Protein Inhibitor of Acid Deoxyribonucleases IMPROVED PURIFICATION PROCEDURE AND PROPERTIES*

A method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate uersus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme The function of acid DNase is unknown, although it may be involved in such important steps as DNA replication, recombi-nation,

The function of acid DNase is unknown, although it may be involved in such important steps as DNA replication, recombination, integration, excision after irradiation, or viral induction. Several studies have shown the presence of acid DNase in nuclear fraction of rat liver (11, mouse liver (21, and HeLa cells (3). However most of acid DNase activity in mammalian cells was found to be associated with lysosomes (4). Therefore it seems likely that acid DNases are involved in the degradation of DNA. In any case, the activity of acid DNases may be regulated by different effecters within the cells.
We have reported (5) the presence, in mouse liver, of a protein inhibitor of this enzyme. This natural inhibitor is also active on beef spleen and Helix aspersa acid DNases (6) but inactive on pancreatic DNase and Escherichia coli endonuclease (5). The present paper reports on the purification of both acid DNase and its inhibitor to homogeneity. For this purpose, we have developed a technique of affinity chromatography (7) which takes advantage of the interaction between the enzyme and its inhibitor.
This method has permitted the isolation of nine proteins interacting with acid DNase. One of these proteins is the inhibitor, another carries

Materials
Beef liver was removed within 1 hour after slaughter, immediately frozen in Dry Ice, and then stored at -20". JH-labeled DNA was prepared from T, phage according to Richardson et al. (81, as modified by Lava1 and Paoletti (9). Its specific activity was 14.2 x IO3 cpm per nmol of nucleotide.

Prepamtion of Immobilized Acid DNase
Coupling techniques were those described by Kato and Anfinsen (11) and Cuatrecasas (12). Sepharose 4B (40 ml) was suspended in the same volume of distilled water after extensive washing. After raising the pH to between 11 and 11.5 with 2 N NaOH, 40 ml of freshly prepared cyanogen bromide (9 g) solution were added and the pH was maintained between 11.0 and 11.5 with 2 N NaOH for 4 to 5 min at room temperature.
The activated Sepharose was then washed with 2 liters of distilled water and 1 liter of 0.1 M NaHCO, solution and finally resuspended in 40 ml of distilled Protein Inhibitors of Acid Deonyribonucleases 117 water. An equal volume containing 80 nmol (11.6 g) of octamethylene diamine was added and the pH was adjusted to 10 with 6 N HCl. After a l&hour reaction at 4', the beads were washed with the following solvent: distilled water. 0.1 M NaHCO, solution, distilled water again, and resuspended in 40 ml of distilled water. An equal volume containing 40 nmol (4 g) of succinic anhydride was added and the pH was raised to and maintained at 6 with 10 N NaOH. The mixture was incubated 5 hours at 4" and the gel was washed as previously described, The succinylaminooctyl-Sepharose was mixed with at least 4 x IO5 units (21.5 mg) of purified acid DNase obtained by a modification of the method described below (two cycles of hydroxylapatite chromatography) and 1 g of N-ethyl-N'-(3.dimethylaminopropyl).carbodiimide, giving a final volume of 160 ml. The pH was adjusted to 4.8 with acetic acid and the mixture was incubated 20 hours at 20". The unit of inhibitory activity was defined as the amount of protein required for 50% inhibition of the acid DNase. In the last stages of inhibitor purification, the latter was thoroughly devoid of acid DNase and the assay of inhibitory activity was easier. To obtain the inhibition kinetics of the enzyme, the procedure described above (Step b) was used with 0.25 unit of acid DNase (Fraction VI) and inhibitor.
An assay was carried out without inhibitor and from the inhibition curve obtained the concentration of inhibitor which causes a 50% decrease of the acid DNase activity was determined.

Other Enzymatic Assays
RNase Assays-Enzyme (0.05 ml) and inhibitor (0.025 ml) were incubated with 1 mg of total yeast RNA in a mixture (1 ml) containing 100 rmol of sodium acetate, pH 5.0, (acid RNase) or 50 pmol of potassium phosphate, 100 rmol of sodium chloride and 10 pmol of magnesium chloride, pH 7.3 (alkaline RNase). After 1 hour at 37", 1 ml of 1 N HCl in ethanol, and 0.1 ml of bovine albumin (4 mg/ml) were added. After 5 min at 0" this mixture was centrifuged at 7,700 x g for 1 hour and the absorbance of the supernatant at 260 nm was determined. One unit is the amount of protein catalyzing the conversion of 1 ymol of RNA into acid-soluble form in 1 min. Phosphatase Assays-Purified preparations (0.02 ml) were assayed by measuring the liberation of p-nitrophenyl from 3 ml of 1 mM sodium p-nitrophenylphosphate according to Chersi  was carried out at 8 ma per tube for 6 hours. The gels were then immersed for 16 hours in Coomassie brilliant blue R (0.05%) dissolved in methanol/acetic acid/water (5/l/5, v/v/v), soaked 30 min in 7.5% acetic acid, 5% methanol, and destained electrophoretitally in the same solvent for 3 hours.

Sucrose Gradient Centrifugation
Samples (0.2 ml) containing cytochrome c as an internal marker were layered on a linear gradient (4 ml) of sucrose (5 to 20% w/v) in 100 mM sodium acetate 10 mM ETDA, pH 5.0, and then were centrifuged at 63,000 rpm for 16 hours in a Spinco SW65 rotor. Two-drop fractions were collected and diluted to 0.5 ml in 100 mM sodium acetate buffer, pH 5.0. Cytochrome c was determined by absorption at 405 nm and acid DNase activity as described above.

Other Measurement
Protein was determined either according to Lowry et al. (19) using bovine serum albumin (Fraction V) as a standard, or spectrophotometrically at 280 nm.

Purification of Acid DNase
A summary of the procedure is presented in Table  I. All procedures were carried out at 4".
Step I: Preparation of Crude Extract-Frozen beef liver (1 kg) was thawed, minced, freed from connective tissue, and homogenized in 5 liters of 100 mM NaCl. Unbroken cells and organelles were disrupted by a lo-min sonication in a Branson

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Protein Inhibitors of Acid Deonyribonucleases S I25 sonic oscillator (the temperature was kept below 6"). The extract was centrifuged 15 min at 11,000 x g and the supernatant was saved (crude extract).
Step II: Ammonium Sulfate Fractionation-Solid ammonium sulfate was slowly added with stirring to the crude extract fraction to 30% saturation. After 30 min the suspension was centrifuged at 11,000 x g for 10 min and the supernatant was brought to 100%' saturation with ammonium sulfate. After 30 min, the solution was centrifuged, the pellet dissolved in 1 Iiter of distilled water, dialyzed overnight against 10 liters of50 mM NaCl solution, then concentrated in a bed of polyethylene glycol (Carbowax 20,000) for 24 hours. The concentrated material was then dialyzed against four changes of 1 liter of 50 mu potassium phosphate, pH 6.0. The precipitate formed during dialysis was eliminated by centrifugation and the supernatant (about 300 ml) was saved.
Elulion volume (ml) Step 111: CM-Sephadex Chromatography-The ammonium sulfate fraction was applied to a column of CM-Sephadex C-50 (20 cm' x 10 cm) equilibrated with 50 mM potassium phosphate, pH 6.0. The column was washed with 1.5 liters of the same buffer, then eluted at a flow rate of 2 ml/min with a 2liter linear gradient: 50 mM potassium phosphate, pH 6.0, to 350 mM potassium phosphate, pH 7.0. The fractions containing the acid DNase, eluted after 250 mM phosphate, were pooled. They are contaminated with the protein inhibitor and other proteins interacting with the acid DNase.
Step IV: Affinity Chromatography-In order to separate DNase from its inhibitor and other interacting proteins, Fraction III was applied to a column (1.75 cm* x 10 cm) of immobilized acid DNase (DSOS) which had been equilibrated with 300 mM potassium phosphate, pH 6.8. The column was then washed with 100 ml of the same buffer. Unadsorbed proteins were collected and dialyzed for 1 hour against three changes of 1 liter of 50 mM potassium phosphate, pH 6.8.
Step V: Hydroxylapatite Chromatography-Fraction IV was applied to a hydroxylapatite column (5 cm* x 10 cm) previously equilibrated with 50 mM potassium phosphate, pH 6.8, The column was eluted at a rate of 50 ml/hour, successively with 200 ml of 100, 200, and 306 mM potassium phosphate, pH 6.8. Fractions of 3 ml were collected. Acid DNase was eluted with 300 mM potassium phosphate following a peak of inactive proteins. The fractions with the highest specific activities were pooled, dialyzed for 1 hour against three l-liter changes of 100 mM sodium phosphate, pH 7.1 and concentrated with Carbowax to reduce its volume to about 2 ml. For other experimental details, see under "Results." inhibitor because of the interference with nonspecific inhibitors of the acid DNase (histones and other basic proteins). Therefore the presence of specific inhibitor was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions containing inhibitor, usually eluted between 200 mM and 250 mM phosphate, were pooled (Fraction III).
Step VI: Gel Filtration on Sephadex G-lOO-Fraction V was applied to a column of Sephadex G-100 (5 cm* x 100 cm) previously equilibrated with 100 mM sodium phosphate, pH 7.1. The column was then eluted with the same buffer at a flow rate of IO ml per hour using a pressure head of 10 cm. Fractions (3 ml) were collected. Peaks of phosphodiesterase and DNase activities were eluted at I90 and 210 mf, respectively, resulting in a partial separation of the two activities (Pig. 1). The peak fractions which contain acid DNase without phosphodiesterase activity were pooled, concentrated with Carbowax and stored in 50% glycerol at -20°.
Step IV: Hydroxy~apatite Chromatography-Fraction III, previously dialyzed four times against 2 liters of 50 mM potassium phosphate, pH 6.8, was applied to a hydroxylapatite column (28 cm2 x 7.5 cm) equilibrated with the same buffer. The column was washed with 100 ml of the same buffer and the proteins eluted with a linear gradient of potassium phosphate 50 to 400 mM, pH 6.8 (total volume 1.5 liters). The flow rate was 70 ml per hour and fractions of 20 ml were collected. The contaminating acid RNase was eluted between 75 and 200 mM potassium phosphate while the acid DNase was eluted between 250 and 350 mM potassium phosphate. The amount of specific inhibitor contained in the fractions may be determined by microdensitometry of sodium dodecyl sulfate-polyacrylamide gels. The fractions located between 170 and 280 mM potassium phosphate were pooled, concentrated with Carbowax to about 70 ml and dialyzed for 1 hour in three l-liter changes of 300 mM potassium phosphate, pH 6.8.

hLrification of the Acid DNase Inhibitor
A summary of the procedure is presented in Table II. Steps I, II, and III of the purification of the acid DNase and its inhibitor were identical. In the CM-Sephadex chromatography (Step III), it was not possible to measure quantitatively the specific Step Ir: Affinity Chromatography-The Fraction IV was applied to an insoluble acid DNase (DSOS) column (5 cm* x 10 cm) equilibrated with 300 mM potassium phosphate, pH 6.8. The column was washed with IO0 ml of the same buffer and the adsorbed proteins were eluted successively (Fig. 2)  acetate, pH 7.4. The protein pattern determined spectrophotometrically at 280 nm showed two peaks; the first one contained the pure inhibitor while the second one contained a small amount of inhibitor along with eight other proteins (interacting directly or indirectly with the insolubilized acid DNase). Both peaks were dialyzed for 1 hour in three l-liter changes of 100 mM sodium phosphate, pH 7.1. The inhibitor was concentrated with Carbowax and stored in the same buffer in 50% glycerol at -20".

Polyacrylamide
Gel Electrophoresis The purity of acid DNase and its inhibitor was followed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown on Fig. 3, A and B, both acid DNase and its protein inhibitor appeared as single bands. The gel pattern of proteins eluted from the insoluble acid DNase with 750 mM guanidine-HC1/500 mM sodium acetate solution, is presented on Fig. 3 C. It shows nine molecular species which were able to interact directly or indirectly with acid DNase in the chromatographic system, one of these proteins being the specific inhibitor, as checked by its influence on acid DNase kinetics (see below). Another protein (molecular weight, 59,000) possesses the phosphodiesterase activity (see below).

Properties of Acid DNase
Tests for Contaminating Enzymes-One milligram of pure acid DNase (which represents about 50,000-fold the amount commonly used in an enzymatic assay) contained practically undetectable activities of the following enzymes: acid phosphatase (less than 0.0025 unit), alkaline phosphatase (let's than 0.001 unit), acid RNase (less than 0.002 unit), alkaline RNase (less than 0.002 unit), and neither exonuclease activity nor phosphodiesterase activity.
Stability-There was no detectable loss of activity for over 6 months when the acid DNase was stored in 50% glycerol at -20". To stabilize the enzyme during incubation at 37" at pH 5, the addition of 100 prg of bovine serum albumin per ml of assay is required. The heat sensitivity of the acid DNase was determined by preincubation at various temperatures for 15 min in 100 mM sodium phosphate, pH 7.1. The enzyme was stable up to 50" but lost 95%' of its activity at 60". After 5 min of incubation at this temperature the percentage of remaining activity was only 40%. Molecular weight-The molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18). A value of 26,500 was obtained. This result is in good agreement with the estimation of molecular weight by gel filtration on Sephadex G-75 (27,000). and by sucrose density gradient zone centrifugation (2.85 S) according to Martin and Ames (20).
Effect of Different Ions, RNA, and Other Reagents-As observed with all acid DNases, divalent cations, and sulfate exhibit inhibitory effects. Hydrolysis of DNA was completely inhibited by 20 mM of either Ca*+, Mg*+, or Mn*+. Total yeast RNA was a potent competitive inhibitor of beef liver acid DNase (K, = 0.33 x 10m8M), as already described for other acid DNases and RNAs (24, 25). Sodium p-hydroxymercuribenzoate (5 mM) and sodium iodoacetate (1 mM) are both strongly inhibitory (97% inhibition).
On the other hand, 2-mercaptoethanol (2%) and EDTA (5 mM) did not affect enzyme activity. Catalytic Properties-The optimal conditions for acid DNase activity were determined. The enzyme exhibited maximum activity at pH 4.9 to 5.1 in reaction mixture containing 100 mM sodium acetate buffer. At lower and higher ionic strength the rate of DNA hydrolysis was reduced. The effect of the secondary structure of DNA on enzymatic rate was studied. The initial velocity of DNA degradation was four times higher for native than for heat-denatured DNA. Experiments were carried out in order to control whether beef liver enzyme, as all acid DNases, yields oligonucleotides bearing :I'-P end groups (21). After extensive hydrolysis of fish sperm DNA (200 mg) during 23 hours at 37' with acid DNase the reaction products were loaded to a Dowex 1 column then eluted with a stepwise gradient of ammonium acetate (pH 4.5). Many small oligonucleotides were produced but not mononucleotides.
All these oligonucleotides were good substrate for spleen phosphodiesterase but not for snake venom phosphodiesterase. These experiments show that beef liver acid DNase produces endmucleolytic breakages giving oligonucleotides with 3'-phosphomonoester end groups.
The kinetics of the purified acid DNase ohey Michaelis-Henry law. The K, is 2 x lo-' M in 100 mM acetate, pH 5.0, 10 mM EDTA. O.Sj

Properties of the Inhibitor
The protein nature of the inhibitor has been documented previously using an impure preparation (5).
Purity and Molecular Weight-The inhibitor (Fraction V) shows a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 3B) and its molecular weight determined in the same way was 21,500. The results obtained after centrifugation in a sucrose density gradient in nondenaturing conditions have confirmed this value and showed consequently that the molecule does not have subunits. The Alcian blue staining used to reveal glycoproteins in acrylamide disc electrophoresis (22) was negative. Contaminating enzyme activities in 1 mg of purified inhibitor (which represents 400 times the amount normally used for inhibitor determinations) were either undetectable (phosphodiesterase, acid RNase, acid DNase), or extremely weak: acid phosphatase (less than 0.001 unit), alkaline phosphatase (less than 0.002 unit), exonuclease (less than 0.0001 unit), and alkaline RNase (less than 0.0004 unit). Influence of Inhibitor on Enzyme Kinetics-We have previously shown that the acid DNase inhibitor modifies the kinetics of pure acid DNase (5). In the presence of the inhibitor a plot of V against S gave a sigmoidal-shaped curve at pH 5.0. With increasing inhibitor concentration, the sigmoidal shape became more pronounced. Moreover, the enzyme-inhibitor interaction disappeared progressively with a small pH shift from 5.00 to 5.57. However, in this previous work, total purification of the inhibitor has not been achieved. As shown in Figs, 4 and 5, the purified inhibitor possessed exactly the same properties. The inhibitor was also active on snail acid DNase and new kinetic studies performed on Helix aspersa enzyme' have confirmed the results from Lava1 and Paoletti (6) likewise obtained with an impure inhibitor. The K, for the inhibitor derived from kinetic data with beef liver acid DNase was 1.82 x lo-'M. Evidence for Acid DNase: Znhibitor Znteraction-As long as the inhibitor was not obtained in a highly purified form the existence of a specific complex between the acid DNase and its inhibitor could not be ascertained, because the association of the molecules could be mediated by other protkins. The affinity chromatography experiments described above revealed that in addition to the inhibitor, eight proteins were able to bind to insoluble acid DNase. This ambiguity was resolved by rechromatography of the highly purified inhibitor on an insoluble acid DNase column, the strong binding of inhibitor, only eluted by a guanidine-HCI solution, attests clearly to the direct and specific interaction between acid DNase and its inhibitor. The latter was monitored by sodium dodecyl sulfateacrylamide gel electrophoresis (Fig. 3D).
Effect of RNA on Acid DNase: Inhibitor Interaction-The kinetic data showed that, at high concentration, the DNA was able to compete with the inhibitor and to reverse its action. Due to its strong affinity for acid DNase (K, = 2 x low5 M), DNA might induce a conformational change in the enzyme with a concomitant disappearance of DNase-inhibitor interaction. According to this hypothesis, it was expected that RNA, which is a competitive inhibitor of acid DNase (24, 25), would produce the ,same effect as the substrate on the acid DNaseinhibitor interaction. We invgstigated the kinetics of inhibition of acid DNase by the inhibitor in the presence and absence of RNA. It can be seen in Fig. 6 that, in the presence of RNA (1 &g/ml) the inhibitory effect could be suppressed while the sigmoidal-shaped curve was changed into a Michaelian form. Binding of the specific inhibitor to CM-Sephadex suggested it is a basic protein. So, controls were carried out with other basic proteins (cytochrome c, protamine) to exclude the possibility of an electrostatic neutralization of the inhibitor by RNA. In our conditions the controls excluded this hypothesis.
The stoichiometry of the acid DNase-inhibitor system was determined by gel filtration of both molecules on Sephadex G-75 according to Andrews (23). A molecular weight of 45,000 I Unpublished data.

Inhibition of Phosphodiesterase
Activity-The effect of inhibitor on phosphodiesterase activity was investigated. We have measured the hydrolysis of bis(p-nitrophenyl) phosphate with a preparation which still contains phosphodiesterase (acid DNase, Fraction V) at different concentrations of substrate in by guest on March 24, 2020 http://www.jbc.org/ Downloaded from the absence and in the presence of inhibitor. Fig. 7 shows that the inhibitor is also able to inhibit the phosphodiesterase. This result will be discussed below.
Other Proteins Interacting with Acid DNase As shown in Fig. 3C the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins eluted from the insoluble acid DNase column with a 750 mM guanidine-HC1/500 mM sodium acetate solution, pH 7.4, revealed that nine molecular species were able to interact in the chromatographic system. Among these proteins, only two were identified. One is the inhibitor, as checked by its influence on acid DNase kinetics, the other is a protein (molecular weight, 59,000) which exhibits phosphodiesterase activity towards bis(p-nitrophenyl) phosphate, as suggested by its chromatographic behavior (Fig. 1) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments (see below). The molecular weights of the seven other proteins were the following: 1, 17,000; 2, 46,000; 3, 71,000; 4, 79,000; 5, 89,000; 6, 100,000; 7, 112,000. Their other properties are unknown.

DISCUSSION
This work was undertaken to purify extensively, from beef liver, large quantities of protein inhibitor of acid DNases that might be suitable for physicochemical and physiological studies of acid DNases. The affinity chromatography method allowed us to isolate both the inhibitor and acid DNase as proteins in homogeneous state. We were unable to detect contaminating activities. The difficulties met in isolating the inhibitor by physicochemical methods (5)  There is some controversy as to the nature of this activity. Bernardi and Griffe (26) concluded that the hog spleen acid DNase, in a highly purified state, possesses this phosphodiesterase activity as an intrinsic property of the molecule. Sicard et al. (27) studying this problem with an acid DNase purified from the same source, confirm the preceding hypothesis but say that, even if the two activities belong to different proteins they cannot be separated by conventional techniques. On the other hand Swenson and Hodes (28) and Slor and Hodes (29) obtain from, respectively, beef spleen and sheep spleen heated crude extract, the pure acid DNases completely free of phosphodiesterase. Dulaney and Touster (4) report that the electrophoretically pure acid DNase of rat liver lysosomes did not show phosphodiesterase activity. Recently Yamanaka and his co-workers (30) succeeded in separating the two activities during the purification of human acid DNases. In our work, before the last step of acid DNase purification, the enzyme contained only a slight phosphodiesterase activity. By gel filtration on Sephadex G-100 a partial separation could be obtained (Fig. 1). The peak of acid DNase was then divided into three parts. Comparison of phosphodiesterase activity and sodium dodecyl sulfate-polyacrylamide gels revealed a clear relationship between enzymatic activity and the amount of the 59,000-dalton protein (Fig. 8). Due to the sharpness of the band of phosphodiesterase with regard to those corresponding to acid DNase, it seems quite possible to obtain an apparent homogeneous acid DNase still contaminated with phosphodiesterase. The affinity chromatography experiments provided a good explanation for difficulties in obtaining acid DNase preparations completely devoid of phosphodiesterase activity. On the insoluble acid DNase column the phosphodiesterase is so firmly bound to the enzyme that only a 500 mM guanidine-HCl solutions was able to break their interaction.
Heat sensitivities of the two enzymes differ significantly. By preincubating the DNase preparation (Fraction V) at various temperatures for 15 min, 50% of acid DNase and phosphodiesterase activities were lost at 52 and 62%, respectively. The rate of thermal inactivation at 60' is higher for acid DNase than for phosphodiesterase.
Their stability to some reagents added directly to the assay mixture was also studied and the results (Table III) show that phosphodiesterase activity is more resistant to p-hydroxymercuribenzoate and iodoacetate denaturation than acid DNase activity. Although these results should not be taken as strong evidence that these activities belong to different molecules, they do support the latter hypothesis. As to the identity of the protein splitting the FIG. 8. Identification of phosphodiesterase as a protein distinct from acid DNase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on samples from the acid DNase peak obtained after gel filtration on Sephadex G-100. (Fig. 1). This peak was divided into three parts and the samples (100 11) Bernardi and Bernardi (31). According to our results, it does not look as if it is the acid exonuclease because no such activity was detectable in our DNase preparation before the elimination of phosphodiesterase activity (Fraction V).
The properties of beef liver acid DNase are comparable with those described for enzymes extracted from various sources.
Concerning the inhibitor, the requirements for the highest efficiency have been defined and the stoichiometric measurements conclude that the enzyme-inhibitor complex consists of one molecule of acid DNase and one molecule of inhibitor. On the other hand the determination of interaction parameters in uitro indicates that the acid DNase possesses a higher affinity for the substrate than for the inhibitor.
These results, as well as the kinetic data, are consistent with the model of an enzyme-inhibitor complex composed of cata-lytic and regulatory subunits as already proposed (5). According to this hypothesis one molecule of inhibitor would be bound to one molecule of acid DNase. The quaternary structure of the latter is not yet completely understood. The peptide mapping of hog spleen acid DNase seemed to indicate that the enzyme was composed of two identical subunits (32) but molecular weight determination of the acid DNase under a variety of dissociating conditions (6 M guanidine with or without &mercaptoethanol) failed to show any dissociation into subunits (33). These results could be reconciled if the assumption could be checked that the acid DNase is either made up of identical subunits bound through covalent linkage or contains large portions of the polypeptide chain in duplicate. The human serum transferrin raises similar problems (34). In all cases the model we suggest for the acid DNase-inhibitor complex would be suitable. Although the structure of the inhibitor is unknown, it can be suggested that, like the acid DNase, the inhibitor molecule might exhibit a second order symmetry. Upon binding to the enzyme, the inhibitor would induce a conformational change to a new state in which the catalytic sites would have less affinity for the substrate. The fact that RNA is able to reverse the action of the inhibitor and, like the DNA substrate, shows a high affinity for catalytic sites (24, 25), provides further support to this model.
The new procedure described herein for the extensive purification of beef liver acid DNase and its inhibitor leads to the availability of large quantities of these proteins. Furthermore affinity chromatography is a convenient means for the isolation of several proteins which directly or indirectly exhibit interacting properties towards acid DNase. The existence of such a system will facilitate studies concerning the role of acid DNase in the cells and the regulation of their activity. In this prospect a lot of various investigations are obviously required. The comparison of specificity of acid DNases in the absence and in the presence of inhibitor would be of a great interest, as mentioned by Lehman (35), concerning the possible involvement of this enzyme in other biological events than DNA degradation (DNA replication, recombination, cell division, etc...). Studies on purification and identification of a number of proteins able to interact with acid DNase are still necessary. The subcellular localization of all the components of the system will be attractive and might give preliminary indications on their physiological role. Inside the cell, they might be part, with acid DNase, phosphodiesterase, and inhibitor, of a multimeric complex including other enzymatic activities as well as structural proteins (36)(37)(38). This could explain the action of the inhibitor on the phosphodiesterase. This hypothesis is suggested by the fact that it is impossible to separate these proteins by polyacrylamide gel electrophoresis under nondenaturing conditions or by gel filtration on Sephadex G-100, the group of proteins being excluded as a large complex. According to another hypothesis, these proteins could be receptors of the enzyme located in subcellular organelles, membranes, or nucleus.