R. LEHMAN, G. G. R o u s s o s AND E. A. PRATt, J. Biol. Chem., 237 (i962) 819. 4 S. ZAMENHOFF AND E. CHARGAFF, Science, lO8 (1948) 628. g S. ZAMENHOFF AND E. CHARGAFF, J. Biol. Chem., 18o (1949) 727 • n VV. DABROWSKA, E. J. COOPER AND M. LASXOWSKI, J. Biol. Chem., 177 (1945) 991. 7 E. J. COOPER, M. J. TRAIJTMAN AND M. LASKOWSKI, Proc. Soc. Exptl. Biol. Med., 73 (195 o) 219. s E. C R u z - C o K E , M. PLAZA DE L o s REYES, J . MARTENS, J. D E L NIDO AND J. ARAYA, Soc. Biol. Santiago de Chili, 9 (1951) 38. 9 E . CRuz-CoKE, M. PLAZA DE LOS REYES, J. MARTENS, J. DEL NIDO AND J. ARAYA, Soc. Biol. Santiago de Chili, 9 (1951) 44. 10 I-t, H. HENSTELL AND R. L. FREEDMAN, Cancer Res., 12 (1952) 341. 11 N. B. KURNICK, L. J. SCHWARTZ, S. PARISER AND S. L. LEE, J. Clin. Invest., 32 (1953) 193. 12 M. NENITZ, J. Gen. Physiol., 33 (195°) 349. la M. KUNITZ, Science, lO8 (1948) 19. 14 R. ~vVILLST[~TTEI1AND H. I(RAUT, Bet. Deut. Chem. Ges., 56 (1923) 1117. 1~ T. BUCHER, Biochim. Biophys. Acla, I (1947) 292. 16 j . S. WlBERa, Arch. Biochem. Biophys., 73 (I958) 337. 17 g. HEPPEL, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, A c a d e m i c Press, N e w Y o r k , 1955, p. 137. as K. SnORTMAN, Biochim. Biophys. Acta, 51 (1961) 37. 19 N. SHORTMAN,Biochim. Biophys. Acta, 55 (1962) 88. 20 H. FRAENKEL-CONRAT, R. C. B~AN, E. D. DUCAY AND H. S. OLCOTT, Arch. Biochem. Biophys., 37 (1952 ) 393. 21 N. M. GREEN, J. Biol. Chem., 205 (1953) 535. 22 N. M. GREEN ANI) E. WORK, Biochem. J., 54 (1953) 34723 A. L. DOUNCE, N. 142. SA.RKAR AND E. I{. NAY, J. Cellular Comp. Physiol., 57 (1961) 47. 24 S. BRODY, Acta Obslet. Gvnecol. Scand., 38 (1959) 424 . 2z R. N. FEINSTEIN, J. Bio~l. Chem., 235 (196o) 733.

Biochim. Biophys. Acta, 82 (1964) 237-248