DEOXYRIBONUCLEASES OF MOUSE TISSUES*

Transcription

1 DEOXYRIBONUCLEASES OF MOUSE TISSUES* BY JOSEPH SHACK (From the National Cancer Institute,t National Institutes of Health, Public Health Service, Bethesda, Maryland) (Received for publication, November 19, 1956) It was observed (1, 2), in the course of the isolation of deoxyribonucleate (DNA) from various tissues of the strain A mouse, that extracts of a number of the tissues contained deoxyribonuclease I (DNase I) as well as the more widely distributed DNase II. The presence of both enzymes in veal kidney has also been reported (3). In view of the importance of controlling DNase action during the preparation of DNA, a further examination was made of the DNase activity of the mouse tissue extracts, particularly with respect to the effects of ph and of the type and concentration of added salts. This paper deals with these studies and also with some observations on the intracellular distribution of the liver DNases and the effect, of DNase II action on the ultraviolet absorption of DNA. Methods and Materials The DNA used in these studies consisted of two samples isolated from the transplantable lymphoma 1 of the strain A mouse; the preparation and some of the physicochemical properties of these samples have been described elsewhere (11). Except as otherwise stated, the tissues were homogenized in several parts of ice-cold 0.14 M NaCl in a Waring blendor and the homogenates were clarified by high speed centrifugation. When blending was thorough, most of the enzymatic activity was found in the clear supernatant fluids. In some cases these fluids were also dialyzed against distilled water. When prepared in this manner, no significant increase of either the DNase I or DNase II activity of the supernatant fluids was ever observed on dialysis or on storage at 2O, at room temperature, or in the frozen state (- 35 ). * A preliminary report of some of this work was given at the Forty-fourth annual meeting of the American Association for Cancer Research, Chicago, April 4-11, 1953 (1). t United States Department of Health, Education, and Welfa.re. 1 The terminology proposed by Cunningham and Laskowski (3) is used. DNase I refers to those DNases which, like bovine pancreatic DNase (4-6), have optima near neutrality and require the presence of bivalent cations; DNase II refers to the class first described by Maver and Greco (7) which have optima near ph 5 (7-10) and are activated by salts of either univalent or bivalent cations, the latter being effective at lower concentrations (8, 10). 573

2 574 DEOXYRIBONUCLEASES DNase activity was estimated (1) from the fall of viscosity essentially as described by McCarty (6) and (2) from spectrophotometric determination (at 260 mp) of the production of material soluble in cold 0.25 N perchloric acid by a method similar to that described by Kurnick (12). R, designates the initial rate of loss of viscosity in viscosity units per minute. In agreement with other reports (13, lo), the production of acid-soluble substances exhibited an initial lag which was longer, the smaller the enzymatic activity. Subsequently, the slopes of the curves relating increase of optical density to time were constant up to about 40 to 60 per cent reaction and were proportional to the concentration of tissue extract in the reaction mixture. The DNase activity was therefore in each case taken as the slope of this linear portion of the curve rather than as the ratio of the increase of absorption to total time of reaction. Except as otherwise stated, the reaction mixtures contained 0.85 mg. of DNA per ml. and the desired salts, buffers, and tissue extracts; the reactions were carried out at The initial relative viscosity of such mixtures was 2.8 to 3.0 in the Ostwald type viscosimeters that were used. Results InJluence of Salts on DNase Activity-The influence of salts on DNase activity was examined for each tissue at both ph 5 and 7. Typical data for dialyzed spleen extract at ph 5 are given in Figs. 1 and 2.* Such spleen extracts had relatively little activity at ph 7 with either NaCl or MgC12, and it appears that they contain considerable DNase II, but little if any DNase I; the data at ph 7 are not included in Figs. 1 and 2, but will be discussed in connection with activity-pa curves. Since it appears from Fig. 1 that full activation of the DNase II of such an extract is not attained with salts of multivalent anions, only salts of univalent anions were employed in further studies. The results with serum are given in Fig. 3. The serum had negligible activity at ph 5 with either MgClz or NaCl, and it can be concluded that mouse serum contains no DNase II but, in common with sera of various other species (14, 12), contains DNase I. Fig. 4 presents the data obtained with dialyzed liver extracts at both ph 5 and 7; the results clearly show that liver extracts contain both DNase I and DNase II. With extracts of kidney, thymus, and lymphoma 1, the salt effects at ph 5 were essentially the same as those shown for spleen and liver in Figs. I, 2, and 4. At, ph 7, cnc*h of these three tissues exhibit,ctl significant 2 For the salts of nnivalent cations the values of log norm:tlitjy given in I igs. 1 t,o 4 include the contribution of the buffer (0.002 Na ); for salts of multivalent, cnt,ions, log normality gives the concentration of the added bivalent cation.

3 J. SHACK 575 activity, the behavior of which, with respect to changing salt concentration, was similar to that shown for serum and liver in Figs. 3 and 4. The DNase activity of the tissue extracts at certain significant conditions is given in Table I. -3 LOG NORiiLITY OF CATlO-& FIG. 1. Effect of salts on activity of spleen DNase at ph 5. A, CaC12; A, MgClz; 0, MgS04; X, NaCl; 0, sodium acetate; f, KCl; 9, sodium citrate; 0, Na6304. LOG NORMALITY OF CATIONS FIG. 2. Comparison of viscosity and acid solubility data with spleen extract at ph 5. Viscosity data, + and X; acid solubility data, l and 0. The inhibition (at ph 5) of the DNase II of spleen, liver, and kidney by high concentrations of salt was further investigated by addition of various amounts of a second salt to reaction mixtures already containing the optimal concentration of a first salt. In every case, addition of the second salt resulted in an inhibition which increased with the concentration. No combination of salts ever resulted in activities greater than those found with a single salt; this provides some evidence that each tissue contains only a single DNase II.

4 576 DEOXYRIBONUCLEASES ph-activity Relations-pH-activity curves were determined for each tissue in the presence of (1) 0.15 M NaCl (or 0.12 M NaCI-0.01 M sodium cit- FIG. 3. Effect of salts on activity of mouse serum DNase at, ph 7 LOG~NORMAilTY OF CATlOiS FIG. 4. Effect of salts on activity of DNases of liver at ph 5.0 and ph 7.0. A dialyzed fraction SZ (see the text) was used. C ) rate) which activates only the DNase II and (2) or 0.01 M MgCL which activates both DNase I and DNase II. Typical curves are given in Figs. 5 and 6; in each case the reaction mixtures contained 0.01 M acetate and 0.01 M Veronal. Data for spleen in 0.01 M MgClz practically superimpose on that shown for 0.15 M NaCl in Fig. 5. In particular, the activity at ph 7 is essentially the same with both salts (see Table I), about

5 J. SHACK to 3 per cent of the maximum at ph 5. By contrast, the curve for lymphoma extract3 in 0.01 M MgC12, but not in 0.15 M NaCI, shows a definite peak near ph 7 which can be attributed to the presence of DNase I. The data given in Fig. 64 clearly show the presence of both DNase I and DNase II in liver extracts; the dotted curves should give an approximation of the ph-activity curve of the DNase I of the liver extracts. Curves for liver extracts based on acid solubility measurements are similar TiSSlR (1) Spleen... Thymus... Lymphoma Liver... Kidney.... Laked red blood cells. Serum... - I TABLE DNase Activity of Strain A Mouse Tissues -_ Viscosity I units per gm. tissue AtpH7 Inhibitionp~,~itrate ar In 0.01 M M&lx 1%:A:*w (3) (4) * Units as defined by McCarty (6), except that T = The values in the case of the tissues refer to centrifuged homogenates prepared as described in the text. The values for red blood cells refer to cells contained in 1 ml. of whole blood, and those for serum refer to 1 ml. of serum. t Activity values in 0.15 M NaCl are in every case within 10 per cent of these. $ Except for that of spleen, the activity values in this column are rough approximations due to the difficulty of measuring such low values accurately. g Citrate 0.01 M; in each case 0.01 of MgCls present. + indicates strong inhibition; -, no inhibition. to those given in Fig. 6. In accordance with expectations from the activity values in Table I, pa-activity curves of extracts of thymus and kidney also show peaks near ph 7 in 0.01 M MgC12 but not in 0.15 M NaCI. The effect of storage of liver extracts at 3 also indicates the presence of two distinct enzymes; in one instance, the DNase I activity was 70, 38, and 14 per cent, respectively, of the original value at 1, 2, and 15 days, and the DNase II activity was unchanged. 3 The results with lymphoma below ph 5 were not considered reliable due to precipitation of DNA-protein complexes. 4 ph-activity curves in 0.15 M NaCl (no added Mg) were practically the same as that given for 0.12 M NaCI-0.01 M citrate.

6 578 DEOXYRIBONUCLEASES The following experiment demonstrates that both DNase I and II activities of the liver are largely associated with the particulate matter of the cell. Fresh liver was first homogenized with a Potter-Elvehjem glass homogenizer into 5 volumes of cold 0.14 M NaCl per gm. of tissue; this homogenate was divided by centrifugation into a clear supernatant fluid (S1) and a sediment. The sediment was then thoroughly homogenized into the original volume of 0.14 M NaCl in a Waring blendor and again separated into a clear supernatant solution (Sz) and a sediment which was then homogenized into the original volume of 0.14 M saline (SJ. The fractions S1, SZ, and Sa contained 35, 18, and 47 per cent, respectively, of % 02- o\o PH PH FIG. 5 FIG. 6 FIG. 5. ph-activity curves of spleen, lymphoma, and serum DNase. 9, spleen in 0.15 M N&l; A, lymphoma in 0.15 M NaCl; 0, lymphoma in 0.01 M MgC12; all are based on viscosity data. 0, serum in M MgC12, viscosity data; X, serum in M MgC12, acid solubility data. FIG. 6. ph-activity curves of liver. 0, in M MgC12; 0, in 0.12 M NaCI-0.01 M sodium citrate. The dotted curve represents the difference between the above curves. the nitrogen of the tissue. The percentages of total recoverable DNase I were 8, 78, and 14 and of DNase II 6, 62, and 32 for the S1, SZ, and S3 fractions, respectively. It is clear that both activities are initially associated with particulate material. The findings with respect to DNase II are in agreement with other reports (15, 16). Changes of Ultraviolet AbSorption-Kunitz (13) showed that the action of bovine pancreatic DNase I on DNA led to an increase of ultraviolet absorption. The results given in Fig. 7 show that a similar increase of ultraviolet absorption results from the action of spleen DNase II, that the rate of increase is proportional to the concentration of extract, and that, the total increase is essentially independent, of the amoulltj of cxtraet. The slow decrease of absorption t]hat, follows the initial rise may result from further degradation, possibly by other enzymes. Essentially the same results were obtained when MgClz was used to activate the enzyme.

7 .I. SHACK 579 Fig. 8 shows that the increase of ultraviolet absorption occurs after the loss of viscosity, and closely parallels the formation of acid-soluble sub- AD TIME IN MINUTES FIG. 7. Effect of spleen DNase II on ultraviolet absorption (at 260 rnp) of DNA DNA P = 6.8 X lo+ M in 0.10 M sodium acetate buffer, ph , 0, and 0, respectively, give the data for mixtures containing, per 3 ml., 0.10,0.20, and 0.40 of a dialyzed spleen extract. The ordinate gives an increase of optical density in 1 cm. cuvettes. Initial optical density, The slopes of the straight lines 1, 2, and 3, respectively, are , , and optical density unit per minute. TIME IN MINUTES FIG. 8. Comparison of the rates of change of viscosity, acid solubility, and optical density during action of spleen DNase. 0, viscosity; 0, acid solubility; A, optical density. Reaction mixture 0.20 M in sodium acetate buffer; ph Optical density measurements were made on a 25-fold dilution of reaction mixture into 0.20 M acetate buffer, ph = 38. stances. Studies of the relative rate of change of these three properties during the action of crystalline bovine pancreatic DNase I have been described by Kunitz (13). DISCUSSION It is evident that the activity of spleen at ph 7 is qualitatively different from that of the other tissues; it is nearly the same in 0.15 M NaCl as in

8 580 DEOXYRIBONUCLEASES 0.01 M MgCL, and is not inhibited by citrate. The 21 units of activity for spleen at ph 7 may be due to its DNase II which, on this assumption, possesses at ph 7 about 3 per cent of the activity exhibited at ph 5. The best estimate of DNase I content of the several tissues appears to be the values of Column 3, Table I (0.01 M MgCL), minus those in Column 4 (0.15 1\4 NaCl); except for spleen, this difference is only slightly less than the total activity in 0.01 M MgCL The DNase II content is, in every case, the value in Column 2. DNase II has been found in all mammalian tissues thus far examined, including pancreas (9), in which, however, the DNase I activity is far greater. Previous failures to detect DNase I in tissues other than pancreas may be due, in some instances, to use of high concentrations of buffer salts, or, in others, to the presence of inhibitors of DNase I in the tissue extracts. It is likely that part of the DNase I of the tissues is derived from serum. However, even if it is assumed that 25 per cent of the volume of a tissue consists of serum, or extracellular fluids having the same DNase I content as serum, it is apparent that they would contribute not, more than about 1.5 units to the values given in Column 3, Table I; the balance presumably represents intracellular DNase I. The association of DNase I and DNase II with the particulate matter of the liver cell supports the conclusion that both are intracellular enzymes. With respect to the preparation of DNA, it is evident that an inhibitor for DNase I should be used routinely; in this connection, it is immaterial whether the DNase I is derived from cells or extracellular fluids. It appears that the present findings necessitate revision of the conclusion (8, 17) that the principal function of citrate in such preparative procedures is to depress activity of DNase II through maintenance of a neutral ph. Our results confirm and extend Webb s conclusions that DNase II is inactive in the absence of electrolytes, that salts of bivalent cations are more effective with respect to activation than those of univalent cations, and that higher concentrations of all salts are inhibitory. The effects of salts on serum and tissue DNase I are quite similar to those found with pancreatic DNase I by others (6, 13). It has been suggested (13, 18) that the activation of bovine pancreatic DNase I is due to the conversion of the DNA to a complex with Mg++ (or other bivalent ion) rather than to specific effects on the enzyme itself. Similar reasoning suggests that activation of DNase II also results from binding of cations by DNA. It is apparent from Figs. 1, 2, and 4 that the extent of activation is not a simple function of either salt concentration or ionic strength, since the salts of Mg++ and Ca++ are far more effective than those of Naf and K at comparable ionic strengths. For example, the ionic strength required for 50 per cent activation of spleen DNase II

9 J. SHACK 581 (Fig. 1) is M with MgClz and 0.04 with NaCl. Such a fmding is in harmony with other evidence (19) that DNA binds bivalent cations far more tightly than univalent cations. SUMMARY Studies of the dependence of DNase activity on ph and on the type and concentration of added salts show that a number of tissues of the strain A mouse contain DNase I as well as DNase II. Both DNase I and DNase II of liver are largely associated with particulate matter of the cell. The action of DNase II results in an increase of the ultraviolet absorption of DNA which occurs after the loss of viscosity and closely parallels the conversion of DNA to acid-soluble material. The relative effectiveness of various salts in the activation of DNase II indicates that such activation results from binding of cations by DNA rather than from a non-specific increase of the ionic strength. BIBLIOGRAPHY 1. Shack, J., Proc. Am. Assn. Cancer Res., 1,49 (1953). 2. Shack, J., and Thompsett, J. M., J. Nat. Cancer Inst., 13, 1425 (1953). 3. Cunningham, L., and Laskowski, M., Biochim. et biophys. acta, 11, 590 (1953). 4. Fischer, F. G., Biittger, I., and Lehmann-Echternacht, H., 2. physiol. Chem., 271, 246 (1941). 5. Laskowski, M., and Seidel, M. K., Arch. Biochem., 7, 465 (1945). 6. McCarty, M., J. Gen. Physiol., 29, 123 (1946). 7. Maver, M. E., and Greco, A. E., J. Biol. Chem., 181, 861 (1949). 8. Webb, M., Nature, 169, 417 (1952). 9. Allfrey, V., and Mirsky, A. E., J. Gen. Physiol., 36, 227 (1952). 10. Webb, M., Ezp. Cell Res., 6, 27 (1953). 11. Shack, J., Jenkins, R. J., and Thompsett, J. M., J. Nat. Cancer Inst., 13, 1435 (1953). 12. Kurnick, N. B., Arch. Biochem. and Biophys., 43, 97 (1953). 13. Kunita, M., J. Gen. Physiol., 33, 363 (1950). 14. Wrbblewski, F., and Bodansky, O., Proc. Sot. Ezp. Biol. and Med., 74,443 (1950). 15. Schneider, W. C., and Hogeboom, G. H., J. BioZ. Chem., 198, 155 (1952). 16. Webb, M., Exp. Cell Res., 6, 16 (1953). 17. Brown, K. D., Jacobs, G., and Laskowski, M., J. BioZ. Chem., 194,445 (1952). 18. Weissman, N., and Fisher, J., J. BioZ. Chem., 178, 1007 (1949). 19. Shack, J., Jenkins, R. J., and Thompsett, J. M., J. BioZ. Chem., 203, 373 (1953).

10 DEOXYRIBONUCLEASES OF MOUSE TISSUES Joseph Shack J. Biol. Chem. 1957, 226: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1