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The inhibition of pancreas deoxyribonuclease
349 THE INHIBITION LISELOTTE
OF PANCREAS M. GILBERT,
DEOXYRIBONUCLEASE
W. G. OVEREND
and M. WEBB
Department of Chemistry, The University, Birmingham and Strangeways Research Laboratory, Cambridge, Great Britain Received
October
10, 1950
RECENT communications (30, 31) have described the results of an investigation of the properties and mode of action of deoxyribonuclease isolated from beef-pancreas by essentially the method of McCarty (28). These and additional studies have shown that the action of this enzyme, which requires magnesium ions for its activation, is analogous to that of the corresponding ribonuclease (32) but is confined specifically to the deoxyribo type of nucleic acid or its partially degraded products (16). In view of the increased use of deoxyribonuclease in cytochemical work (8, 24), together with the possible significance of enzymes of this type in a detailed investigation of the inhibition of the processes of cell division, Certain preliminary observations have been the enzyme appeared justified. mentioned briefly in a previous communication (31), and the present paper describes fully the extension of this work. In addition, a study has been made of the action of deoxyribonuclease on the deoxyribonucleohistone of calf thymus cell nuclei alone and in the presence of substances known to inhibit mitosis. MATERIAL
AND
METHODS
was isolated from beef-pancreas by McCarty’s (28) method, Deoxyribonuclease and was purified by repeated fractional precipitation with ammonium sulphate (31). Deoxyribonucleic acid. Highly polymeric preparations of deoxyribonucleic acid were isolated from calf-thymus and herring sperm according to the method of Mirsky and Pollister (29). The chemical and physical properties of these preparations N/P (= = 1.42 & 1.56 respectively) will be described elsewhere. In addition to these preparations, commercial (B. D. H.) deoxyribonucleic acid was also used as substrate. After purification as previously described (31) the nucleic acid had N/P : 1.6. Deoxyribonucleohistone was isolated with 0.002 M sodium arsenate (33) from the gelatinous mass of thymus cell nuclei remaining after the extraction of the minced tissue with 0.14 M sodium chloride. The nucleoprotein was precipitated by the addition of sodium chloride to 0.14 M concentration, and purified by repeated solution in 0.002 M sodium arsenate and precipitation with sodium chloride. Finally, it was dissolved in distilled water, precipitated with cold ethanol (1 vol.) and dried with
350
L. M.
Gilberf,
W. G. Overend,
and M.
Webb
ethanol and ether. The residual white powder (N, 15.6; P, 5.05 per cent), which contained a negligible amount of arsenate (Marsh test and potas;Llm iodide reaction), dissolved readily in water to give a viscous, opalescent solution. The product gave a positive reaction for sulphur after sodium fusion, but the nitroprusside reaction for -SH groups was negative hefore, and only weakly positive after, treatment -rrith potassium cyanide (22). EXPERIMENTAL
The inhibition
of the action of Deoxyribonuclease Deoxyribonucleic acid
on Thymus
The inhibitory action of various substances on rhe activity of deoxyribonuclease was determined initially by comparative measurements of the formation of “acid-soluble” polynucleotide material as follows:Method I. A series of test-tubes each containing 2 per cent (“‘I,) purified commercial thymus sodium deoxyribonucleate (2.0 ml), 0.1 M magnesium sulphate (1.0 ml), 0.1 M Verona1 buffer pH 7.0 (1.0 ml), 0.01 per cent (“/“) deoxyribonuclease (0.5 ml) and the solution (0.5 ml) of the substance under examination, was incubated at 37’. After 16 hrs the tubes were removed from the incubator, cooled to 0” and aliquots (4.0 ml) of the contents transferred to tared centrifuge tubes. 5 N hydrochloric acid (0.1 ml) was then added to each and the resulting “acid insoluble” precipitate collected at the centrifuge. The deposit was washed at the centrifuge with ethanol, ethanol and ether and finally with ether. After standing overnight in vacua at room temperature, the tubes were re-weighed and the amount of “acid insoluble” material determined by difference. Each determination was carried out in triplicate. By this method the following substances were examined for inhibitory action:-0.1 A4 sodium arsenate, 0.1 &I sodium selenite, 0.5 M thioglycollic acid neutralised to pH 8.0, 0.3 M potassium fluoride (in the presence of 0.2 M phosphate buffer pH 7.9), 0.1 M hydroxylamine, 0.1 M cysteine, and 0.03 M iodoacetic acid. The results obtained (Table I) show that the enzyme was almost completely inhibited by arsenate, selenite and fluoride ions, and partially inhibited by thioglycollate. Method II. Since the use of the above method was only suitable in cases where the inhibitory substances remained in solution in the presence of dilute acid, the following procedure proved of more general application:A series of test-tubes, each containing 1 .O per cent (“lV) purified sodium deoxyribonucleate (2.0 ml), 0.1 M magnesium sulphate (1.0 ml), 0.01 per cent (“/“) deoxyribonuclease (1 .O ml) and a solution (1.0 ml) of the inhibitory
Inhibition
of Pancreas deoxyribonmlease TABLE
The inhibition
of deoxyribonuclease
I
by various substances Method I (see Text).
Inhibitor
0.1 A4 Sodium selenite 0.1 M Sodium arsenate 0.5 M Thioglycollic acid 0.3 M Potassium fluoride 0.1 M Hydroxylamine 0.1 M Cysteine 0.03 M Iodoacetic acid Control (No Inhibitor)
351
under
the conditions
described
in
.--Percentage of deoxyrihonucleic acid converted to acid soluble material in 16 hours at 37’ c. 5 5 44 10 95 94 91 95
C.
substance, was incubated at 37”. At suitable time intervals (Fig. I) the contents of one tube were treated with 5 N hydrochloric acid (0.1 ml) in order to precipitate the residual “acid-insoluble” material. After centrifuging, the extent of the hydrolysis, in terms of the increase in acid soluble phosphorus, was determined by Allen’s (1) method in an aliquot of either the precipitate in 0.05 clear supernatant, or a solution of the ‘“acid insoluble” M sodium carbonate. The following substances were examined for inhibitory action by this method.(a) 0.01 M copper sulphate; (b) 0.1 11/1zinc sulphate; (c) 0.01 M zinc sulphate; (d) 0.125 M sodium sulphide; (e) 0.1 ill potassium fluoride; (f) 0.01 M potassium fluoride; (g) 0.1 M sodium citrate adjusted to pH 7.0; (h) 0.01 M sodium citrate adjusted to pH 7.0; (i) 0.1 M sodium borate: (j) 0.01 M hydroxylamine hydrochloride; (1~) 0.01 M semicarbazide hydrochloride; (1) 0.01 M sodium bisulphite; (m) 6 X 10F4 M iodoacetic acid: (n) 6 X lop4 M iodoacetamide. Of these substances a, b, c, d, e, g and i completely inhibited, and substances f and h partially inhibited the action of the enzyme (Fig. 1). The inhibitory action of fluoride and citrate ions on the activity of systems containing deoxyribonuclease and magnesium was reported by McCarty (28), whilst the inhibition of the enzyme by arsenate was noted by Fischer, Bottger and Lehmann-Echternacht (14). Since dilute sodium arsenate (or sodium citrate) has been proposed as a solvent for the isolation of undegraded deoxyribonucleoproteins (33), it was of interest to determine the
L. M. Gilbert, W. G. Ouerend, and M. Webb
352
Fig. 1. Effect
of various
subsfances upon the hydrolysis
of thymus deoxyribonucleic
acid by deoxyribo-
Conditions as described in Method II. e---d control (no inhibitor), A---A 0.1 ~34zinc sulphate, 0-0 0.01 M zinc s&hate, a----(> 0.1 M sodium citrate, l __ l 0.01 M sodium citrate, O--O 0.01 M -semicarbazide~ hydrochloride, @---a 0.01 M potassium fluoride, 0 - -- 0 0.01 M hydroxylamine hydrochloride, $3~@ 0.01 M sodium bisulphite, l - - - - l 6 x lo-’ M iodoacetic acid. @--a 6 x lo-4 M iodoacetic acid. Curve A, 0.01 M copper sulphate, 0.125 M sodium sulphide, 0.1 M potassium fluoride and 0.1 M sodium borate.
n&ease.
relationship between the concentration tion, in systems containing constant and enzyme.
of arsenate and the degree of inhibiamounts of nucleic acid, magnesium,
E$ect of concentration of sodium arsenate on the activity of deoxyribonuclease A series of tubes containing a buffered solution of deoxyribonucleic acid, concentrations of sodium arsenate deoxyribonuclease, Mg+f and varying (10-l to 10-C M) was prepared according to Method I (above), and incubated at 37’. After 16 hours, the “acid-soluble” material remaining in each tube The results (Fig. 2) show that was determined as previously described. under the conditions of the experiment a final concentration of 1 X lop3 M sodium arsenate was required to inhibit the enzyme completely. It has been shown (30, 31) that the action of deoxyribonuclease on highly polymerised preparations of deoxyribonucleic acid results initially in a rapid depolymerisation of the substrate as evidenced by a striking decrease in During this period the products the relative viscosity of the solution. which result from the action o.f the enzyme are completely insoluble in dilute acids, and only when the relative viscosity has reached a constant
Inhibifion
of Pancreas deoxyribonuclease
353
Fig. 2. The effect of varying concentrations of sodium arsenate on the activity of deoxyribonuclease. Measurements of acid insoluble material (recovered nucleic acid) were made after 16 hours at 37” according to Method I.
value does the liberation of acid-soluble material occur to any marked extent. From then onwards, the formation of products soluble in dilute mineral acids follows a course typical of enzymic hydrolysis. The action of the enzyme on various commercial samples of deoxyribonucleic acid follows a similar course, but because these preparations are already partially degraded, the initial change in relative viscosity occurs between narrower limits. Recently Jungner, Jungner and AllgCn (25) and Vercauteren (34) independently have studied the enzymic degradation of deoxyribonucleic acid by different techniques with closely similar results, whereas Greenstein, Carter and Chalkley (19) conclude that the initial disaggregation of the nucleic acid molecule, as shown by the changes in relative viscosity, may be induced by a variety of inorganic and organic electrolytes, and is not due specifically to the action of the enzyme. In consequence of the mode of action of deoxyribonuclease, as outlined above, and in particular, of the conclusions of Greenstein et al. (19), it was of interest to determine whether the substances previously shown to inhibit the overall action of the enzyme, inhibited both the depolymerisation and the hydrolysis of the substrate. Influence
of inhibitors
on the clepolymerisation
of deoxyribonucleic
acid
The enzymic depolymerisation of the deoxyribonucleic acid was followed viscosimetrically at 38.5”. Measurements were made either in Ostwald viscometers or in viscometers of the type described by Koch, Orthmann and
L. M. Gilbert, W. G. Overend, and M. Webb
Fig. 3 A.
Fig. 3 B.
Fig. 3 A. The action of inhibiiors on the depolymerisaiion of calf thymus and herring sperm deoxyribonucleic acids. The relative viscosities (Q) of the solutions are plotted as the ordinate, and are taken as a measure of the polymerisation of the substrate. All solutions contained the deoxyribonucleic acid (0.2 per cent, 2 ml), 0.2 M Verona1 buffer pH 6.92 (1.0 ml), 0.1 M magnesium sulphate, deoxyribonuclease (0.002 per cent, 0.5 ml) and the inhibitor (0.5 ml). A---A Herring sperm deoxyribonucleic acid substrate, no inhibitor. (l-6) Herring sperm deoxyribonucleic acid substrate. 0.01 M zinc sulphate. l ~ l Thymus deoxyribonucleic acid substrate. No inhibitor. A-A Thymus deoxyribonucleic acid substrate. 0.01 M sodium arsenate. O-0 Thymus deoxyribonucleic acid substrate. 0.01 M sodium selenite. Fig. 3 B. The a&ion of inhibitors on the depolymerisation of purified (commercial) thymus deoxyribonucleic acid. All solutions contained the deoxyribonucleic acid solution (8 per cent, 2 ml), 0.2 M Verona1 buffer pH 6.92 (1.0 ml), 0.1 M magnesium sulphate (1.0 ml), deoxyribonuclease (0.002 per cent, 0.05 ml) and the inhibitor (0.5 ml). A---A control (no inhibitor), @--CD 0.01 M semicarbazide hydrochloride, A-A l ~ l 0.01 M sodium arsenate, V---V 0.01 M zinc sul0.01 M copper sulphate, phate, O-0 0.01 M sodium citrate.
Degenfelder (26). Both the highly polymerised preparations of the deoxyribonucleic acids from thymus and herring sperm (0.2 per cent (“I,) solutions) and the purified commercial deoxyribonucleic acid (8.0 per cent (“I,) solution) were used in these experiments. The buffered substrate mixture containing the deoxyribonucleic acid solution (2.0 ml), 0.2 M Verona1 buffer, pH 6.92 (1.0 ml), 0.1 M magnesium sulphate (1.0 ml) and the inhibitor (0.5 ml), was introduced into the viscometer, and the temperature allowed to equilibrate. The deoxyribonuclease solution (0.002 per cent (“I,). 0.5 ml, preheated
Inhibition
of Pancreas deoxyribonucleease
to 38.5”) was then added and viscosity measurements commenced immediThe following substances were examined for inhibitory ately after mixing. action on the depolymerisation under the above conditions: sodium arsenate, sodium selenite, zinc sulphate, copper sulphate, sodium citrate (adjusted to pH 7.0), semicarbazide hydrochloride and hydroxylamine hydrochloride (Figs. 3 A and 3 B). The fact that arsenate, selenite, citrate, copper and zinc ions inhibit the enzymic depolymerisation of deoxyribonucleic acid, proves that the change in viscosity observed in the control experiments (without inhibitor) is due specifically to the action of deoxyribonuclease, and not to the non-specific action of metallic ions (19). Influence
of inhibitors on the hydrolysis deoxyribonucleic acid
of depolymerised
The experiments outlined under Method II above, were repeated with the exception that the addition of substances (a) to (n) was not made until 20 minutes after the addition of the enzyme (i.e. until depolymerisation From then on the liberation of acid soluble was more or less complete). polynucleotide material was followed by phosphorus estimations in the usual way. Under these conditions the action of the enzyme was arrested by those substances which inhibited the depolymerisation of the deoxyribonucleic acid (Fig. 3). The Hydrolysis
of Deoxyribonucleohistone
by Deoxyribonuclease
The action of deoxyribonuclease on the deoxyribonucleohistone from calf thymus nuclei was studied viscosimetrically in Ostwald viscometers (flow time for water (4.5 ml) 85-90 sets.) at 34.5”* 0.25”. The deoxyribonucleohistone was dissolved in water by stirring mechanically, and the resulting opalescent, viscous solution filtered through a plug of cotton wool. The filtrate was suitably diluted with water until a test sample (2.0 ml) together with 0.1 M Verona1 buffer pH 6.9 (1.0 ml) and distilled water (1.5 ml) had a flow time in the viscometers of 3 to 4 minutes. The concentration of the nucleoprotein solution (0.9 per cent “IV) was then determined by phosphorus analyses and by drying and weighing the precipitate produced by the addition of ethanol (2 ~01s) to an aliquot (10 ml) of the solution. A gradual decrease in the relative viscosity occurred with time in mixtures of the nucleoprotein (2.0 ml), Verona1 buffer (1.0 ml) and distilled water (1.5 ml), (Fig. 4). Since this change was not observed when the nu-
356
L. M. Gilbert, W. G. Ouerend, and M. Webb 1.1 2.6
Fig. 4. Self hydrolysis (depolymerisation) of thymus deoxyribonucleohistone at pH 6.9 ( l __ and its inhibition by 2 A4 sodium chloride (O-O), 0.01 M sodium arsenate (D-CD,) and 0.1 M sodium fluoride (A--A).
l
)
cleoprotein solution (2.0 ml) was diluted with 2.0 M sodium chloride (2.0 ml), 0.05 A4 sodium arsenate (2.0 ml) or 0.05 M sodium fluoride (2.0 ml), (Fig. 4); it is concluded that a small amount of thymus deoxyribonuclease (37) was associated with the nucleoprotein. In initial experiments it was observed that the addition of magnesium ions (essential for the action of deoxyribonuclease with deoxyribonucleic acid as substrate) to the deoxyribonucleohistone solution, resulted in the precipitation of the nucleoprotein. Qualitative experiments in which the nucleoprotein solution (1 .O ml) was added to 0.2 per cent (“/“) solutions of various metallic salts, showed that precipitation occurred with all the metallic ions examined with the exception of sodium and potassium. From a visual estimation it was possible to arrange the metallic ions in the following order according to the bulk of precipitate produced and the rate at which it separated:-
Ag+ > Hg ++ > Fe+++ > Be++ > Pb++ > Cu++ > Ba++ = Gaff = = Mn++ > Fe++ > Mg++. The effect was limited to cations, and was not a non-specific effect due to ionic concentration, since the precipitates failed to dissolve when diluted with water (10 ~01s.). Furthermore, it was not a peculiar property of the
Inhibition
of Pancreas deoxyribonuclease
357
Fig. 5. The action of deoxyribonuclease on thymus deoxyrihonucleohistone (O--O) and its inhibition by the addition of 1.0 ml 0.1 M sodium fluoride ( l ~--. l ) or 0.1 M sodium citrate (a:---(D). Hydrolysis of the substrate shown by the decrease in relative viscosity (TV). Each solution contained the deoxyribonucleohistone solution (2.0 ml), 0.2 A4 Verona1 buffer pH 6.9 (1.2 ml) and 0.002 deoxyribonuclease (0.3 ml). Fig. G. The action of 0.1 M sodium arsenate (@ ~~ a,), 0.1 M sodium selenite ( a I_ l ) and 0.1 M hydroxylamine or cysteine (O---O) on the hydrolysis of deoxyribonucleohistone and cysteine proby deoxyribonuclease. Conditions were as in Fig. 5. Hydroxylamine duced no inhibition of the enzyme. Thus, the curve (O-O) coincides with that showing the action of the enzyme alone on thymus deoxyribonucleohistone.
deoxyrihonucleohistone isolated by Stern’s (33) method, since the same effect vvas observed when solutions of fibrous nucleoprotein, isolated from thymus with 1.0 M sodium chloride according to the method of Mirsky and Pollister (29), were treated with the metallic ions listed above. In the few cases examined, precipitation of the nucleoprotein appeared to be complete, since the clear solutions obtained after removing the insoluble (Ag+ Be++ and RIg+f) “salts” (centrifuge) gave negative reactions for deoxyribose with the Dische (9) dephenylamine reagent. It was observed that the Ca+f, Fe++, Be++ and Mg++ “salts” of the nucleohistone were completely soluble in 2 &1 sodium chloride. In this solvent, the Cu++ and Ba++ “salts” were partially soluble and yielded suspensions of high viscosity, whereas the Ag+, Hg++ and Fe+++ “salts” remained undissolved.
358
L. M. Gilbert, W. G. Ouerend, and M. Webb
Since the precipitating effect of Mg+f and other metallic ions on thymus nuclrohistone rendered it impossible to follow viscosimetrically the action of magnesium-activated deoxyrihonuclease on this substrate, the action of the enzyme on the nucleoprotein was studied in the absence of free magnesium ions. Under these conditions the enzyme exhibited high activity and caused the rapid hydrolysis of the substrate (Fig. 5). Since this hydrolysis was inhibited when either 0.1 A4 sodium fluoride (1.0 ml) or 0.1 M sodium citrate (1 .O ml) was added to solutions containing deoxyribonucleohistone (2.0 ml), 0.2 Afveronal buffer (1.2 ml) and 0.002 per cent (“‘/“) deoxyribonuclease (0.3 ml), (Fig. 5), it appeared that the enzyme-substrate system was activated by magnesium present in the nucleohistone. In this connection the nucleoprotein yielded 10.55 per cent ash on combustion which, when analysed according to the method previously described (35), was found to contain 0.15 per cent magnesium (= a magnesium content of 0.016 per cent in the nucleohistone). In addition to sodium citrate and sodium fluoride, 0.1 M sodium arsenate (1 .O ml) and 0.1 A1 sodium selenite (1 .O ml) were found to inhibit the action of deoxyribonuclease (0.002 per cent, 0.3 ml) on deoxyribonucleohistone (2.0 ml) in 0.1 Al Verona1 buffer pH 6.92 (1.2 ml), (Fig. S), whereas equivalent concentrations of hydroxylamine hydrochloride (in 0.1 iVf sodium acetate) and cysteine were without effect upon the activity of the enzyme (Fig. 6). It will be noted from these results (Fig. 6) that the increased ionic strength, due to the presence of the “inhibitors,” reduced the initial relative viscosity of the nucleoprotein solution.
The injluence of mitotic inhibitors on the hydrolysis of deoxyribonucleohistone by deoxyribonuclease It appeared possible that certain inhibitors of mitosis exert their action by reacting with nuclear constituents and thereby preventing subsequent enzymic processes. It was of some interest, therefore, to determine whether such substances exerted any effect upon the degradation of the nuclear deoxyribonucleohistonc by deoxyribonuclease. Particular attention was directed to the -SH reactants chloroacetophenone, iodoacetic acid and iodoaretamide (Group I) which cause telophase inhibition in cells in chick tissue cultures (Hughes 23). In addition, the action of colchicine and aminopterin (4-amino-pteroylglutamic acid) (Group 2) and certain nitrogen mustards (Group 3) was studied. For the evaluation of the action of compounds of groups 1 and 2, the experimental technique was as follows:-
Inhibition
of Pancreas deoxyribonuclease
TIME
Fig. 7.
359
(“M,
Fig. 8.
of deoxyribonucleohistone with deoxyribonuclease alone ( l -l ) and in Fig. 7. The hydrolysis the presence of 1.0 ml 0.01 M iodoacetamide (0 - - - - 0) under the conditions recorded in the text. The curves showing the hydrolysis of the substrate with time in the presence of aminopterin (1 mg) and chloroacetophenone (1 ml of a saturated aqueous solution) coincide with the above, and are not recorded in the figure. ufter treatment wifh nilroFig. 8. The action of deoxyribonuclease on thymus deoxyribonucleohisfone yen musfards. cI)--CD control-deoxyribonucleohistone with deoxyribonuclease as in Fig. 5. Deoxyribonucleohistone treated directly with (1) N.N. di-(2-chloroethyl)-p-amino benzoic acid (O--O) and (2) P-naphthyl-di-(2-chloroprophyl)amine ( l ~~- 6 ) according to method (a) (p. 359). The curves (e-5) and ( l - - - - l ) show the action of deoxyribonuclease on the deoxyribonucleohistone after treatment with buffered aqueous solutions of P-naphthyl-di-$2-chloropropyl)amine and N.N. di-(2-chloroethyl)-p-aminobenzoic acid respectively, which had been kept at room temperature for 20 mins.
A mixture of the nucleoprotein solution (2.0 ml) 0.1 M Verona1 buffer pH 6.92 (1.2 ml) and the inhibitor solution (1.0 ml) was allowed to stand at room temperature for 20-60 minutes. It was then introduced into the viscometer, the temperature allowed to equilibrate, and deoxyribonuclease (0.002 per cent (“/“) 0.3 ml) added. Measurements of relative viscosity were made at frequent intervals during the following 30-60 minutes. The action of compounds of group 3 was determined according to the following methods:(a) To the mechanically stirred nucleoprotein solution (8.0 ml) was added a solution of the nitrogen mustard (8.0 mg) in the minimum volume of ethThe resulting turbid solution was anol necessary for complete solution. stirred for a further 15 minutes and then allowed to stand at room temper-
360
L. M. Gilbert, W. G. Overend, and M. Webb
Fig. 9. Fig. 9. The hydrolysis by deoxyribonucleose of thymus deoxyribonucleohistone treated with nitrogen mustards. Hydrolysis measured by the liberation of acid soluble phosphorus, expressed as a percentage of the total phosphorus of the substrate, according to method (c) (p. 360). O----O. The action of deoxyribonuclease on thymus deoxyribonucleohistone alone (control). Q)---CE and l __ l , the action of deoxyribonuclease on deoxyribonucleohistone treated with N.N. di-(2-chloroethyl)-p-aminobenzoic acid and P-naphthyl-di(2-chloropropyl)-amine respectively.
ature for GO minutes, (marked precipitation occurred in such solutions on prolonged standing). At the end of this period an aliquot of the solution lvas diluted with 0.1 M Verona1 buffer pH 6.9 (2.0 ml) and then introduced into the viscometer. After equilibration of temperature, deosgribonuclease (0.002 per cent, 0.5 ml) was added and viscosity measurements commenced. (b) The ethanolic solution of the nitrogen mustard (4 mg) was added with stirring to 0.1 AI veronal buffer pH 6.9 (4.0 ml). This was follo\ved after 20 minutes by the addition of the nucleoprotein solution (4.0 ml). After a further 60 minutes, 4 ml of the mixture were placed in the viscometer, the temperature brought to 34.5” and the deosyribonuclease added. (c) An ethanolic solution of the nitrogen mustard (4.0 mg) \vas diluted with buffer (4.0 ml) and the nucleoprotein solution (8.0 ml) added \vith mechanical stirring. After 2 hours at room temperature, ethanol (3 vols) was added and the resulting precipitate collected at the centrifuge. A solution of the precipitate in distilled water (16.0 ml) and 0.1 M Verona1 bufrer pH 7.1 (10.0 ml) was brought to 37” and deoxyribonuclease (0.02 per cent, (“/“) 4 ml) added. At suitable intervals, aliquots (3 ml) of the solution were withdrawn, cooled to 0” and 5 N hydrochloric acid (0.1 ml) added. The resulting precipitate was removed (centrifuge) and the total “acid soluble”
Inhibition
of Pancreas deoxyribonuclease
organic phosphorus determined in the clear supernatant as previously described. The results of these experiments show that no inhibition of the enzymic degradation of the deoxyribonucleohistone resulted from the presence of colchicine, aminopterin or the -SH reactants (Fig. 7). On the other hand, by reaction with the nitrogen mustards, became less the nucleoprotein, susceptible to hydrolysis with deoxyribonuclease, although, under the conditions of the experiments, neither the depolymerisation (Fig. 8) nor the hydrolysis (Fig. 9) of the substrate were completely inhibited. Furthermore, the inhibitory ac.tivities of these substances were reduced considerably when they were kept in aqueous solution for 20 minutes before reaction with the nucleoprotein (Fig. 8). DISCUSSION
The fact that the magnesium ion is essential for the activity of deoxyribonuclease from pancreas, has been observed by Fischer, Lehmann-Echternacht and Bottger (15), McCarty (28), Greenstein, Carter and Chalkley (19) and by Overend and Webb (31), (but cf. B. 2.). It follows therefore, that substances which inhibit the activity of deoxyribonuclease may be divided into two groups, namely those which exert their action through their ability to remove, by coordination or precipitation, the activating magnesium ion, and those which directly interact with the functional groups of the enzyme protein. Among substances of the first group, we have confirmed in agreement with McCarty (28) and others, the inhibitory action of fluorides, citrates and borates. Reference to Fig. 1 shows that a final concentration of 1.67 >: 10e2 M potassium fluoride or sodium borate completely inhibits the action of the enzyme on deoxyribonucleic acid in the presence of 1.67 X lop2 ill magnesium sulphate. Sodium citrate at the same molar concentration is only slightly less effective and gives 90 per cent inhibition during the same interval of time. Lower concentrations of potassium fluoride or sodium citrate (Fig. l), which only partially remove the magnesium ion, decrease the activity of the enzyme without causing complete inhibition. The inhibitory action of arsenate and selenite (Table I) on the activity of the enzyme, may also reside in the ability of these ions to form sparingly soluble magnesium salts. Addition of a solution of sodium selenite to magnesium sulphate, for example, results in rapid crystallisation of magnesium selenite from solution. 21-513703
362
L. M. Gilbert, W. G. Ouerend, and M. Webb
Among substances of the second group, sodium sulphide and thioglycollic acid (Table I) were found to decrease markedly the activity of deoxyribonuclease. On the other hand, cysteine, sodium bisulphite, hydroxylamine and semicarbazide had no influence upon the activity of the enzyme. Similar negative results were obtained with the sulphydryl reactants, iodoacetamide and iodoacetic acid. These substances have been shown to arrest mitosis in telophase in cells in chick tissue cultures (23) and were, in consequence, of particular interest in this investigation. The nature of the marked inhibitory action of copper and zinc ions upon the activity of the enzyme remains to be determined. In view of the lack of effect of iodoacetamide and iodoacetic acid (Fig. 1 and Table I) it does not appear that the action of these metals can be due to their reaction with essential -SH groupings of the enzyme. It is possible that these ions exert their effect on the nucleic acid rather than on the enzyme, since these and other metallic ions are known to precipitate deoxyribonucleic acid (15). The observation that zinc inhibits the enzymic degradation of deoxyribonucleic acid is of particular interest when considered in relation to the findings that significant quantities of zinc are concentrated in the deoxyribonucleoproteins isolated from thymus and tumour tissue (20). It is also interesting to recall that both copper and zinc inhibit the action of ribonuclease on ribonucleic acid, although this enzyme is inactivated by -SH reactants and presumably contains essential, functional sulphydryl groups (38). The action of the enzyme on deoxyribonucleohistone proceeds in the absence of free magnesium ion. The inhibition of the enzyme by fluoride citrate, arsenate and selenite ions (Figs. 5 and 6) under these conditions, however, suggests that the enzyme-substrate system is activated by the magnesium present in the nucleoprotein. This observation is in accordance with the suggestion of Weissman and Fisher (36) that magnesium does not directly activate deoxyribonuclease, but alters the substrate in some way such that the enzyme may function. Cohen (7) has shown that when deoxyribonucleohistone is digested with trypsin, there is an increase in the viscosity of the solution due to the liberated deoxyribonucleic acid exhibiting a higher viscosity than the nucleoprotein. Iri the present experiments, the rapid decrease in viscosity which occurred when deoxyribonuclease was added to the deoxyribonucleohistone solution commenced instantaneously, and an initial rise in viscosity was never observed. Thus, it has not been possible to determine whether the enzyme acts directly upon the nucleoprotein, or whether the degradation
Inhibition
of Pancreas deoxgribonuclease
363
of the deoxyribonucleic acid moiety is preceded by the dissociation of the nucleohistone. The action of deoxyribonuclease on deoxyribonucleohistone was not inhibited by pre-treatment of the substrate with colchicine, 4-aminopteroylglutamic acid, or the -SH reactants chloroacetophenone, iodoacetic acid and iodoacetamide. The absence of any inhibitory action of the latter group of substances is in accordance with the apparent low sulphydryl content of the nucleohistone, and suggests that, under the given experimental conditions, these agents do not react with essential amino groupings (3). The nitrogen mustards, which in sub-lethal concentrations cause mitotic abnormalities similar to those produced by mustard gas (17, 5, 12, 13), were observed to react with the nucleohistone and render the latter partially resistant to enzymic degradation. These compounds were added to the substrate in concentrated ethanolic solution; a method similar to that used by Dixon and Needham (lo), in their studies on the action of mustard gas on various enzyme systems (21). Under these conditions some precipitation of the nucleoprotein occurred. Precipitation also results from the application of mustard gas to nucleoproteins (4) and nucleic acids (11). In the latter case, it is known from electrometric titration that reaction occurs between the mustard gas and primary and secondary phosphoryl groups as well as amino groups of the nucleic acid (11). It is probable that the effect of the nitrogen mustards is due to the alkylating effect of the halogenoalkyl groups on similar groups of the nucleic acid or nucleoprotein (18, 27, 6). In view of the high order of reactivity of the nitrogen mustards with various functional constituents of the cell, little importance can be attached to the observations that the products from the interaction of deoxyribonucleohistone with these substances are resistant to enzymic attack. Indeed, with regard to the inhibition of mitosis produced by these compounds, their precipitating action on the nucleoprotein may be of greater significance than the fact that they render the latter partially resistant to degradation with deoxyribonuclease. Apart from fluoride and selenite ions, which inhibit both deoxyribonuclease and mitosis in tissue cultures (23), the lack of effect of the various mitotic inhibitors on the degradation of deoxyribonucleohistone with deoxyribonuclease suggests that if an enzyme of the deoxyribonuclease-type exists in cell nuclei and plays any part in the sequence of changes which occur during mitosis, the properties of such an enzyme must differ considerably from those of the exocellular, pancreatic deoxyribonuclease.
L. M. Gilbert, W. G. Overend, and M. Webb SUMMARY
The action of inhibitors on the enzymic degradation of deoxyribonucleic acid and deoxyribonucleohistone by pancreatic deoxyribonuclease has been studied. The results show that the inhibitors may be divided broadly into two classes, namely those which remove the activating magnesium ion, and those which directly interact with the enzyme protein. Active inhibitors prevent both stages (i.e. depolymerisation and hydrolysis) of the degradation of the deoxyribonucleic acid. The products which result from the reaction of nitrogen mustards with deoxyribonucleohistone are partially resistant to the action of deoxyribonuclease. Pre-treatment of deoxyribonucleohistone with other mitotic inhibitors has no effect upon its subsequent degradation with deoxyribonuclease. Thanks are due to Professor M. Stacey, F.R.S. and to Dr. Honor B. Fell for their interest in this work, and to Dr. A. F. W. Hughes for suggesting the study of the action of mitotic inhibitors on the enzymic degradation of deoxyribonucleohistone. REFERENCES ALLEN, R. J. L., Biochem. J., 34, 858 (1940). BARGONI, N., Boll. sot. ital. sper., 20, 534, 1945. Chem. Abs. 40, 6513 (1946). BARRON, E. G. S., and SINGER, T. P., J. biol. Chem. 157, 221 (1945). BERENBLUM, I., and SCHOENTAL, R., Nature, 159, 727 (1947). BODENSTEIN, D., J. Exptt. Zool., 104, 311 (1947). BUTLER, J. A. V., Nature, 166, 18 (1950). COHEN, S. S., J. Biol. Chem., 158, 255 (1945). DAVIDSON. J. N.. Ann. Rev. Biochem.. 18, 155 (1949). DISCHE, i., Mikrochemie, 8, 4 (1930): ’ DIXON, M., and NEEDHAM, D. M., Nature, 158, 432 (1946). 11. ELMORE, D. E., GULLAND, J. M., JORDAN, D. O., and TAYLOR, H. F. W., Biochem.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
308
(1948).
12. FELL, H. B., and ALLSOPP, C. B., Cancer Res., 8, 145 13. -Cancer Res., 8, 177 (1948). 14. FISCHER, F. G., BOTTGER, I., and LEHMANN-ECHTERNACHT,
(1941).
15. FISCHER,
F.
G.,
LEHMANN-ECHTERNACHT,
(1941).
H.,
and
J.
42,
(1948). H., Z. physiol. Chem., 271,246
BOTTGER,
I., J. prakt.
Chem., 158, 79
L. M., OVEREND, W. G., and WEBB, iV., ExptI. Cell. Res, 2, 138 (1951). R., and BODENSTEIN, D., J. Exptt. Zool., 103, 1 (1946). GOLDACRE, R. J., LOVELESS, A., and Ross, W. C. .J., Nature, 163, 667 (1949). GREENSTEIN, J. P., CARTER, C. E., and CHALKLEY, H. W., Cold Spring Harbor Symp. Quant.
16. GILBERT, 17. GILLETTE, 18. 19.
20. HEATH,
21.
BioZ., 12, 64 (1947). J. C., Nature, 164, 1055
HERRIOT, 22. HOLMES, 23. HUGHES,
(1949).
R. M., ANSON, M. L., and NORTHROP, J. H., J. Gen. Physiol., 30, 185 B. E., Ann. Rep. Brit. Empire Cancer Campaign, 26, 191 (1948). A. F. W., J. Roy. Microscop. Sot., 69, 215 (1949); Quant. J. Micro. Sot.
press).
(1946). (in the
Inhibition
of Pancreas deoxyribonuclease
24. JACOBSON, W., and WEBB, M., 25. JUNGNER, G., JUNGNER, I., and 26. KOCH, J. R., ORTHMANN, A. C., 34, 489 (1939). 27. KON, G. A. R., and ROBERTS, 28. MCCARTY, M., J. Gen. Physiot., 29.
30. 31.
32. 33. 34. 35.
36. 37. 38.
J. Physiol., 112, 29 (1951). ALLGEN, L. G., Nature, 164, 1009 (1949). and DEGENFELDER, M. A., J. Amer. Leather.
Chem. Assoc.,
J. Chem. Sot., 978 (1950). 29, 123 (1946). MIRSKY, A. E., and POLLISTER, A. W., Proc. Naft. Acad. Sci., 28, 344 (1942). OVEREND, W. G., and WEBB, M., Research, 2, 99 (1949). __ J. Chem. Sot., 2746 (1950). Research, 3, 238 (1950). STERN, K. G., Exptl. Cell Res. Suppl. I, 97 (1949). VERCAUTEREN, R., Nature, 165, 603 (1950). WEBB, M., J. Gen. Microbial., 2, 275 (1948). WEISSMAN, N., and FISCHER, J., J. biol. Chem., 178, 1007 (1949). ZAMENHOF, S., and CHARGAFF, E., J. biol. Chem., 180, 727 (1949). ZITTLE, C. A., J. biol. Chem., 163, 111 (1946). J. J.,
OF PANCREAS M. GILBERT,
DEOXYRIBONUCLEASE
W. G. OVEREND
and M. WEBB
Department of Chemistry, The University, Birmingham and Strangeways Research Laboratory, Cambridge, Great Britain Received
October
10, 1950
RECENT communications (30, 31) have described the results of an investigation of the properties and mode of action of deoxyribonuclease isolated from beef-pancreas by essentially the method of McCarty (28). These and additional studies have shown that the action of this enzyme, which requires magnesium ions for its activation, is analogous to that of the corresponding ribonuclease (32) but is confined specifically to the deoxyribo type of nucleic acid or its partially degraded products (16). In view of the increased use of deoxyribonuclease in cytochemical work (8, 24), together with the possible significance of enzymes of this type in a detailed investigation of the inhibition of the processes of cell division, Certain preliminary observations have been the enzyme appeared justified. mentioned briefly in a previous communication (31), and the present paper describes fully the extension of this work. In addition, a study has been made of the action of deoxyribonuclease on the deoxyribonucleohistone of calf thymus cell nuclei alone and in the presence of substances known to inhibit mitosis. MATERIAL
AND
METHODS
was isolated from beef-pancreas by McCarty’s (28) method, Deoxyribonuclease and was purified by repeated fractional precipitation with ammonium sulphate (31). Deoxyribonucleic acid. Highly polymeric preparations of deoxyribonucleic acid were isolated from calf-thymus and herring sperm according to the method of Mirsky and Pollister (29). The chemical and physical properties of these preparations N/P (= = 1.42 & 1.56 respectively) will be described elsewhere. In addition to these preparations, commercial (B. D. H.) deoxyribonucleic acid was also used as substrate. After purification as previously described (31) the nucleic acid had N/P : 1.6. Deoxyribonucleohistone was isolated with 0.002 M sodium arsenate (33) from the gelatinous mass of thymus cell nuclei remaining after the extraction of the minced tissue with 0.14 M sodium chloride. The nucleoprotein was precipitated by the addition of sodium chloride to 0.14 M concentration, and purified by repeated solution in 0.002 M sodium arsenate and precipitation with sodium chloride. Finally, it was dissolved in distilled water, precipitated with cold ethanol (1 vol.) and dried with
350
L. M.
Gilberf,
W. G. Overend,
and M.
Webb
ethanol and ether. The residual white powder (N, 15.6; P, 5.05 per cent), which contained a negligible amount of arsenate (Marsh test and potas;Llm iodide reaction), dissolved readily in water to give a viscous, opalescent solution. The product gave a positive reaction for sulphur after sodium fusion, but the nitroprusside reaction for -SH groups was negative hefore, and only weakly positive after, treatment -rrith potassium cyanide (22). EXPERIMENTAL
The inhibition
of the action of Deoxyribonuclease Deoxyribonucleic acid
on Thymus
The inhibitory action of various substances on rhe activity of deoxyribonuclease was determined initially by comparative measurements of the formation of “acid-soluble” polynucleotide material as follows:Method I. A series of test-tubes each containing 2 per cent (“‘I,) purified commercial thymus sodium deoxyribonucleate (2.0 ml), 0.1 M magnesium sulphate (1.0 ml), 0.1 M Verona1 buffer pH 7.0 (1.0 ml), 0.01 per cent (“/“) deoxyribonuclease (0.5 ml) and the solution (0.5 ml) of the substance under examination, was incubated at 37’. After 16 hrs the tubes were removed from the incubator, cooled to 0” and aliquots (4.0 ml) of the contents transferred to tared centrifuge tubes. 5 N hydrochloric acid (0.1 ml) was then added to each and the resulting “acid insoluble” precipitate collected at the centrifuge. The deposit was washed at the centrifuge with ethanol, ethanol and ether and finally with ether. After standing overnight in vacua at room temperature, the tubes were re-weighed and the amount of “acid insoluble” material determined by difference. Each determination was carried out in triplicate. By this method the following substances were examined for inhibitory action:-0.1 A4 sodium arsenate, 0.1 &I sodium selenite, 0.5 M thioglycollic acid neutralised to pH 8.0, 0.3 M potassium fluoride (in the presence of 0.2 M phosphate buffer pH 7.9), 0.1 M hydroxylamine, 0.1 M cysteine, and 0.03 M iodoacetic acid. The results obtained (Table I) show that the enzyme was almost completely inhibited by arsenate, selenite and fluoride ions, and partially inhibited by thioglycollate. Method II. Since the use of the above method was only suitable in cases where the inhibitory substances remained in solution in the presence of dilute acid, the following procedure proved of more general application:A series of test-tubes, each containing 1 .O per cent (“lV) purified sodium deoxyribonucleate (2.0 ml), 0.1 M magnesium sulphate (1.0 ml), 0.01 per cent (“/“) deoxyribonuclease (1 .O ml) and a solution (1.0 ml) of the inhibitory
Inhibition
of Pancreas deoxyribonmlease TABLE
The inhibition
of deoxyribonuclease
I
by various substances Method I (see Text).
Inhibitor
0.1 A4 Sodium selenite 0.1 M Sodium arsenate 0.5 M Thioglycollic acid 0.3 M Potassium fluoride 0.1 M Hydroxylamine 0.1 M Cysteine 0.03 M Iodoacetic acid Control (No Inhibitor)
351
under
the conditions
described
in
.--Percentage of deoxyrihonucleic acid converted to acid soluble material in 16 hours at 37’ c. 5 5 44 10 95 94 91 95
C.
substance, was incubated at 37”. At suitable time intervals (Fig. I) the contents of one tube were treated with 5 N hydrochloric acid (0.1 ml) in order to precipitate the residual “acid-insoluble” material. After centrifuging, the extent of the hydrolysis, in terms of the increase in acid soluble phosphorus, was determined by Allen’s (1) method in an aliquot of either the precipitate in 0.05 clear supernatant, or a solution of the ‘“acid insoluble” M sodium carbonate. The following substances were examined for inhibitory action by this method.(a) 0.01 M copper sulphate; (b) 0.1 11/1zinc sulphate; (c) 0.01 M zinc sulphate; (d) 0.125 M sodium sulphide; (e) 0.1 ill potassium fluoride; (f) 0.01 M potassium fluoride; (g) 0.1 M sodium citrate adjusted to pH 7.0; (h) 0.01 M sodium citrate adjusted to pH 7.0; (i) 0.1 M sodium borate: (j) 0.01 M hydroxylamine hydrochloride; (1~) 0.01 M semicarbazide hydrochloride; (1) 0.01 M sodium bisulphite; (m) 6 X 10F4 M iodoacetic acid: (n) 6 X lop4 M iodoacetamide. Of these substances a, b, c, d, e, g and i completely inhibited, and substances f and h partially inhibited the action of the enzyme (Fig. 1). The inhibitory action of fluoride and citrate ions on the activity of systems containing deoxyribonuclease and magnesium was reported by McCarty (28), whilst the inhibition of the enzyme by arsenate was noted by Fischer, Bottger and Lehmann-Echternacht (14). Since dilute sodium arsenate (or sodium citrate) has been proposed as a solvent for the isolation of undegraded deoxyribonucleoproteins (33), it was of interest to determine the
L. M. Gilbert, W. G. Ouerend, and M. Webb
352
Fig. 1. Effect
of various
subsfances upon the hydrolysis
of thymus deoxyribonucleic
acid by deoxyribo-
Conditions as described in Method II. e---d control (no inhibitor), A---A 0.1 ~34zinc sulphate, 0-0 0.01 M zinc s&hate, a----(> 0.1 M sodium citrate, l __ l 0.01 M sodium citrate, O--O 0.01 M -semicarbazide~ hydrochloride, @---a 0.01 M potassium fluoride, 0 - -- 0 0.01 M hydroxylamine hydrochloride, $3~@ 0.01 M sodium bisulphite, l - - - - l 6 x lo-’ M iodoacetic acid. @--a 6 x lo-4 M iodoacetic acid. Curve A, 0.01 M copper sulphate, 0.125 M sodium sulphide, 0.1 M potassium fluoride and 0.1 M sodium borate.
n&ease.
relationship between the concentration tion, in systems containing constant and enzyme.
of arsenate and the degree of inhibiamounts of nucleic acid, magnesium,
E$ect of concentration of sodium arsenate on the activity of deoxyribonuclease A series of tubes containing a buffered solution of deoxyribonucleic acid, concentrations of sodium arsenate deoxyribonuclease, Mg+f and varying (10-l to 10-C M) was prepared according to Method I (above), and incubated at 37’. After 16 hours, the “acid-soluble” material remaining in each tube The results (Fig. 2) show that was determined as previously described. under the conditions of the experiment a final concentration of 1 X lop3 M sodium arsenate was required to inhibit the enzyme completely. It has been shown (30, 31) that the action of deoxyribonuclease on highly polymerised preparations of deoxyribonucleic acid results initially in a rapid depolymerisation of the substrate as evidenced by a striking decrease in During this period the products the relative viscosity of the solution. which result from the action o.f the enzyme are completely insoluble in dilute acids, and only when the relative viscosity has reached a constant
Inhibifion
of Pancreas deoxyribonuclease
353
Fig. 2. The effect of varying concentrations of sodium arsenate on the activity of deoxyribonuclease. Measurements of acid insoluble material (recovered nucleic acid) were made after 16 hours at 37” according to Method I.
value does the liberation of acid-soluble material occur to any marked extent. From then onwards, the formation of products soluble in dilute mineral acids follows a course typical of enzymic hydrolysis. The action of the enzyme on various commercial samples of deoxyribonucleic acid follows a similar course, but because these preparations are already partially degraded, the initial change in relative viscosity occurs between narrower limits. Recently Jungner, Jungner and AllgCn (25) and Vercauteren (34) independently have studied the enzymic degradation of deoxyribonucleic acid by different techniques with closely similar results, whereas Greenstein, Carter and Chalkley (19) conclude that the initial disaggregation of the nucleic acid molecule, as shown by the changes in relative viscosity, may be induced by a variety of inorganic and organic electrolytes, and is not due specifically to the action of the enzyme. In consequence of the mode of action of deoxyribonuclease, as outlined above, and in particular, of the conclusions of Greenstein et al. (19), it was of interest to determine whether the substances previously shown to inhibit the overall action of the enzyme, inhibited both the depolymerisation and the hydrolysis of the substrate. Influence
of inhibitors
on the clepolymerisation
of deoxyribonucleic
acid
The enzymic depolymerisation of the deoxyribonucleic acid was followed viscosimetrically at 38.5”. Measurements were made either in Ostwald viscometers or in viscometers of the type described by Koch, Orthmann and
L. M. Gilbert, W. G. Overend, and M. Webb
Fig. 3 A.
Fig. 3 B.
Fig. 3 A. The action of inhibiiors on the depolymerisaiion of calf thymus and herring sperm deoxyribonucleic acids. The relative viscosities (Q) of the solutions are plotted as the ordinate, and are taken as a measure of the polymerisation of the substrate. All solutions contained the deoxyribonucleic acid (0.2 per cent, 2 ml), 0.2 M Verona1 buffer pH 6.92 (1.0 ml), 0.1 M magnesium sulphate, deoxyribonuclease (0.002 per cent, 0.5 ml) and the inhibitor (0.5 ml). A---A Herring sperm deoxyribonucleic acid substrate, no inhibitor. (l-6) Herring sperm deoxyribonucleic acid substrate. 0.01 M zinc sulphate. l ~ l Thymus deoxyribonucleic acid substrate. No inhibitor. A-A Thymus deoxyribonucleic acid substrate. 0.01 M sodium arsenate. O-0 Thymus deoxyribonucleic acid substrate. 0.01 M sodium selenite. Fig. 3 B. The a&ion of inhibitors on the depolymerisation of purified (commercial) thymus deoxyribonucleic acid. All solutions contained the deoxyribonucleic acid solution (8 per cent, 2 ml), 0.2 M Verona1 buffer pH 6.92 (1.0 ml), 0.1 M magnesium sulphate (1.0 ml), deoxyribonuclease (0.002 per cent, 0.05 ml) and the inhibitor (0.5 ml). A---A control (no inhibitor), @--CD 0.01 M semicarbazide hydrochloride, A-A l ~ l 0.01 M sodium arsenate, V---V 0.01 M zinc sul0.01 M copper sulphate, phate, O-0 0.01 M sodium citrate.
Degenfelder (26). Both the highly polymerised preparations of the deoxyribonucleic acids from thymus and herring sperm (0.2 per cent (“I,) solutions) and the purified commercial deoxyribonucleic acid (8.0 per cent (“I,) solution) were used in these experiments. The buffered substrate mixture containing the deoxyribonucleic acid solution (2.0 ml), 0.2 M Verona1 buffer, pH 6.92 (1.0 ml), 0.1 M magnesium sulphate (1.0 ml) and the inhibitor (0.5 ml), was introduced into the viscometer, and the temperature allowed to equilibrate. The deoxyribonuclease solution (0.002 per cent (“I,). 0.5 ml, preheated
Inhibition
of Pancreas deoxyribonucleease
to 38.5”) was then added and viscosity measurements commenced immediThe following substances were examined for inhibitory ately after mixing. action on the depolymerisation under the above conditions: sodium arsenate, sodium selenite, zinc sulphate, copper sulphate, sodium citrate (adjusted to pH 7.0), semicarbazide hydrochloride and hydroxylamine hydrochloride (Figs. 3 A and 3 B). The fact that arsenate, selenite, citrate, copper and zinc ions inhibit the enzymic depolymerisation of deoxyribonucleic acid, proves that the change in viscosity observed in the control experiments (without inhibitor) is due specifically to the action of deoxyribonuclease, and not to the non-specific action of metallic ions (19). Influence
of inhibitors on the hydrolysis deoxyribonucleic acid
of depolymerised
The experiments outlined under Method II above, were repeated with the exception that the addition of substances (a) to (n) was not made until 20 minutes after the addition of the enzyme (i.e. until depolymerisation From then on the liberation of acid soluble was more or less complete). polynucleotide material was followed by phosphorus estimations in the usual way. Under these conditions the action of the enzyme was arrested by those substances which inhibited the depolymerisation of the deoxyribonucleic acid (Fig. 3). The Hydrolysis
of Deoxyribonucleohistone
by Deoxyribonuclease
The action of deoxyribonuclease on the deoxyribonucleohistone from calf thymus nuclei was studied viscosimetrically in Ostwald viscometers (flow time for water (4.5 ml) 85-90 sets.) at 34.5”* 0.25”. The deoxyribonucleohistone was dissolved in water by stirring mechanically, and the resulting opalescent, viscous solution filtered through a plug of cotton wool. The filtrate was suitably diluted with water until a test sample (2.0 ml) together with 0.1 M Verona1 buffer pH 6.9 (1.0 ml) and distilled water (1.5 ml) had a flow time in the viscometers of 3 to 4 minutes. The concentration of the nucleoprotein solution (0.9 per cent “IV) was then determined by phosphorus analyses and by drying and weighing the precipitate produced by the addition of ethanol (2 ~01s) to an aliquot (10 ml) of the solution. A gradual decrease in the relative viscosity occurred with time in mixtures of the nucleoprotein (2.0 ml), Verona1 buffer (1.0 ml) and distilled water (1.5 ml), (Fig. 4). Since this change was not observed when the nu-
356
L. M. Gilbert, W. G. Ouerend, and M. Webb 1.1 2.6
Fig. 4. Self hydrolysis (depolymerisation) of thymus deoxyribonucleohistone at pH 6.9 ( l __ and its inhibition by 2 A4 sodium chloride (O-O), 0.01 M sodium arsenate (D-CD,) and 0.1 M sodium fluoride (A--A).
l
)
cleoprotein solution (2.0 ml) was diluted with 2.0 M sodium chloride (2.0 ml), 0.05 A4 sodium arsenate (2.0 ml) or 0.05 M sodium fluoride (2.0 ml), (Fig. 4); it is concluded that a small amount of thymus deoxyribonuclease (37) was associated with the nucleoprotein. In initial experiments it was observed that the addition of magnesium ions (essential for the action of deoxyribonuclease with deoxyribonucleic acid as substrate) to the deoxyribonucleohistone solution, resulted in the precipitation of the nucleoprotein. Qualitative experiments in which the nucleoprotein solution (1 .O ml) was added to 0.2 per cent (“/“) solutions of various metallic salts, showed that precipitation occurred with all the metallic ions examined with the exception of sodium and potassium. From a visual estimation it was possible to arrange the metallic ions in the following order according to the bulk of precipitate produced and the rate at which it separated:-
Ag+ > Hg ++ > Fe+++ > Be++ > Pb++ > Cu++ > Ba++ = Gaff = = Mn++ > Fe++ > Mg++. The effect was limited to cations, and was not a non-specific effect due to ionic concentration, since the precipitates failed to dissolve when diluted with water (10 ~01s.). Furthermore, it was not a peculiar property of the
Inhibition
of Pancreas deoxyribonuclease
357
Fig. 5. The action of deoxyribonuclease on thymus deoxyrihonucleohistone (O--O) and its inhibition by the addition of 1.0 ml 0.1 M sodium fluoride ( l ~--. l ) or 0.1 M sodium citrate (a:---(D). Hydrolysis of the substrate shown by the decrease in relative viscosity (TV). Each solution contained the deoxyribonucleohistone solution (2.0 ml), 0.2 A4 Verona1 buffer pH 6.9 (1.2 ml) and 0.002 deoxyribonuclease (0.3 ml). Fig. G. The action of 0.1 M sodium arsenate (@ ~~ a,), 0.1 M sodium selenite ( a I_ l ) and 0.1 M hydroxylamine or cysteine (O---O) on the hydrolysis of deoxyribonucleohistone and cysteine proby deoxyribonuclease. Conditions were as in Fig. 5. Hydroxylamine duced no inhibition of the enzyme. Thus, the curve (O-O) coincides with that showing the action of the enzyme alone on thymus deoxyribonucleohistone.
deoxyrihonucleohistone isolated by Stern’s (33) method, since the same effect vvas observed when solutions of fibrous nucleoprotein, isolated from thymus with 1.0 M sodium chloride according to the method of Mirsky and Pollister (29), were treated with the metallic ions listed above. In the few cases examined, precipitation of the nucleoprotein appeared to be complete, since the clear solutions obtained after removing the insoluble (Ag+ Be++ and RIg+f) “salts” (centrifuge) gave negative reactions for deoxyribose with the Dische (9) dephenylamine reagent. It was observed that the Ca+f, Fe++, Be++ and Mg++ “salts” of the nucleohistone were completely soluble in 2 &1 sodium chloride. In this solvent, the Cu++ and Ba++ “salts” were partially soluble and yielded suspensions of high viscosity, whereas the Ag+, Hg++ and Fe+++ “salts” remained undissolved.
358
L. M. Gilbert, W. G. Ouerend, and M. Webb
Since the precipitating effect of Mg+f and other metallic ions on thymus nuclrohistone rendered it impossible to follow viscosimetrically the action of magnesium-activated deoxyrihonuclease on this substrate, the action of the enzyme on the nucleoprotein was studied in the absence of free magnesium ions. Under these conditions the enzyme exhibited high activity and caused the rapid hydrolysis of the substrate (Fig. 5). Since this hydrolysis was inhibited when either 0.1 A4 sodium fluoride (1.0 ml) or 0.1 M sodium citrate (1 .O ml) was added to solutions containing deoxyribonucleohistone (2.0 ml), 0.2 Afveronal buffer (1.2 ml) and 0.002 per cent (“‘/“) deoxyribonuclease (0.3 ml), (Fig. 5), it appeared that the enzyme-substrate system was activated by magnesium present in the nucleohistone. In this connection the nucleoprotein yielded 10.55 per cent ash on combustion which, when analysed according to the method previously described (35), was found to contain 0.15 per cent magnesium (= a magnesium content of 0.016 per cent in the nucleohistone). In addition to sodium citrate and sodium fluoride, 0.1 M sodium arsenate (1 .O ml) and 0.1 A1 sodium selenite (1 .O ml) were found to inhibit the action of deoxyribonuclease (0.002 per cent, 0.3 ml) on deoxyribonucleohistone (2.0 ml) in 0.1 Al Verona1 buffer pH 6.92 (1.2 ml), (Fig. S), whereas equivalent concentrations of hydroxylamine hydrochloride (in 0.1 iVf sodium acetate) and cysteine were without effect upon the activity of the enzyme (Fig. 6). It will be noted from these results (Fig. 6) that the increased ionic strength, due to the presence of the “inhibitors,” reduced the initial relative viscosity of the nucleoprotein solution.
The injluence of mitotic inhibitors on the hydrolysis of deoxyribonucleohistone by deoxyribonuclease It appeared possible that certain inhibitors of mitosis exert their action by reacting with nuclear constituents and thereby preventing subsequent enzymic processes. It was of some interest, therefore, to determine whether such substances exerted any effect upon the degradation of the nuclear deoxyribonucleohistonc by deoxyribonuclease. Particular attention was directed to the -SH reactants chloroacetophenone, iodoacetic acid and iodoaretamide (Group I) which cause telophase inhibition in cells in chick tissue cultures (Hughes 23). In addition, the action of colchicine and aminopterin (4-amino-pteroylglutamic acid) (Group 2) and certain nitrogen mustards (Group 3) was studied. For the evaluation of the action of compounds of groups 1 and 2, the experimental technique was as follows:-
Inhibition
of Pancreas deoxyribonuclease
TIME
Fig. 7.
359
(“M,
Fig. 8.
of deoxyribonucleohistone with deoxyribonuclease alone ( l -l ) and in Fig. 7. The hydrolysis the presence of 1.0 ml 0.01 M iodoacetamide (0 - - - - 0) under the conditions recorded in the text. The curves showing the hydrolysis of the substrate with time in the presence of aminopterin (1 mg) and chloroacetophenone (1 ml of a saturated aqueous solution) coincide with the above, and are not recorded in the figure. ufter treatment wifh nilroFig. 8. The action of deoxyribonuclease on thymus deoxyribonucleohisfone yen musfards. cI)--CD control-deoxyribonucleohistone with deoxyribonuclease as in Fig. 5. Deoxyribonucleohistone treated directly with (1) N.N. di-(2-chloroethyl)-p-amino benzoic acid (O--O) and (2) P-naphthyl-di-(2-chloroprophyl)amine ( l ~~- 6 ) according to method (a) (p. 359). The curves (e-5) and ( l - - - - l ) show the action of deoxyribonuclease on the deoxyribonucleohistone after treatment with buffered aqueous solutions of P-naphthyl-di-$2-chloropropyl)amine and N.N. di-(2-chloroethyl)-p-aminobenzoic acid respectively, which had been kept at room temperature for 20 mins.
A mixture of the nucleoprotein solution (2.0 ml) 0.1 M Verona1 buffer pH 6.92 (1.2 ml) and the inhibitor solution (1.0 ml) was allowed to stand at room temperature for 20-60 minutes. It was then introduced into the viscometer, the temperature allowed to equilibrate, and deoxyribonuclease (0.002 per cent (“/“) 0.3 ml) added. Measurements of relative viscosity were made at frequent intervals during the following 30-60 minutes. The action of compounds of group 3 was determined according to the following methods:(a) To the mechanically stirred nucleoprotein solution (8.0 ml) was added a solution of the nitrogen mustard (8.0 mg) in the minimum volume of ethThe resulting turbid solution was anol necessary for complete solution. stirred for a further 15 minutes and then allowed to stand at room temper-
360
L. M. Gilbert, W. G. Overend, and M. Webb
Fig. 9. Fig. 9. The hydrolysis by deoxyribonucleose of thymus deoxyribonucleohistone treated with nitrogen mustards. Hydrolysis measured by the liberation of acid soluble phosphorus, expressed as a percentage of the total phosphorus of the substrate, according to method (c) (p. 360). O----O. The action of deoxyribonuclease on thymus deoxyribonucleohistone alone (control). Q)---CE and l __ l , the action of deoxyribonuclease on deoxyribonucleohistone treated with N.N. di-(2-chloroethyl)-p-aminobenzoic acid and P-naphthyl-di(2-chloropropyl)-amine respectively.
ature for GO minutes, (marked precipitation occurred in such solutions on prolonged standing). At the end of this period an aliquot of the solution lvas diluted with 0.1 M Verona1 buffer pH 6.9 (2.0 ml) and then introduced into the viscometer. After equilibration of temperature, deosgribonuclease (0.002 per cent, 0.5 ml) was added and viscosity measurements commenced. (b) The ethanolic solution of the nitrogen mustard (4 mg) was added with stirring to 0.1 AI veronal buffer pH 6.9 (4.0 ml). This was follo\ved after 20 minutes by the addition of the nucleoprotein solution (4.0 ml). After a further 60 minutes, 4 ml of the mixture were placed in the viscometer, the temperature brought to 34.5” and the deosyribonuclease added. (c) An ethanolic solution of the nitrogen mustard (4.0 mg) \vas diluted with buffer (4.0 ml) and the nucleoprotein solution (8.0 ml) added \vith mechanical stirring. After 2 hours at room temperature, ethanol (3 vols) was added and the resulting precipitate collected at the centrifuge. A solution of the precipitate in distilled water (16.0 ml) and 0.1 M Verona1 bufrer pH 7.1 (10.0 ml) was brought to 37” and deoxyribonuclease (0.02 per cent, (“/“) 4 ml) added. At suitable intervals, aliquots (3 ml) of the solution were withdrawn, cooled to 0” and 5 N hydrochloric acid (0.1 ml) added. The resulting precipitate was removed (centrifuge) and the total “acid soluble”
Inhibition
of Pancreas deoxyribonuclease
organic phosphorus determined in the clear supernatant as previously described. The results of these experiments show that no inhibition of the enzymic degradation of the deoxyribonucleohistone resulted from the presence of colchicine, aminopterin or the -SH reactants (Fig. 7). On the other hand, by reaction with the nitrogen mustards, became less the nucleoprotein, susceptible to hydrolysis with deoxyribonuclease, although, under the conditions of the experiments, neither the depolymerisation (Fig. 8) nor the hydrolysis (Fig. 9) of the substrate were completely inhibited. Furthermore, the inhibitory ac.tivities of these substances were reduced considerably when they were kept in aqueous solution for 20 minutes before reaction with the nucleoprotein (Fig. 8). DISCUSSION
The fact that the magnesium ion is essential for the activity of deoxyribonuclease from pancreas, has been observed by Fischer, Lehmann-Echternacht and Bottger (15), McCarty (28), Greenstein, Carter and Chalkley (19) and by Overend and Webb (31), (but cf. B. 2.). It follows therefore, that substances which inhibit the activity of deoxyribonuclease may be divided into two groups, namely those which exert their action through their ability to remove, by coordination or precipitation, the activating magnesium ion, and those which directly interact with the functional groups of the enzyme protein. Among substances of the first group, we have confirmed in agreement with McCarty (28) and others, the inhibitory action of fluorides, citrates and borates. Reference to Fig. 1 shows that a final concentration of 1.67 >: 10e2 M potassium fluoride or sodium borate completely inhibits the action of the enzyme on deoxyribonucleic acid in the presence of 1.67 X lop2 ill magnesium sulphate. Sodium citrate at the same molar concentration is only slightly less effective and gives 90 per cent inhibition during the same interval of time. Lower concentrations of potassium fluoride or sodium citrate (Fig. l), which only partially remove the magnesium ion, decrease the activity of the enzyme without causing complete inhibition. The inhibitory action of arsenate and selenite (Table I) on the activity of the enzyme, may also reside in the ability of these ions to form sparingly soluble magnesium salts. Addition of a solution of sodium selenite to magnesium sulphate, for example, results in rapid crystallisation of magnesium selenite from solution. 21-513703
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L. M. Gilbert, W. G. Ouerend, and M. Webb
Among substances of the second group, sodium sulphide and thioglycollic acid (Table I) were found to decrease markedly the activity of deoxyribonuclease. On the other hand, cysteine, sodium bisulphite, hydroxylamine and semicarbazide had no influence upon the activity of the enzyme. Similar negative results were obtained with the sulphydryl reactants, iodoacetamide and iodoacetic acid. These substances have been shown to arrest mitosis in telophase in cells in chick tissue cultures (23) and were, in consequence, of particular interest in this investigation. The nature of the marked inhibitory action of copper and zinc ions upon the activity of the enzyme remains to be determined. In view of the lack of effect of iodoacetamide and iodoacetic acid (Fig. 1 and Table I) it does not appear that the action of these metals can be due to their reaction with essential -SH groupings of the enzyme. It is possible that these ions exert their effect on the nucleic acid rather than on the enzyme, since these and other metallic ions are known to precipitate deoxyribonucleic acid (15). The observation that zinc inhibits the enzymic degradation of deoxyribonucleic acid is of particular interest when considered in relation to the findings that significant quantities of zinc are concentrated in the deoxyribonucleoproteins isolated from thymus and tumour tissue (20). It is also interesting to recall that both copper and zinc inhibit the action of ribonuclease on ribonucleic acid, although this enzyme is inactivated by -SH reactants and presumably contains essential, functional sulphydryl groups (38). The action of the enzyme on deoxyribonucleohistone proceeds in the absence of free magnesium ion. The inhibition of the enzyme by fluoride citrate, arsenate and selenite ions (Figs. 5 and 6) under these conditions, however, suggests that the enzyme-substrate system is activated by the magnesium present in the nucleoprotein. This observation is in accordance with the suggestion of Weissman and Fisher (36) that magnesium does not directly activate deoxyribonuclease, but alters the substrate in some way such that the enzyme may function. Cohen (7) has shown that when deoxyribonucleohistone is digested with trypsin, there is an increase in the viscosity of the solution due to the liberated deoxyribonucleic acid exhibiting a higher viscosity than the nucleoprotein. Iri the present experiments, the rapid decrease in viscosity which occurred when deoxyribonuclease was added to the deoxyribonucleohistone solution commenced instantaneously, and an initial rise in viscosity was never observed. Thus, it has not been possible to determine whether the enzyme acts directly upon the nucleoprotein, or whether the degradation
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363
of the deoxyribonucleic acid moiety is preceded by the dissociation of the nucleohistone. The action of deoxyribonuclease on deoxyribonucleohistone was not inhibited by pre-treatment of the substrate with colchicine, 4-aminopteroylglutamic acid, or the -SH reactants chloroacetophenone, iodoacetic acid and iodoacetamide. The absence of any inhibitory action of the latter group of substances is in accordance with the apparent low sulphydryl content of the nucleohistone, and suggests that, under the given experimental conditions, these agents do not react with essential amino groupings (3). The nitrogen mustards, which in sub-lethal concentrations cause mitotic abnormalities similar to those produced by mustard gas (17, 5, 12, 13), were observed to react with the nucleohistone and render the latter partially resistant to enzymic degradation. These compounds were added to the substrate in concentrated ethanolic solution; a method similar to that used by Dixon and Needham (lo), in their studies on the action of mustard gas on various enzyme systems (21). Under these conditions some precipitation of the nucleoprotein occurred. Precipitation also results from the application of mustard gas to nucleoproteins (4) and nucleic acids (11). In the latter case, it is known from electrometric titration that reaction occurs between the mustard gas and primary and secondary phosphoryl groups as well as amino groups of the nucleic acid (11). It is probable that the effect of the nitrogen mustards is due to the alkylating effect of the halogenoalkyl groups on similar groups of the nucleic acid or nucleoprotein (18, 27, 6). In view of the high order of reactivity of the nitrogen mustards with various functional constituents of the cell, little importance can be attached to the observations that the products from the interaction of deoxyribonucleohistone with these substances are resistant to enzymic attack. Indeed, with regard to the inhibition of mitosis produced by these compounds, their precipitating action on the nucleoprotein may be of greater significance than the fact that they render the latter partially resistant to degradation with deoxyribonuclease. Apart from fluoride and selenite ions, which inhibit both deoxyribonuclease and mitosis in tissue cultures (23), the lack of effect of the various mitotic inhibitors on the degradation of deoxyribonucleohistone with deoxyribonuclease suggests that if an enzyme of the deoxyribonuclease-type exists in cell nuclei and plays any part in the sequence of changes which occur during mitosis, the properties of such an enzyme must differ considerably from those of the exocellular, pancreatic deoxyribonuclease.
L. M. Gilbert, W. G. Overend, and M. Webb SUMMARY
The action of inhibitors on the enzymic degradation of deoxyribonucleic acid and deoxyribonucleohistone by pancreatic deoxyribonuclease has been studied. The results show that the inhibitors may be divided broadly into two classes, namely those which remove the activating magnesium ion, and those which directly interact with the enzyme protein. Active inhibitors prevent both stages (i.e. depolymerisation and hydrolysis) of the degradation of the deoxyribonucleic acid. The products which result from the reaction of nitrogen mustards with deoxyribonucleohistone are partially resistant to the action of deoxyribonuclease. Pre-treatment of deoxyribonucleohistone with other mitotic inhibitors has no effect upon its subsequent degradation with deoxyribonuclease. Thanks are due to Professor M. Stacey, F.R.S. and to Dr. Honor B. Fell for their interest in this work, and to Dr. A. F. W. Hughes for suggesting the study of the action of mitotic inhibitors on the enzymic degradation of deoxyribonucleohistone. REFERENCES ALLEN, R. J. L., Biochem. J., 34, 858 (1940). BARGONI, N., Boll. sot. ital. sper., 20, 534, 1945. Chem. Abs. 40, 6513 (1946). BARRON, E. G. S., and SINGER, T. P., J. biol. Chem. 157, 221 (1945). BERENBLUM, I., and SCHOENTAL, R., Nature, 159, 727 (1947). BODENSTEIN, D., J. Exptt. Zool., 104, 311 (1947). BUTLER, J. A. V., Nature, 166, 18 (1950). COHEN, S. S., J. Biol. Chem., 158, 255 (1945). DAVIDSON. J. N.. Ann. Rev. Biochem.. 18, 155 (1949). DISCHE, i., Mikrochemie, 8, 4 (1930): ’ DIXON, M., and NEEDHAM, D. M., Nature, 158, 432 (1946). 11. ELMORE, D. E., GULLAND, J. M., JORDAN, D. O., and TAYLOR, H. F. W., Biochem.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
308
(1948).
12. FELL, H. B., and ALLSOPP, C. B., Cancer Res., 8, 145 13. -Cancer Res., 8, 177 (1948). 14. FISCHER, F. G., BOTTGER, I., and LEHMANN-ECHTERNACHT,
(1941).
15. FISCHER,
F.
G.,
LEHMANN-ECHTERNACHT,
(1941).
H.,
and
J.
42,
(1948). H., Z. physiol. Chem., 271,246
BOTTGER,
I., J. prakt.
Chem., 158, 79
L. M., OVEREND, W. G., and WEBB, iV., ExptI. Cell. Res, 2, 138 (1951). R., and BODENSTEIN, D., J. Exptt. Zool., 103, 1 (1946). GOLDACRE, R. J., LOVELESS, A., and Ross, W. C. .J., Nature, 163, 667 (1949). GREENSTEIN, J. P., CARTER, C. E., and CHALKLEY, H. W., Cold Spring Harbor Symp. Quant.
16. GILBERT, 17. GILLETTE, 18. 19.
20. HEATH,
21.
BioZ., 12, 64 (1947). J. C., Nature, 164, 1055
HERRIOT, 22. HOLMES, 23. HUGHES,
(1949).
R. M., ANSON, M. L., and NORTHROP, J. H., J. Gen. Physiol., 30, 185 B. E., Ann. Rep. Brit. Empire Cancer Campaign, 26, 191 (1948). A. F. W., J. Roy. Microscop. Sot., 69, 215 (1949); Quant. J. Micro. Sot.
press).
(1946). (in the
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of Pancreas deoxyribonuclease
24. JACOBSON, W., and WEBB, M., 25. JUNGNER, G., JUNGNER, I., and 26. KOCH, J. R., ORTHMANN, A. C., 34, 489 (1939). 27. KON, G. A. R., and ROBERTS, 28. MCCARTY, M., J. Gen. Physiot., 29.
30. 31.
32. 33. 34. 35.
36. 37. 38.
J. Physiol., 112, 29 (1951). ALLGEN, L. G., Nature, 164, 1009 (1949). and DEGENFELDER, M. A., J. Amer. Leather.
Chem. Assoc.,
J. Chem. Sot., 978 (1950). 29, 123 (1946). MIRSKY, A. E., and POLLISTER, A. W., Proc. Naft. Acad. Sci., 28, 344 (1942). OVEREND, W. G., and WEBB, M., Research, 2, 99 (1949). __ J. Chem. Sot., 2746 (1950). Research, 3, 238 (1950). STERN, K. G., Exptl. Cell Res. Suppl. I, 97 (1949). VERCAUTEREN, R., Nature, 165, 603 (1950). WEBB, M., J. Gen. Microbial., 2, 275 (1948). WEISSMAN, N., and FISCHER, J., J. biol. Chem., 178, 1007 (1949). ZAMENHOF, S., and CHARGAFF, E., J. biol. Chem., 180, 727 (1949). ZITTLE, C. A., J. biol. Chem., 163, 111 (1946). J. J.,