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15 Pancreatic DNase
Pancreatic DNase STANFORD MOORE
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Chemical Structure . . . . . . . . . . . . . A . Sequence, . . . . . . . . . . . . . . . B . Essentiality of Specific Residues . . . . IV. Catalytic Properties . . . . . . . . . . . . . A . Roles of Divalent Metal Ions . . . . . . B . Substrate Specificity . . . . . . . . . . . V. Actin as an Inhibitor of DNase I . . . . . . VI. Research Applications . . . . . . . . . . . 11. Purification
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281 282 285 285 286 288 288 290 293 295
Introduction
Bovine pancreatic deoxyribonuclease is the most thoroughly studied of the enzymes of the DNase I class (EC 3.1.21. l), defined as enzymes that cleave the substrate endonucleolytically to yield primarily S-phosphodiand oligonucleotide end products. In this chapter, the term DNase will refer to the bovine enzyme, unless otherwise specified. Bovine pancreatic DNase I can be resolved into four components of similar catalytic activity, DNases A, B, C, and D. For most purposes the mixture of the four is a suitable catalyst, subject to the degree of freedom from contaminating proteases or ribonucleases : when one of the subfractions has been studied, the letter designation will be added. The information on DNase to 1970 has been summarized by Laskowski 28 1 THE ENZYMES, Vol. XIV Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved ISBN 0-12-122714-6
282
STANFORD MOORE
(I) in Volume IV of this series. This chapter will cover primarily the information that has been gathered in the subsequent decade. Comparative studies on pancreatic DNases from different species have shown that the ovine and human enzymes are closely similar to the bovine catalyst, as cited in the following section on purification. The characterization of DNases from other tissues has included the finding that the DNase I secreted by the bovine parotid gland ( 2 , 3 )is very similar but not identical, chemically (4), to the bovine pancreatic enzyme. The results provide one of the several examples of enzymes built to the same basic design via different genes in different tissues of the same species. Moving further afield in terms of biological source, the DNase in germinating barley that Brawerman and Chargaff (5) showed had some enzymatic properties in common with bovine pancreatic DNase, has been reinvestigated by ‘iao (6). By applying current techniques, he has succeeded in purifying and characterizing the enzyme from malt diastase and finds it to be remarkably homologous in all of its chemical and enzymatic properties with the mammalian enzyme. Thus, the detailed picture of the chemical structure and catalytic properties of bovine pancreatic deoxyribonuclease summarized in the following pages applies to a broader spectrum of enzymes than was envisaged at the outset of the research. II. Purification
The starting material for the chromatographic purification of pancreatic DNase has usually been the amorphous enzyme prepared from bovine pancreas by ammonium sulfate fractionation according to the procedure of Kunitz (7). In an extension of the chromatographic experiments of Price et al. (8) on sulfoethyl-Sephadex, Salnikow et al. (9) observed four active components by chromatography on phosphocellulose (Fig. 1). Pancreatic juice submitted to initial chromatography on DEAE-cellulose gave a simi1. M . Laskowski, Sr., “The Enzymes,” 3rd. Ed., Vol. 4, p. 289, 1971. 2. W. D. Bail and W. J. Rutter, J . Exp. Z o d . 178, I (1971). 3. W. D. Ball, BBA 341, 305 (1974). 4. R. L. Lundblad, S . Hoffman, C. M. Noyes, and H. S. Kingdon,J. Dent. Res. 56, 320 (1977). 5. G . Brawerman and E. Chargaff, JBC 210, 445 (1954). 6. T.-H. Liao, Phytochernistry 16, 1469 (1977). 7. M. Kunitz, J . Gen. Physiof. 33, 349 (1950). 8 . P. A. Price, T.-Y. Liu, W. H. Stein, and S. Moore, JBC 244, 917 (1%9). 9. J. Salnikow, S. Moore, and W. H. Stein, JBC 245, 5685 (1970).
15.
283
PANCREATIC DNase
1
1
-
’M f” > .-
u
a 0
300
Effluent
600
(ml)
900 ...
FIG. 1. Chromatography of bovine pancreatic DNase (Worthington DP grade) on phosphocellulose. Column, 2 x 75 cm: temperature, 25”; flow rate, 35 ml per hour: 4-mI fractions were collected: column equilibrated with 0.25 M sodium acetate at pH 4.7: initial eluent, 150 ml of 0.38 M sodium acetate buffer, pH 4.7; linear gradient with 400 ml each of the initial and the limit buffer, 0.7 M sodium acetate, pH 4.7: (---) absorbance at 280 nm: (0-0) enzymatic activity. From Salnikow ei nl. (Y), reproduced with permission. lar pattern of DNases on phosphocellulose. The major enzyme, DNase A, was the protein taken for detailed sequence analysis; it is a glycoprotein with a neutral carbohydrate side chain. The protein moiety of DNase B is indistinguishable from that of DNase A; the carbohydrate side chain contains sialic acid. From amino acid analyses, DNase C was characterized as being the same as A except for a proline residue substituted for a histidine. This conclusion was confirmed by peptide maps (10) interpreted in the light of the sequence studies. Liao ( 1 1 ) has shown that DNase D has the same sequence as C but contains sialic acid in the carbohydrate portion. In order to obtain the small amount of DNase D in a stable form ( / I ) , the diisopropyl fluorophosphate-treated protein fraction was rechromatographed on DEAE-cellulose with use of a CaC1, gradient, thus combining the experience of Hugli (f2)on the chromatographic removal of traces of chymotrypsin and chymotrypsinogen B and of Price et nl. (8) on the stabilization of DNase against proteolysis by Ca2+. Liao ( I / ) has summarized the differences in the four enzymes (Table I). In Fig. 1 it can be noted that the activity-to-protein ratio may be slightly lower for the sialylated DNases B and D than for A and C; this observation is borne out by the specific activities in Table I. When DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, the purification prob10. J. Salnikow and D. Murphy, JBC 248, 1499 (1973). 11. T.-H. Liao, JBC 249, 2354 (1974). 12. T. E. Hugli, JBC 248, 1712 (1973).
284
STANFORD MOORE TABLE 1
ANALYSESOF DNASESA, B ,
c, A N D D“
Analysis for
DNase A”
DNase B”
DNase C”
DNase Dh
Mannose Galactose N - Acet ylglucosamine Sialic acid Proline Histidine Specific activity (unitsimg)
5.8 0.0 I .9 0.0 9.0 6.2 1158
4.5 I .O 3.4 1 .O 9.2 5.7 92 1
4.7 0.0 1.9 0.0 10.1 4.9 I045
4.3 I .O 3.3 0.8 9.9 5.1 837
“ From Liao ( / I ) ,
reproduced with permission.
’ Constituents expressed as residues per molecule
lem is a special one. The separation of DNases A , B, C, and D from one another is not necessary, but freedom from traces of ribonucleases or proteases is essential. Wang and Moore (13) have described a procedure that effects complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose, an extension of the method of Otsuka and Price (14). DNase is extremely sensitive to inactivation by proteases in the absence of Ca2+; the protease-free preparation retains full stability in the absence of Ca’+ for more than 10 days at pH 8 and 37”. Removal of the last traces of RNase has been accomplished (7) by affinitychromatography on a long (72 cm) column of 5-(4-aminophenylphosphoryl)uridine2’(3’)-phosphate-Sepharose (15, 16). The fully active product, obtained in quantitative yield, has less than I part of RNase per 10 million parts of DNase. Wadano et (11. (171, in the course of isolating DNases from ovine pancreas, have found chromatography on conconavalin A-agarose to be an effectivestep in the purification of DNases with neutral carbohydrate side chains: their method of isolation of DNase, which includes the use of phenylmethanesulfonyl chloride (18) as a protease inhibitor and chromatography on CM-cellulose with Ca2+-containingbuffers, offers the possibility of preparing bovine DNases A and C in higher yield than that obtained in the initial steps of the Kunitz (7) procedure. They also found 13. 14. 15. 16. 17. 18.
D. Wang and S. Moore, JBC 253, 7216 (1978). A. S. Otsuka and P. A. Price, A I I ~Biochem. . 62, 180 (1974). M . Wilchek and M . Gorecki, “Methods in Enzymology,” Vol. 34, p. 492, 1974. 0 . Brison and P. Chambon, Anti/. Biockern. 75, 402 (1976). A. Wadano, P. A. Hobus, and T.-H. Liao, Biocliemisrry 18, 4124 (1979). D. E. Fahrney and A . M. Gold, JACS 85, 997 (1963).
15. PANCREATIC DNase
285
that adsorption on Con A-agarose provided a means of obtaining a protease-free preparation of bovine pancreatic DNase. Funakoshi et a/. (19) have isolated from human duodenaljuice a DNase I that has properties very similar to those of the bovine pancreatic enzyme. Love and Hewitt (2U) have purified the human pancreatic enzyme with similar results. In their experiments they introduced a fluorometric DNase assay based upon the use of a circular DNA substrate and the binding of the denatured split products to ethidium bromide. In the other papers covered in this chapter, authors define their own modifications of the assays ( 1) based upon hyperchromicity, proton release, or acid-soluble nucleotides determined by absorbance or radioactivity. 111.
Chemical Structure
A.
SEQUENCE
Through study of the peptides yielded by tryptic or chymotryptic hydrolysis and by cyanogen bromide cleavage, Salnikowet al. (21)and Liaoet LII. (22) derived a sequence for the amino acid residues in reduced and carboxymethylated DNase A. The disulfide bridges were characterized by drawing upon the observation of Price et ril. (23) that in the presence of Ca2+one of the two S-S bonds could be selectively reduced by mercaptoethanol ; cyanogen bromide cleavage of the alkylated derivative provided data on the pairings. The result of the sequence study is given in Fig. 2. The sequence is a working hypothesis based upon all of the data available at this time. The molecule corresponds to a protein of 257 residues with carbohydrate attached through an aspartamido-hexose linkage at one position (Asn-18 in an Asn-X-Thr sequence). The amide -NH3 value is 21, which agrees with the determination by Lindberg (24) recalculated for the present molecular size. The molecular weight of DNase A, calculated from the amino acid and carbohydrate composition, is 30,072. The numbers of individual residues are Lys-9, His-6, Arg-11, Asp-20, Asn-12, Thr-15, Ser30, Glu-10, Gln-9, Pro-9, Gly-9, Ala-22, +Cys-4, Val-24, Met-4, Ile-11, Leu19. A. Funakoshi, Y. Tsubota, H. Wakasugi, H. Ibayashi, and Y. Takagi, J . Biochem. (Tokyo) 82, 1771 (1977). 20. J. D. Love and R. R. Hewitt, JBC 254, 12588 (1979). 21. J. Salnikow, T.-H. Liao, S . Moore, and W. H. Stein,JBC 248, 1480 (1973). 22. T.-H. Liao, J. Salnikow, S. Moore, and W. H. Stein, JBC 248, 1489 (1973). 23. P. A. Price, W. H. Stein, and S . Moore, JBC 244, 929 (1969). 24. U . Lindberg, Biochemistry 6, 335 (1967).
286
STANFORD MOORE
Leu-Lys-Ile-Ala-Ah-Phe-Asn-I
10 Corb. le-Arg-Thr-Phe-Gly- Glu-Thr-Lys-Met-Ser-Asn-
20
30
Alo -Thr-Leu- Alo-Ser-Tyr -1le-Vol- Arg-Arg -Tyr-Asp-Ile-Val-Leu-Ile-Glu-Gln-Val40
Arg-Asp-Ser-His-Leu-Val-
50 Ala- Val-Gly-Lys-Leu-Leu-Asp-Tyr
- Leu-Asn-Gln- Asp-Asp70
60
Pro - As n -T hr - Ty r - H i s -Ty r - Vo I- Vo I- Ser - GI u - Pro - Leu- GIy - Arg - Asn- Ser -Ty r - Lys -G Iu 80
YO
- Gln-Tyr -
Arg-Tyr-Leu-Phe-Leu-Phe-Arg-Pro-Asn-Lys-Val-Ser-Val-Leu-Asp-Thr-Tyr I00
110
Asp-Asp-Gly-Cys- Glu- Ser-Cys-Gly -Am-Asp-Ser-Phe-Ser- Arg-Glu-Pro- Alo-vai-vai -
u
I30
Lys-Phe-Ser-Ser-His-Ser-Thr-Lys-Val-Lys-Glu-Phe-Ala-IleAlo-Pro-Ser-Asp-Ala-Val-
140
Val-Alo-Leu-His-SerI50
Ala-Glu-lle-Asn-Ser-Leu-Tyr
-Asp-Val-Tyr -Leu-Asp-Val
160
Gln-Gln- Lys -Trp-His-Leu-Asn-Asp-Vol-Met-Leu-Met-Gly-Asp-Phe-Asn-Alo-Asp-CysSer-Tyr- Vol-Thr-Ser-Ser-Gln-Trp-
I80
Ser- Ser- Ile- Arq-Leu- Arg-Thr- Ser-Ser -Thr-Phe200
190
Gln-Trp-Leu- I l e - Pro-Asp-Ser-Alo-Asp-Thr-Thr-Alo-Thr-Ser-Thr-Asn-Cys-Aia 210
220
Asp-Arg-Ile- Val-Val- Alo-Gly-Ser-Leu-Leu-Gln-Ser-Ser-Vol230 A lo-Pro-Phe-Asp-Phe-GIn250
Ser-Asp-His-Tyr-Pro-Val-
-
I70
-Tyr-
Vol- Gly-Pro-Ser- Ala-
240
Ala-Ah-Tyr-Gly- Leu-Ser- Asn-Glu-Mel-Ala-Leu-Alo-Ile
-
2 57
Glu-Vol-Thr-Leu-Thr
FIG. 2. Sequence of bovine pancreatic deoxyribonuclease A. From Sainikow et rrf. (21) and Liao et a / . ( 2 )reproduced . with permission. 23, Tyr-15, Phe-11, Trp-3, Man-5.8, and GlcNAc-1.9. The carbohydrate side chain (25) is probably a mixture of oligosaccharides of slightly different chain lengths. The pl is about 5 (7).
B. ESSENTIALITY OF SPECIFIC RESIDUES
The enzyme is inactivated by iodoacetate at pH 7.2 in the presence of Mn2+ or Cuz+ with the formation of one residue of 3carboxymethylhistidine (26). The sequence of the tryptic peptide that con25. B. J. Catley, s. Moore, and W. H. Stein, JBC 244, 933 (1969). 26. P. A. Price, S. Moore, and W. H. Stein, JBC 244, 924 (1969).
15. PANCREATIC DNase
287
tained the modified amino acid permits the assignment of the crucial histidine residue to position 13 1 (22). The conclusion from studies with small substrates (I) that there may be a positive charge near the active center histidine residue fits with the fact that this imidazole ring is alkylated by iodoacetate but not by iodoacetamide (16). The histidine residue in DNase A that is substituted by proline in DNase C is His- 118 (10): this residue is thus not essential to the activity of the enzyme. Loss of activity by nitration has been found to parallel the formation of a single 3-nitrotyrosine residue per enzyme molecule (27). By reference to Fig. 1, the composition and sequence of the Tyr-(3-N02)-containing peptide isolated from an enzymatic digest permits assignment of the tyrosine residue to position 62 (2). Ca2+ was not able to stabilize the nitrated enzyme toward chymotryptic digestion, thermal denaturation, or mercaptoethanol reduction of the essential disulfide linkage. Tyr-62 may contribute to the formation of a Cazf binding site on the molecule that is not coincident with the region of His- 131, since the latter remains sensitive to specific carboxymethylation in the nitrated enzyme (27). The disulfide bond that can be reduced without loss of activity (13)is the one involved in the small loop between residues 98 and 101 (22). The disulfide bond between residues 170 and 206 is essential for maintenance of the active molecule. By controlled proteolysis with chymotrypsin, Hugli (28) was able to split the bond between Trp and Ser at positions 178 and 179; the product retained nearly the full activity of the native enzyme. When the cleaved molecule was further digested with carboxypeptidase-Y, the residues Thr-Ser-Ser-Gln-Trp (residues 1 7 4 178) were removed and the molecule still retained 80% of its activity. Thus, five residues h a t e d in the central portion of the peptide chain are functionally expendable. The COOH-terminal residues of DNase, which are normally unavailable to carboxypeptidases, become susceptible to removal when the enzyme is denatured in 0.005% sodium dodecyl sulfate (29). Study of the effect of carboxypeptidase action upon the enzyme required development of a procedure for restoring activity to DNase that has been denatured by the detergent. The inactivation of DNase could be completely reversed by diluting the enzyme solution tenfold into 6 M guanidinium chloride before a 100-fold dilution for assay. A loss of regenerable activity could be correlated with the removal of 1 or 2 amino acid residues (-Leu-Thr) from the 27. T. E. Hugli and W. H. Stein,JBC 246, 7191 (1971). 28. T. E. Hugli, JBC 248, 1712 (1973). 29. T.-H. Liao,JBC 250, 3831 (1975).
288
STANFORD MOORE
COOH-terminal sequence. DNase thus resembles RNase (30,31) in being one of the several enzymes in which the residues at the COOH terminus have a determining effect upon the folding of the chain into the active conformation. By following the kinetics of the reaction of N-bromosuccinimide with DNase by amino acid analysis for tryptophan, Sartin er ul. (32)have been able to show that modification at Trp-155 is the change most crucial to inactivation by that reagent. Methanesulfonyl chloride at pH 5 inactivated DNase by modification of the hydroxyl group of an as yet unidentified serine residue (3.3). DNase is inactivated by 2-nitro-5-thiocyanobenzoicacid (34)by a reaction that has been shown to involve cleavage of the peptide chain at the hydroxyamino acids at positions 14, 40, 72, and 135 (35). In more general derivatization experiments, guanidination of the nine E-NH, groups or picolinimidylation of the a- and €-amino groups yields active derivatives (36). The NH, groups are thus not essential per se, but when positive charges on the enzyme are removed by carbamylation with cyanate, activity is progressively lost. Modification of the carboxyl groups by condensation with glycine ethyl ester in the presence of a carbodiimide causes major inactivation in the absence of Ca2+(-?3).The presence of the bivalent cation slows the rate of the inactivation.
IV.
Catalytic Properties
A. ROLESOF DIVALENT METALIONS The conformation and the activity of the molecule are markedly dependent upon the presence of metal ions. The resistance to proteolysis conveyed by Ca2+ (8) has been important in the purification of the protein. The role of Caz+ in the refolding of the reduced enzyme to the active conformation is crucial (23). The apparent molecular weight of DNase by gel filtration in Tris buffer increases with pH in the range from pH 7.5 to 9 30. 31. 32. 33. 34. 35. 36.
C. B. Anfinsen, J5C 221, 405 (1956). M . C. Lin,JBC 245. 6726 (1970). J . L. Sartin, T. E. Hugh, and T.-H. Liao, JBC 255, 8633 (1980). T. L. Poulos and P. A. Price, JBC 249, 1453 (1974). T.-H. Liao and L. J . McKenzie, JBC 254, 9598 (1979). T.-H. Liao and A. Wadano, JBC 254, 9602 (1979). B . V. Plapp, S. Moore, and W. H. Stein, JEC 246, 939 (1971).
15.
289
PANCREATIC DNase
TABLE I1
CA" REQUIREMENTFOR DNASEACTIVITYAT P H 8 '* H yperchromicity assay Specific activity Metal ions in assay
midmg)
Maxi mu m specific activity (9%)
2.5 mM MgCI, 2.5 mM MgCI2, Dowex-purified 2.5 mM MgCI, + EGTA lo-' M CaC12b 2.5 mM MgCI, + IO-'M CaC12b
10-18 3-6 0.1 8 710'
1.4-2.5 0.4-0.9 0 1.1 100
(&so
I
pH-stat assay Specific activity (NaOHI min/DNase) 10
3 0 5
650'
Maximum specific activity (%)
1.5 0.5
0 0.8 100
" From Price (42), reproduced with permission.
Each value represents the average or range of values for three or more assays of metal ion-free DNase A. Hyperchromicity assays were in 5 mM ionic strength Tris-chloride buffer at pH 8 and 25". pH-stat assays were in buffer-free solution at 25". DNA concentration is 0.04 mg/ml. ' Addition of lo-" M EGTA has no effect on these activities.
(37,38), a hydrodynamic change that is reversed by the presence of Caz+.
By gel filtration of DNase A at pH 7.5 with 45Ca2+,Price (39) found 2 Ca2+ bound with an average Kd of 1.4 x and 3 bound with a K d of 2 x With Mg2+the Kd for two sites was 2.3 x One of the two strong Ca2+ binding sites is not subject to competition from Mg2+.Fifty percent of the M CaCl, and maximum transition in the CD spectrum occurs near half-maximum protection against action by trypsin is achieved near 1.3 x M CaCl, (40).The uv spectra show a conformational change indicative of increased interiorization of tryptophan and tyrosine residues in the presence of Ca'+ (41). Price (42) has made the key observation (Table 11) that with reagents that have been purified to reduce the Ca2+contamination to a minimum, DNase in the presence of Mg2+is about 99.5% inactive. In the presence of B . Librraga, C . Bustamante, A. Gil, and E. Melgar, BBA 579, 298 (1979). B . Lizarraga, D. Sanchez-Romero, A. Gil, and E. Melgar,JBC 253, 3191 (1978) P. A. Price, JBC 247, 2895 (1972). T. L. Poulos and P. A. Price, JBC 247, 2900 (1972). 41. R . Tbllis and P. A. Price, JEC 249, 5033 ( 1974). 42. P. A. Price, JBC 250, 1981 (1975).
37. 38. 39. 40.
290
STANFORD MOORE
a very low concentration (0.01 mM) of EGTA, a chelating agent that binds Ca*+about lo6 times more strongly than it binds Mg2+,DNase activity in the presence of 2.5 mM Mg2+becomes undetectable. A Ca2+concentration of 0. I mM yields maximum activity; concentrations as high as 1 mM are inhibitory. Earlier experiments on the low activity of DNase when only MgCl, is added are attributed to the effect of traces of Ca2+in the reagents. Bivalent metal ions serve two essential roles; Ca2+ must be bound to the enzyme and ions such as Mg2+to the substrate. The concento 3 x tration of Ca2+in bovine pancreatic juice is 4 x M ( 4 3 ,a range that can contribute to keeping DNase functional in its physiological environment. The effects of various bivalent metal ions are summarized in Table I11 (42). Sr2+and Ba2+can substitute for Ca2+,but are leass effective. Mn2+ and Co2+ can substitute for Mg2+, the latter with only about 10% efficiency. The results permit earlier studies on metal ion effects [see, e.g., Refs. (44,431 to be interpreted in more operational terms. The degree to which the enzyme functions optimally depends upon two factors, the Ca2+concentration and the Mg2+ or Mn2+ concentration, with the former being subject to variation from trace amounts of Ca2+as a contaminant in the latter. Double-strand scission [see ( I ) ] can be expected when the affinity of the enzyme for substrate is at a maximum in the presence of both CaLf and Mg2+;single-strand scission and changes in specificity are likely to be associated with suboptimal concentrations of Ca2+ present as contaminants when Ca2+is not deliberately added (42). Douvas and Price (46) have shown that 1 Mg2+ per 2 DNA-phosphorus is optimum. Na+-DNA is inhibitory and maximum rates are obtained with Mg2+-DNAas substrate rather than with Na+-DNA plus MgC1,. Preincubation of DNase with Ca2+ before addition to the substrate increases the initial rate of hydrolysis twofold over that obtained with Ca2+-free DNase with double-stranded calf thymus DNA as substrate. B. SUBSTRATE SPECIFICITY
Junowicz and Spencer (45) have conducted an extensive enzymatic and chromatographic study of the terminal purines and pyrimidines in the oligonucleotides liberated by the digestion of calf thymus DNase A with a variety of bivalent ion mixtures for different times; the experiments were 43. 44. 45. 46.
A. Frouin and P. Gerard, C.R. SOC.Bid. 72, 98 (1912). E. Junowicz and J. H. Spencer, BBA 312, 72 (1973). E. Junowicz and J . H. Spencer, BBA 312, 85 (1973). A. Douvas and P. A. Price, BBA 395, 201 (1975).
IS.
29 1
PANCREATIC DNase TABLE 111
ACTIVITYO F DNASEWITH DIFFERENT METAL IoNs"*~ Activities under following conditions of assay*
Metal ion
2.5 mM MgCI,, lo-' M metal ion
2.5 m M Metal ion, M Caz+
9 0 700 440 220 50 30
700 700 8 0.9 4.2 750 81 0 0
MgCI, MgCI, + 10-5M EGTA CaCI, SrCI, BaCI, MnCI,' COCI," CdCI,, SnCI,, FeCI,, NiCI,, CuCI, ZnCI,, EuCI,, SmCI,, NdCI,
II 0
From Price (42), reproduced with permission. Hyperchromicity assays in 5 rnM Tris, pH 8, 25". with 0.04 mdml of DNA. Activity values are AA,,, per midmg of DNase. The possibility exists that traces of CaZ+in MnCI, and CoCI, account for these values.
conducted before the effects of traces of Ca2+in most bivalent metal salts (42) were fully appreciated. Under near optimal conditions (e.g., with Mg2+plus C a 2 + ) , the enzyme gives the molar yields of the four nucleosides in the end positions listed in Table IV; in these experiments the enzyme is impressive for its versatility rather than its selectivity. The results did not vary greatly with the time of hydrolysis. However, with several bivalent TABLE 1V
DEOXYRIBONUCLEOTIDES AT T H E 5' AND 3' ENDSOF T H E OLIGONUCLEOTIDES RELEASEDFROM C A L F THYMUS DNAovb Mole% nucleosides ~
~~
5' end
3' end
Digestion time (rnin)
dT
dC
dG
dA
dT
dC
dG
dA
1.5 30.0
29.0 34.8
18.2 22.2
23.8 17.4
29.0 25.5
29.0 27.7
10.5 8.5
28.4 30.1
32.1 33.7
" From Junowicz and Spencer (4.5), reproduced with permission. Activation by 33 m M Mg2+ and 0.1 mM Ca'+.
292
STANFORD MOORE
metal additions that gave less than maximum specific activity, the molar ratios of the terminal nucleosides varied markedly and the proportions changed with the time of digestion. At the 5’ end, the ratio of dT or dC to dG or dA varied from 1 : 50 to 1 : 1; at the 3’ end, the ratio of dA or dC to dG or dT varied from 1 : 5-25 to 1 : 1. In these digests and with the singlestranded DNA from E. coli K12, consistent yields were obtained of long oligonucleotides lacking dA at the 3’ end. Simon et al. (47) have extended to DNase their use of crab d(A-T) polymer as a substrate for nucleases. This unique polymer, which is composed predominently of alternating A and T but contains about 3% of G and C residues integrated into its structure, was submitted to controlled hydrolysis by DNase in the presence of varying concentrations of MgCI, to obtain a hexanucleotide fraction enriched in C and G. Pruch and Laskowski (48) have subsequently undertaken to determine whether the I-3% of ribonucleotides, still detectable in preparations of crab d(A-T), that have been exhaustively treated with RNases, are built-in components. Digestion with DNase A to dinucleotides led to chromatographic evidence for the mixed dinucleotides dC-rG, dT-rA, and dT-rG. The authors conclude that crab d(A-T) polymer from C . borealis contains covalently bound ribonucleotides and that, as a corollary premise, sugar specificity of DNase may be limited to the nucleotide following the point of cleavage. The result will stimulate experimentation; the finding fits the function of DNase as an enzyme with which dinucleotides are key end products. Pancreatic DNase has been an enzyme for which no small synthetic substrate is hydrolyzed rapidly. p-Nitrophenyl esters have proved to be convenient synthetic substrates in spectrophotometric assays for use in studies on the kinetics and mechanisms of action of a number of phosphodiesterases. With DNase I, Liao (49) has examined deoxythymidine, 3’, 5’-di-p-nitrophenyl phosphate, a substrate that Razzell and Khorana (50) studied with snake venom phosphodiesterase, and that Cuatrecasas et cil. (51) found to be highly susceptible to the action of staphylococcal nuclease. The compound is rapidly hydrolyzed at a single bond by DNase at pH 7 . 2 with the liberation of p-nitrophenol measurable at 400 nm. The binding is not strong: at 10 mM substrate, the enzyme is not saturated, but the initial rate of hydrolysis (in the first 1 to 5 min) varies linearly with 47. M . Simon, H.-C. Chang, and M . Laskowski, Sr. BBA 232, 462 (1971). 48. J. M. Pruch and M. Laskowski, Sr. JBC 255, 9409 (1980). 49. T.-H. Liao, JBC 250, 3721 (1975). 50. W. E. Razzell and H. G . Khorana, JBC 234, 2105 (1959). 51. P. Cuatrecasas, ‘M. Wilchek, and C. B . Anfinsen, Biochemistry 8, 2277 (1969).
15.
PANCREATIC DNase
293
enzyme concentration in the range from 1 to 6 pg of DNase in 110 pl. Bivalent metals are essential; the maximum facilitation was obtained with 10 mM MnCI, and 1 mM CaCl,. The enzymatic activities toward DNA and N02Ph-pdTp-NOzPhwere lost in parallel upon carboxymethylation of His-131 (26). Since the photometric response is not specific for DNase, the assay has use only with the purified enzyme. Examination of the products of hydrolysis by paper electrophoresis gave the unexpected result that DNase liberates p-nitrophenol from the 3’-ester group. Snake venom phosphodiesterase, which like DNase yields 5’-nucleotides from DNA, liberates p-nitrophenol from the 5’-ester group of this substrate (SO).Staphylococcal nuclease (51 ) gives predominantly p-nitrophenyl phosphate from the 5’ position. Thus, these three diesterases hydrolyze N0,Ph-pdTp-N0,Ph in three different ways. DNase A has been cross-linked to RNase by Wang (52) to prepare a bifunctional enzyme. The coupling was via the thiolation of each protein with N-acetyl-DL-homocysteinethiolactone to yield a disulfide bridge between the two enzymes. The product, which contained one molecule each of DNase and RNase, hydrolyzed thymus DNA and yeast RNA with 75 and 40%, respectively, of the efficiencies of the parent catalysts. The RNA strand of the hybrid substrate phage f l DNA * [3H]RNA was hydrolyzed rapidly by the Mn2+-activatedhybrid enzyme; the RNA strand in the complementary combination was not hydrolyzed significantly by linkage may not be the most practical RNase alone. Although an -S-Slinkage, the concept of the conjugation offers the possibility of delivering in vivo two enzymes that differ in size, charge, and biological function to the same site at the same time. V.
Actin as a n Inhibitor of DNase I
The presence in most mammalian cells of a protein that will inhibit pancreatic DNase has been known for over thirty years (I). Lazarides and Lindberg (S3)found in 1974 that the protein is cytoplasmic actin. Purified actin from chicken skeletal muscle inhibits DNase; the DNase inhibitor isolated from various tissues and cells is found to be closely similar to actin in its physical and chemical properties. Antibodies to the purified DNase inhibitor show reactivity toward actin-containing fibers in human skin fibroblasts. The inhibitory protein is thus a major cellular component that usually constitutes 5-10% of the soluble protein. The attribution of a 52. D. Wang, Biochemistry 18, 4449 (1979). 53. E. Lazarides and U . Lindberg, PNAS 71, 4742 (1974).
294
STANFORD MOORE
physiological role to the DNase-inhibitory action of actin is limited by the paucity of information on the existence of DNase I-type enzymes in nonpancreatic cells. The situation is very different from that which pertains to the highly specific cytoplasmic RNase inhibitor (54), K i= 3 X lo-’’’ M, which is present in trace amounts (about 0.01-0.02% of the soluble protein) along with a one-fifth to one-tenth molar quantity of a pancreatictype RNase, the activity of which is thus modulated. In line with Laskowski’s (I) suggestion, inhibition by actin can be a key criterion for characterization of intracellular nucleases of the DNase I type. Actin can be removed from cellular extracts by adsorption on a column of DNase coupled to agarose (55); so far, it has not been possible to elute the actin from the affinity column in a way that will preserve the inhibitory activity. Elution with 3 M guanidinium chloride, 1 M in sodium acetate (pH 6 3 , and 30% in glycerol yields the actin-like protein, but with more than 90% of the inhibitory activity lost. A pH 2.8 buffer did not release the actin from the adsorbent. Hitchcock et al. (56) and Mannherz et al. (57) have shown that DNase I causes depolymerization of filamentous actin to form a stable complex of 1 mole of DNase I with 1 mole of globular actin (MW -49,000). Wang and Goldberg (58) have utilized the affinity of DNase for actin-containing fibers to visualize microfilament bundles in nonmuscle cells: DNase I was added to formaldehyde-fixed and acetone-extracted chick or human fibroblasts followed by antibody to DNase for indirect immunofluorescence microscopy, or rhodamine-conjugated DNase was used for direct fluorescent microscopy. A selective assay for monomeric and filamentous actin in cell extracts has been developed by Blikstad et al. (59); the inhibition of DNase activity by G-actin is measured a few seconds after the addition of the enzyme and also after depolymerization of F-actin by 0.75 M guanidinium chloride, 0.5 M in sodium acetate, 0.5 mM in CaCl,, 0.5 mM in ATP, and 10 mM in Tris-HC1 (pH 7.5). Under this condition for 5 min at O”, the F-actin is converted to G-actin without loss of the DNase-inhibitory activity in the monomer. With fluorescently labeled DNase, Mannherz et al. (60) estimated the 54. P. Blackburn and S. Moore, “The Enzymes,” 3rd Ed., Vol. 15, in press. 55. U. Lindberg and S. Eriksson, EJB 18, 474 (1971). 56. S. E. Hitchock, L. Carlsson, and U . Lindberg, Cell 7, 531 (1976). 57. H. G. Mannherz, J. Barrington Leigh, R. Leberman, and H. Pfrang,FEBS (Fed.Eur. Biochem. S o c . ) Lett. 60, 34 (1975). 58. E. Wang and A. R. Goldberg, J . Histochem. Cytochem. 26,745 (1978). 59. I. Blikstad, F. Markey, L. Carlsson, T. Persson, and U. Lindberg, Cell 15,935 (1978). 60. H. G. Mannherz, R . S. Goody, M. Konrad, and E. Nowak, EJB 104, 367 (1980).
15. PANCREATIC DNase
295
binding constant for DNase I and monomeric rabbit skeletal muscle actin to be 5 x 10*M-' and the inhibition to be competitive. With fluorescently labeled actin, Ikkai et al. (61) obtained a K b value of 1 x lo6 M-I. A binding constant of 1.2 x 104 M-' was obtained with filamentous actin (60). Crystals of actin . DNase I complexes have been obtained for cystallographic studies (62, 6 3 ) . When Rohr and Mannherz (64)examined rat pancreatic juice by SDS gel electrophoresis for bands coinciding with actin and DNase actin complex, both were observed. When the juice was treated with 0.25 N H2S04, as in the first steps of the Kunitz (7) procedure for the isolation of pancreatic nucleases, the DNase activity doubled; the inhibitory action of actin is destroyed in this step but DNase is stable. The complex could also be dissociated under physiological conditions by rat or human bile; an activating component has been found to be 5'-nucleotidase (65). The enzyme from snake venom can produce the same results; the process is slow (14 hours at 23") with 10 mM DNase * actin complex and 2 mM nucleotidase. Since 5'-nucleotidase is a constituent of plasma membranes, they were tested (66) and found to effect a slow liberation of DNase from the complex; Grazi and Magri (67) suggest that phosphorylation of actin may have a role in the process. It has been proposed (60) that the interaction of DNase and actin may be a physiological process in the extracellular space of the gastrointestinal tract. VI.
Research Applications
DNase I has received wide use as a probe in the study of the structure of chromatin. Active genes are expected to be more readily accessible to digestion by DNase than the transcriptionally inert segments. Experiments in several laboratories (68-71 ) have demonstrated that limited 61. T. Ikkai, K . Mihashi, andT. Kouyarna,FEBS (Fed. Eur.Eiochem. S o c . ) L e t t . 109,216 (1980). 62. H. G. Mannherz, W. Kabsch, and R . Leberman, FEBS (Fed. E w . Biochem. S o c . ) Lett. 73, 141 (1977). 63. H . Sugino, N. Sakabe, K. Sakabe, S. Hatano, F. Oosawa, T. Mikawa, and S. Ebashi, J . Biochem. (Tokyo) 86, 257 (1979). 64. G. Rohr and H. G. Mannherz, EJB 89, 151 (1978). 65. H. G. Mannherz and G . Rohr, FEBS (Fed. E r r . Eiochem. Soc.) Lett. 95, 284 (1978). 66. G . Rohr and H . G . Mannherz, FEBS (Fed. Eur. Eiochem. Soc.) Lett. 99, 351 (1979). 67. E. Grazi and E. Magri, FEES (Fed. Eur. Eiochem. S o c . ) Lett. 104, 284 (1979). 68. H. Weintraub and M. Groudine, Stience 193, 848 (1976). 69. A. Garel and R. Axel, PNAS 73, 3966 (1976). 70. B . Sollner-Webb and G. Felsenfeld, Cell 10, 537 (1977). 71. S. Weisbrod and H. Weintraub, PNAS 76, 630 (1979).
296
STANFORD MOORE
digestion by DNase can provide information on the structure of the nucleosome. High resolution electrophoretic separation of solubilized oligonucleotides has provided evidence (72-74) for a repetitive internal structure of the chromatin subunit. The periodicity in the fragment lengths can be correlated with helical and super helical orientation of DNA in the nucleosome core (75). In a study of the role of postsynthetic modification of histones in gene activation, Vidali et NI. (76) have used DNase I digestion to show that DNA is more readily liberated from chromatin that contains an increased quantity of acetylated histones; Kastern et al. (77) have used DNase coupled to Sepharose in the preparation of DNA-dependent RNA polymerase 11; the use of an immobilized enzyme makes it possible to release RNA polymerase from actively transcribing genes without having the product contaminated by DNase. The interaction of DNase and actin has formed the basis of a procedure for visualizing cellular microfilaments by fluorescent microscopy (58) and for a differential assay for monomeric and filamentous actin in cell extracts (59).
Thoroughly RNase-free DNase (13) has proved useful (78, 79) to digest chromatin in the preparation of nuclear RNA by the procedure of Penman (80).The protease-free nature of the same DNase preparation is of potential value in the isolation of nuclear proteins. On the basis that the process of replication in virus-infected cells is preceded by liberation of the viral nucleic acid from its protective protein coating, Trukhachev and Salganik (81) studied the inhibition of viral synthesis by DNase in cell cultures. In a clinical study, favorable results (82) have been reported from the local application of DNase to patients suffering from herpes infections of the eye. M . NOH, Nitcleic Acids Res. 1, 1573 (1974). L. C. Lutter, J M B 117, 53 (1977). L. C. Lutter, Nircleic Acids Res. 6, 41 (1979). A. Prunell, R. D. Kornberg, L. Lutter, A. Klug, M. Levitt, and F. H. C. Crick, Science 204, 855 (1979). 76. G. Vidali, L. C. Boffa. E. M . Bradbury, and V. G . Allfrey, /“AS 75, 2239 (1978). 77. W. H. Kastern, J. D. Eldridge, and K. P. Mullinix, JBC 254, 7368 (1979). 78. I. Tamm and T. Kikuchi, P N A S 76, 5750 (1979). 79. I. Tamm, T. Kikuchi, J. E. Darnell, Jr., and M. Salditt-Georgieff, Biockemisrry 19, 2743 (1980). 80. S. Penman, in “Fundamental Techniques in Virology” ( K. Habel and N. P. Salzman, eds.), p. 35. Academic Press, New York, 1969. 81. A . A. Trukhachev and R. 1. Salganik, Virology 33, 552 (1967). 82. A . A. Colain, R . 1. Salganik, I . E . Mikhailovskaya, and I . M. Gorban,Ann. h i / . 203, 371 (1970). 72. 73. 74. 75.
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Chemical Structure . . . . . . . . . . . . . A . Sequence, . . . . . . . . . . . . . . . B . Essentiality of Specific Residues . . . . IV. Catalytic Properties . . . . . . . . . . . . . A . Roles of Divalent Metal Ions . . . . . . B . Substrate Specificity . . . . . . . . . . . V. Actin as an Inhibitor of DNase I . . . . . . VI. Research Applications . . . . . . . . . . . 11. Purification
1.
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .
281 282 285 285 286 288 288 290 293 295
Introduction
Bovine pancreatic deoxyribonuclease is the most thoroughly studied of the enzymes of the DNase I class (EC 3.1.21. l), defined as enzymes that cleave the substrate endonucleolytically to yield primarily S-phosphodiand oligonucleotide end products. In this chapter, the term DNase will refer to the bovine enzyme, unless otherwise specified. Bovine pancreatic DNase I can be resolved into four components of similar catalytic activity, DNases A, B, C, and D. For most purposes the mixture of the four is a suitable catalyst, subject to the degree of freedom from contaminating proteases or ribonucleases : when one of the subfractions has been studied, the letter designation will be added. The information on DNase to 1970 has been summarized by Laskowski 28 1 THE ENZYMES, Vol. XIV Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved ISBN 0-12-122714-6
282
STANFORD MOORE
(I) in Volume IV of this series. This chapter will cover primarily the information that has been gathered in the subsequent decade. Comparative studies on pancreatic DNases from different species have shown that the ovine and human enzymes are closely similar to the bovine catalyst, as cited in the following section on purification. The characterization of DNases from other tissues has included the finding that the DNase I secreted by the bovine parotid gland ( 2 , 3 )is very similar but not identical, chemically (4), to the bovine pancreatic enzyme. The results provide one of the several examples of enzymes built to the same basic design via different genes in different tissues of the same species. Moving further afield in terms of biological source, the DNase in germinating barley that Brawerman and Chargaff (5) showed had some enzymatic properties in common with bovine pancreatic DNase, has been reinvestigated by ‘iao (6). By applying current techniques, he has succeeded in purifying and characterizing the enzyme from malt diastase and finds it to be remarkably homologous in all of its chemical and enzymatic properties with the mammalian enzyme. Thus, the detailed picture of the chemical structure and catalytic properties of bovine pancreatic deoxyribonuclease summarized in the following pages applies to a broader spectrum of enzymes than was envisaged at the outset of the research. II. Purification
The starting material for the chromatographic purification of pancreatic DNase has usually been the amorphous enzyme prepared from bovine pancreas by ammonium sulfate fractionation according to the procedure of Kunitz (7). In an extension of the chromatographic experiments of Price et al. (8) on sulfoethyl-Sephadex, Salnikow et al. (9) observed four active components by chromatography on phosphocellulose (Fig. 1). Pancreatic juice submitted to initial chromatography on DEAE-cellulose gave a simi1. M . Laskowski, Sr., “The Enzymes,” 3rd. Ed., Vol. 4, p. 289, 1971. 2. W. D. Bail and W. J. Rutter, J . Exp. Z o d . 178, I (1971). 3. W. D. Ball, BBA 341, 305 (1974). 4. R. L. Lundblad, S . Hoffman, C. M. Noyes, and H. S. Kingdon,J. Dent. Res. 56, 320 (1977). 5. G . Brawerman and E. Chargaff, JBC 210, 445 (1954). 6. T.-H. Liao, Phytochernistry 16, 1469 (1977). 7. M. Kunitz, J . Gen. Physiof. 33, 349 (1950). 8 . P. A. Price, T.-Y. Liu, W. H. Stein, and S. Moore, JBC 244, 917 (1%9). 9. J. Salnikow, S. Moore, and W. H. Stein, JBC 245, 5685 (1970).
15.
283
PANCREATIC DNase
1
1
-
’M f” > .-
u
a 0
300
Effluent
600
(ml)
900 ...
FIG. 1. Chromatography of bovine pancreatic DNase (Worthington DP grade) on phosphocellulose. Column, 2 x 75 cm: temperature, 25”; flow rate, 35 ml per hour: 4-mI fractions were collected: column equilibrated with 0.25 M sodium acetate at pH 4.7: initial eluent, 150 ml of 0.38 M sodium acetate buffer, pH 4.7; linear gradient with 400 ml each of the initial and the limit buffer, 0.7 M sodium acetate, pH 4.7: (---) absorbance at 280 nm: (0-0) enzymatic activity. From Salnikow ei nl. (Y), reproduced with permission. lar pattern of DNases on phosphocellulose. The major enzyme, DNase A, was the protein taken for detailed sequence analysis; it is a glycoprotein with a neutral carbohydrate side chain. The protein moiety of DNase B is indistinguishable from that of DNase A; the carbohydrate side chain contains sialic acid. From amino acid analyses, DNase C was characterized as being the same as A except for a proline residue substituted for a histidine. This conclusion was confirmed by peptide maps (10) interpreted in the light of the sequence studies. Liao ( 1 1 ) has shown that DNase D has the same sequence as C but contains sialic acid in the carbohydrate portion. In order to obtain the small amount of DNase D in a stable form ( / I ) , the diisopropyl fluorophosphate-treated protein fraction was rechromatographed on DEAE-cellulose with use of a CaC1, gradient, thus combining the experience of Hugli (f2)on the chromatographic removal of traces of chymotrypsin and chymotrypsinogen B and of Price et nl. (8) on the stabilization of DNase against proteolysis by Ca2+. Liao ( I / ) has summarized the differences in the four enzymes (Table I). In Fig. 1 it can be noted that the activity-to-protein ratio may be slightly lower for the sialylated DNases B and D than for A and C; this observation is borne out by the specific activities in Table I. When DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, the purification prob10. J. Salnikow and D. Murphy, JBC 248, 1499 (1973). 11. T.-H. Liao, JBC 249, 2354 (1974). 12. T. E. Hugli, JBC 248, 1712 (1973).
284
STANFORD MOORE TABLE 1
ANALYSESOF DNASESA, B ,
c, A N D D“
Analysis for
DNase A”
DNase B”
DNase C”
DNase Dh
Mannose Galactose N - Acet ylglucosamine Sialic acid Proline Histidine Specific activity (unitsimg)
5.8 0.0 I .9 0.0 9.0 6.2 1158
4.5 I .O 3.4 1 .O 9.2 5.7 92 1
4.7 0.0 1.9 0.0 10.1 4.9 I045
4.3 I .O 3.3 0.8 9.9 5.1 837
“ From Liao ( / I ) ,
reproduced with permission.
’ Constituents expressed as residues per molecule
lem is a special one. The separation of DNases A , B, C, and D from one another is not necessary, but freedom from traces of ribonucleases or proteases is essential. Wang and Moore (13) have described a procedure that effects complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose, an extension of the method of Otsuka and Price (14). DNase is extremely sensitive to inactivation by proteases in the absence of Ca2+; the protease-free preparation retains full stability in the absence of Ca’+ for more than 10 days at pH 8 and 37”. Removal of the last traces of RNase has been accomplished (7) by affinitychromatography on a long (72 cm) column of 5-(4-aminophenylphosphoryl)uridine2’(3’)-phosphate-Sepharose (15, 16). The fully active product, obtained in quantitative yield, has less than I part of RNase per 10 million parts of DNase. Wadano et (11. (171, in the course of isolating DNases from ovine pancreas, have found chromatography on conconavalin A-agarose to be an effectivestep in the purification of DNases with neutral carbohydrate side chains: their method of isolation of DNase, which includes the use of phenylmethanesulfonyl chloride (18) as a protease inhibitor and chromatography on CM-cellulose with Ca2+-containingbuffers, offers the possibility of preparing bovine DNases A and C in higher yield than that obtained in the initial steps of the Kunitz (7) procedure. They also found 13. 14. 15. 16. 17. 18.
D. Wang and S. Moore, JBC 253, 7216 (1978). A. S. Otsuka and P. A. Price, A I I ~Biochem. . 62, 180 (1974). M . Wilchek and M . Gorecki, “Methods in Enzymology,” Vol. 34, p. 492, 1974. 0 . Brison and P. Chambon, Anti/. Biockern. 75, 402 (1976). A. Wadano, P. A. Hobus, and T.-H. Liao, Biocliemisrry 18, 4124 (1979). D. E. Fahrney and A . M. Gold, JACS 85, 997 (1963).
15. PANCREATIC DNase
285
that adsorption on Con A-agarose provided a means of obtaining a protease-free preparation of bovine pancreatic DNase. Funakoshi et a/. (19) have isolated from human duodenaljuice a DNase I that has properties very similar to those of the bovine pancreatic enzyme. Love and Hewitt (2U) have purified the human pancreatic enzyme with similar results. In their experiments they introduced a fluorometric DNase assay based upon the use of a circular DNA substrate and the binding of the denatured split products to ethidium bromide. In the other papers covered in this chapter, authors define their own modifications of the assays ( 1) based upon hyperchromicity, proton release, or acid-soluble nucleotides determined by absorbance or radioactivity. 111.
Chemical Structure
A.
SEQUENCE
Through study of the peptides yielded by tryptic or chymotryptic hydrolysis and by cyanogen bromide cleavage, Salnikowet al. (21)and Liaoet LII. (22) derived a sequence for the amino acid residues in reduced and carboxymethylated DNase A. The disulfide bridges were characterized by drawing upon the observation of Price et ril. (23) that in the presence of Ca2+one of the two S-S bonds could be selectively reduced by mercaptoethanol ; cyanogen bromide cleavage of the alkylated derivative provided data on the pairings. The result of the sequence study is given in Fig. 2. The sequence is a working hypothesis based upon all of the data available at this time. The molecule corresponds to a protein of 257 residues with carbohydrate attached through an aspartamido-hexose linkage at one position (Asn-18 in an Asn-X-Thr sequence). The amide -NH3 value is 21, which agrees with the determination by Lindberg (24) recalculated for the present molecular size. The molecular weight of DNase A, calculated from the amino acid and carbohydrate composition, is 30,072. The numbers of individual residues are Lys-9, His-6, Arg-11, Asp-20, Asn-12, Thr-15, Ser30, Glu-10, Gln-9, Pro-9, Gly-9, Ala-22, +Cys-4, Val-24, Met-4, Ile-11, Leu19. A. Funakoshi, Y. Tsubota, H. Wakasugi, H. Ibayashi, and Y. Takagi, J . Biochem. (Tokyo) 82, 1771 (1977). 20. J. D. Love and R. R. Hewitt, JBC 254, 12588 (1979). 21. J. Salnikow, T.-H. Liao, S . Moore, and W. H. Stein,JBC 248, 1480 (1973). 22. T.-H. Liao, J. Salnikow, S. Moore, and W. H. Stein, JBC 248, 1489 (1973). 23. P. A. Price, W. H. Stein, and S . Moore, JBC 244, 929 (1969). 24. U . Lindberg, Biochemistry 6, 335 (1967).
286
STANFORD MOORE
Leu-Lys-Ile-Ala-Ah-Phe-Asn-I
10 Corb. le-Arg-Thr-Phe-Gly- Glu-Thr-Lys-Met-Ser-Asn-
20
30
Alo -Thr-Leu- Alo-Ser-Tyr -1le-Vol- Arg-Arg -Tyr-Asp-Ile-Val-Leu-Ile-Glu-Gln-Val40
Arg-Asp-Ser-His-Leu-Val-
50 Ala- Val-Gly-Lys-Leu-Leu-Asp-Tyr
- Leu-Asn-Gln- Asp-Asp70
60
Pro - As n -T hr - Ty r - H i s -Ty r - Vo I- Vo I- Ser - GI u - Pro - Leu- GIy - Arg - Asn- Ser -Ty r - Lys -G Iu 80
YO
- Gln-Tyr -
Arg-Tyr-Leu-Phe-Leu-Phe-Arg-Pro-Asn-Lys-Val-Ser-Val-Leu-Asp-Thr-Tyr I00
110
Asp-Asp-Gly-Cys- Glu- Ser-Cys-Gly -Am-Asp-Ser-Phe-Ser- Arg-Glu-Pro- Alo-vai-vai -
u
I30
Lys-Phe-Ser-Ser-His-Ser-Thr-Lys-Val-Lys-Glu-Phe-Ala-IleAlo-Pro-Ser-Asp-Ala-Val-
140
Val-Alo-Leu-His-SerI50
Ala-Glu-lle-Asn-Ser-Leu-Tyr
-Asp-Val-Tyr -Leu-Asp-Val
160
Gln-Gln- Lys -Trp-His-Leu-Asn-Asp-Vol-Met-Leu-Met-Gly-Asp-Phe-Asn-Alo-Asp-CysSer-Tyr- Vol-Thr-Ser-Ser-Gln-Trp-
I80
Ser- Ser- Ile- Arq-Leu- Arg-Thr- Ser-Ser -Thr-Phe200
190
Gln-Trp-Leu- I l e - Pro-Asp-Ser-Alo-Asp-Thr-Thr-Alo-Thr-Ser-Thr-Asn-Cys-Aia 210
220
Asp-Arg-Ile- Val-Val- Alo-Gly-Ser-Leu-Leu-Gln-Ser-Ser-Vol230 A lo-Pro-Phe-Asp-Phe-GIn250
Ser-Asp-His-Tyr-Pro-Val-
-
I70
-Tyr-
Vol- Gly-Pro-Ser- Ala-
240
Ala-Ah-Tyr-Gly- Leu-Ser- Asn-Glu-Mel-Ala-Leu-Alo-Ile
-
2 57
Glu-Vol-Thr-Leu-Thr
FIG. 2. Sequence of bovine pancreatic deoxyribonuclease A. From Sainikow et rrf. (21) and Liao et a / . ( 2 )reproduced . with permission. 23, Tyr-15, Phe-11, Trp-3, Man-5.8, and GlcNAc-1.9. The carbohydrate side chain (25) is probably a mixture of oligosaccharides of slightly different chain lengths. The pl is about 5 (7).
B. ESSENTIALITY OF SPECIFIC RESIDUES
The enzyme is inactivated by iodoacetate at pH 7.2 in the presence of Mn2+ or Cuz+ with the formation of one residue of 3carboxymethylhistidine (26). The sequence of the tryptic peptide that con25. B. J. Catley, s. Moore, and W. H. Stein, JBC 244, 933 (1969). 26. P. A. Price, S. Moore, and W. H. Stein, JBC 244, 924 (1969).
15. PANCREATIC DNase
287
tained the modified amino acid permits the assignment of the crucial histidine residue to position 13 1 (22). The conclusion from studies with small substrates (I) that there may be a positive charge near the active center histidine residue fits with the fact that this imidazole ring is alkylated by iodoacetate but not by iodoacetamide (16). The histidine residue in DNase A that is substituted by proline in DNase C is His- 118 (10): this residue is thus not essential to the activity of the enzyme. Loss of activity by nitration has been found to parallel the formation of a single 3-nitrotyrosine residue per enzyme molecule (27). By reference to Fig. 1, the composition and sequence of the Tyr-(3-N02)-containing peptide isolated from an enzymatic digest permits assignment of the tyrosine residue to position 62 (2). Ca2+ was not able to stabilize the nitrated enzyme toward chymotryptic digestion, thermal denaturation, or mercaptoethanol reduction of the essential disulfide linkage. Tyr-62 may contribute to the formation of a Cazf binding site on the molecule that is not coincident with the region of His- 131, since the latter remains sensitive to specific carboxymethylation in the nitrated enzyme (27). The disulfide bond that can be reduced without loss of activity (13)is the one involved in the small loop between residues 98 and 101 (22). The disulfide bond between residues 170 and 206 is essential for maintenance of the active molecule. By controlled proteolysis with chymotrypsin, Hugli (28) was able to split the bond between Trp and Ser at positions 178 and 179; the product retained nearly the full activity of the native enzyme. When the cleaved molecule was further digested with carboxypeptidase-Y, the residues Thr-Ser-Ser-Gln-Trp (residues 1 7 4 178) were removed and the molecule still retained 80% of its activity. Thus, five residues h a t e d in the central portion of the peptide chain are functionally expendable. The COOH-terminal residues of DNase, which are normally unavailable to carboxypeptidases, become susceptible to removal when the enzyme is denatured in 0.005% sodium dodecyl sulfate (29). Study of the effect of carboxypeptidase action upon the enzyme required development of a procedure for restoring activity to DNase that has been denatured by the detergent. The inactivation of DNase could be completely reversed by diluting the enzyme solution tenfold into 6 M guanidinium chloride before a 100-fold dilution for assay. A loss of regenerable activity could be correlated with the removal of 1 or 2 amino acid residues (-Leu-Thr) from the 27. T. E. Hugli and W. H. Stein,JBC 246, 7191 (1971). 28. T. E. Hugli, JBC 248, 1712 (1973). 29. T.-H. Liao,JBC 250, 3831 (1975).
288
STANFORD MOORE
COOH-terminal sequence. DNase thus resembles RNase (30,31) in being one of the several enzymes in which the residues at the COOH terminus have a determining effect upon the folding of the chain into the active conformation. By following the kinetics of the reaction of N-bromosuccinimide with DNase by amino acid analysis for tryptophan, Sartin er ul. (32)have been able to show that modification at Trp-155 is the change most crucial to inactivation by that reagent. Methanesulfonyl chloride at pH 5 inactivated DNase by modification of the hydroxyl group of an as yet unidentified serine residue (3.3). DNase is inactivated by 2-nitro-5-thiocyanobenzoicacid (34)by a reaction that has been shown to involve cleavage of the peptide chain at the hydroxyamino acids at positions 14, 40, 72, and 135 (35). In more general derivatization experiments, guanidination of the nine E-NH, groups or picolinimidylation of the a- and €-amino groups yields active derivatives (36). The NH, groups are thus not essential per se, but when positive charges on the enzyme are removed by carbamylation with cyanate, activity is progressively lost. Modification of the carboxyl groups by condensation with glycine ethyl ester in the presence of a carbodiimide causes major inactivation in the absence of Ca2+(-?3).The presence of the bivalent cation slows the rate of the inactivation.
IV.
Catalytic Properties
A. ROLESOF DIVALENT METALIONS The conformation and the activity of the molecule are markedly dependent upon the presence of metal ions. The resistance to proteolysis conveyed by Ca2+ (8) has been important in the purification of the protein. The role of Caz+ in the refolding of the reduced enzyme to the active conformation is crucial (23). The apparent molecular weight of DNase by gel filtration in Tris buffer increases with pH in the range from pH 7.5 to 9 30. 31. 32. 33. 34. 35. 36.
C. B. Anfinsen, J5C 221, 405 (1956). M . C. Lin,JBC 245. 6726 (1970). J . L. Sartin, T. E. Hugh, and T.-H. Liao, JBC 255, 8633 (1980). T. L. Poulos and P. A. Price, JBC 249, 1453 (1974). T.-H. Liao and L. J . McKenzie, JBC 254, 9598 (1979). T.-H. Liao and A. Wadano, JBC 254, 9602 (1979). B . V. Plapp, S. Moore, and W. H. Stein, JEC 246, 939 (1971).
15.
289
PANCREATIC DNase
TABLE I1
CA" REQUIREMENTFOR DNASEACTIVITYAT P H 8 '* H yperchromicity assay Specific activity Metal ions in assay
midmg)
Maxi mu m specific activity (9%)
2.5 mM MgCI, 2.5 mM MgCI2, Dowex-purified 2.5 mM MgCI, + EGTA lo-' M CaC12b 2.5 mM MgCI, + IO-'M CaC12b
10-18 3-6 0.1 8 710'
1.4-2.5 0.4-0.9 0 1.1 100
(&so
I
pH-stat assay Specific activity (NaOHI min/DNase) 10
3 0 5
650'
Maximum specific activity (%)
1.5 0.5
0 0.8 100
" From Price (42), reproduced with permission.
Each value represents the average or range of values for three or more assays of metal ion-free DNase A. Hyperchromicity assays were in 5 mM ionic strength Tris-chloride buffer at pH 8 and 25". pH-stat assays were in buffer-free solution at 25". DNA concentration is 0.04 mg/ml. ' Addition of lo-" M EGTA has no effect on these activities.
(37,38), a hydrodynamic change that is reversed by the presence of Caz+.
By gel filtration of DNase A at pH 7.5 with 45Ca2+,Price (39) found 2 Ca2+ bound with an average Kd of 1.4 x and 3 bound with a K d of 2 x With Mg2+the Kd for two sites was 2.3 x One of the two strong Ca2+ binding sites is not subject to competition from Mg2+.Fifty percent of the M CaCl, and maximum transition in the CD spectrum occurs near half-maximum protection against action by trypsin is achieved near 1.3 x M CaCl, (40).The uv spectra show a conformational change indicative of increased interiorization of tryptophan and tyrosine residues in the presence of Ca'+ (41). Price (42) has made the key observation (Table 11) that with reagents that have been purified to reduce the Ca2+contamination to a minimum, DNase in the presence of Mg2+is about 99.5% inactive. In the presence of B . Librraga, C . Bustamante, A. Gil, and E. Melgar, BBA 579, 298 (1979). B . Lizarraga, D. Sanchez-Romero, A. Gil, and E. Melgar,JBC 253, 3191 (1978) P. A. Price, JBC 247, 2895 (1972). T. L. Poulos and P. A. Price, JBC 247, 2900 (1972). 41. R . Tbllis and P. A. Price, JEC 249, 5033 ( 1974). 42. P. A. Price, JBC 250, 1981 (1975).
37. 38. 39. 40.
290
STANFORD MOORE
a very low concentration (0.01 mM) of EGTA, a chelating agent that binds Ca*+about lo6 times more strongly than it binds Mg2+,DNase activity in the presence of 2.5 mM Mg2+becomes undetectable. A Ca2+concentration of 0. I mM yields maximum activity; concentrations as high as 1 mM are inhibitory. Earlier experiments on the low activity of DNase when only MgCl, is added are attributed to the effect of traces of Ca2+in the reagents. Bivalent metal ions serve two essential roles; Ca2+ must be bound to the enzyme and ions such as Mg2+to the substrate. The concento 3 x tration of Ca2+in bovine pancreatic juice is 4 x M ( 4 3 ,a range that can contribute to keeping DNase functional in its physiological environment. The effects of various bivalent metal ions are summarized in Table I11 (42). Sr2+and Ba2+can substitute for Ca2+,but are leass effective. Mn2+ and Co2+ can substitute for Mg2+, the latter with only about 10% efficiency. The results permit earlier studies on metal ion effects [see, e.g., Refs. (44,431 to be interpreted in more operational terms. The degree to which the enzyme functions optimally depends upon two factors, the Ca2+concentration and the Mg2+ or Mn2+ concentration, with the former being subject to variation from trace amounts of Ca2+as a contaminant in the latter. Double-strand scission [see ( I ) ] can be expected when the affinity of the enzyme for substrate is at a maximum in the presence of both CaLf and Mg2+;single-strand scission and changes in specificity are likely to be associated with suboptimal concentrations of Ca2+ present as contaminants when Ca2+is not deliberately added (42). Douvas and Price (46) have shown that 1 Mg2+ per 2 DNA-phosphorus is optimum. Na+-DNA is inhibitory and maximum rates are obtained with Mg2+-DNAas substrate rather than with Na+-DNA plus MgC1,. Preincubation of DNase with Ca2+ before addition to the substrate increases the initial rate of hydrolysis twofold over that obtained with Ca2+-free DNase with double-stranded calf thymus DNA as substrate. B. SUBSTRATE SPECIFICITY
Junowicz and Spencer (45) have conducted an extensive enzymatic and chromatographic study of the terminal purines and pyrimidines in the oligonucleotides liberated by the digestion of calf thymus DNase A with a variety of bivalent ion mixtures for different times; the experiments were 43. 44. 45. 46.
A. Frouin and P. Gerard, C.R. SOC.Bid. 72, 98 (1912). E. Junowicz and J. H. Spencer, BBA 312, 72 (1973). E. Junowicz and J . H. Spencer, BBA 312, 85 (1973). A. Douvas and P. A. Price, BBA 395, 201 (1975).
IS.
29 1
PANCREATIC DNase TABLE 111
ACTIVITYO F DNASEWITH DIFFERENT METAL IoNs"*~ Activities under following conditions of assay*
Metal ion
2.5 mM MgCI,, lo-' M metal ion
2.5 m M Metal ion, M Caz+
9 0 700 440 220 50 30
700 700 8 0.9 4.2 750 81 0 0
MgCI, MgCI, + 10-5M EGTA CaCI, SrCI, BaCI, MnCI,' COCI," CdCI,, SnCI,, FeCI,, NiCI,, CuCI, ZnCI,, EuCI,, SmCI,, NdCI,
II 0
From Price (42), reproduced with permission. Hyperchromicity assays in 5 rnM Tris, pH 8, 25". with 0.04 mdml of DNA. Activity values are AA,,, per midmg of DNase. The possibility exists that traces of CaZ+in MnCI, and CoCI, account for these values.
conducted before the effects of traces of Ca2+in most bivalent metal salts (42) were fully appreciated. Under near optimal conditions (e.g., with Mg2+plus C a 2 + ) , the enzyme gives the molar yields of the four nucleosides in the end positions listed in Table IV; in these experiments the enzyme is impressive for its versatility rather than its selectivity. The results did not vary greatly with the time of hydrolysis. However, with several bivalent TABLE 1V
DEOXYRIBONUCLEOTIDES AT T H E 5' AND 3' ENDSOF T H E OLIGONUCLEOTIDES RELEASEDFROM C A L F THYMUS DNAovb Mole% nucleosides ~
~~
5' end
3' end
Digestion time (rnin)
dT
dC
dG
dA
dT
dC
dG
dA
1.5 30.0
29.0 34.8
18.2 22.2
23.8 17.4
29.0 25.5
29.0 27.7
10.5 8.5
28.4 30.1
32.1 33.7
" From Junowicz and Spencer (4.5), reproduced with permission. Activation by 33 m M Mg2+ and 0.1 mM Ca'+.
292
STANFORD MOORE
metal additions that gave less than maximum specific activity, the molar ratios of the terminal nucleosides varied markedly and the proportions changed with the time of digestion. At the 5’ end, the ratio of dT or dC to dG or dA varied from 1 : 50 to 1 : 1; at the 3’ end, the ratio of dA or dC to dG or dT varied from 1 : 5-25 to 1 : 1. In these digests and with the singlestranded DNA from E. coli K12, consistent yields were obtained of long oligonucleotides lacking dA at the 3’ end. Simon et al. (47) have extended to DNase their use of crab d(A-T) polymer as a substrate for nucleases. This unique polymer, which is composed predominently of alternating A and T but contains about 3% of G and C residues integrated into its structure, was submitted to controlled hydrolysis by DNase in the presence of varying concentrations of MgCI, to obtain a hexanucleotide fraction enriched in C and G. Pruch and Laskowski (48) have subsequently undertaken to determine whether the I-3% of ribonucleotides, still detectable in preparations of crab d(A-T), that have been exhaustively treated with RNases, are built-in components. Digestion with DNase A to dinucleotides led to chromatographic evidence for the mixed dinucleotides dC-rG, dT-rA, and dT-rG. The authors conclude that crab d(A-T) polymer from C . borealis contains covalently bound ribonucleotides and that, as a corollary premise, sugar specificity of DNase may be limited to the nucleotide following the point of cleavage. The result will stimulate experimentation; the finding fits the function of DNase as an enzyme with which dinucleotides are key end products. Pancreatic DNase has been an enzyme for which no small synthetic substrate is hydrolyzed rapidly. p-Nitrophenyl esters have proved to be convenient synthetic substrates in spectrophotometric assays for use in studies on the kinetics and mechanisms of action of a number of phosphodiesterases. With DNase I, Liao (49) has examined deoxythymidine, 3’, 5’-di-p-nitrophenyl phosphate, a substrate that Razzell and Khorana (50) studied with snake venom phosphodiesterase, and that Cuatrecasas et cil. (51) found to be highly susceptible to the action of staphylococcal nuclease. The compound is rapidly hydrolyzed at a single bond by DNase at pH 7 . 2 with the liberation of p-nitrophenol measurable at 400 nm. The binding is not strong: at 10 mM substrate, the enzyme is not saturated, but the initial rate of hydrolysis (in the first 1 to 5 min) varies linearly with 47. M . Simon, H.-C. Chang, and M . Laskowski, Sr. BBA 232, 462 (1971). 48. J. M. Pruch and M. Laskowski, Sr. JBC 255, 9409 (1980). 49. T.-H. Liao, JBC 250, 3721 (1975). 50. W. E. Razzell and H. G . Khorana, JBC 234, 2105 (1959). 51. P. Cuatrecasas, ‘M. Wilchek, and C. B . Anfinsen, Biochemistry 8, 2277 (1969).
15.
PANCREATIC DNase
293
enzyme concentration in the range from 1 to 6 pg of DNase in 110 pl. Bivalent metals are essential; the maximum facilitation was obtained with 10 mM MnCI, and 1 mM CaCl,. The enzymatic activities toward DNA and N02Ph-pdTp-NOzPhwere lost in parallel upon carboxymethylation of His-131 (26). Since the photometric response is not specific for DNase, the assay has use only with the purified enzyme. Examination of the products of hydrolysis by paper electrophoresis gave the unexpected result that DNase liberates p-nitrophenol from the 3’-ester group. Snake venom phosphodiesterase, which like DNase yields 5’-nucleotides from DNA, liberates p-nitrophenol from the 5’-ester group of this substrate (SO).Staphylococcal nuclease (51 ) gives predominantly p-nitrophenyl phosphate from the 5’ position. Thus, these three diesterases hydrolyze N0,Ph-pdTp-N0,Ph in three different ways. DNase A has been cross-linked to RNase by Wang (52) to prepare a bifunctional enzyme. The coupling was via the thiolation of each protein with N-acetyl-DL-homocysteinethiolactone to yield a disulfide bridge between the two enzymes. The product, which contained one molecule each of DNase and RNase, hydrolyzed thymus DNA and yeast RNA with 75 and 40%, respectively, of the efficiencies of the parent catalysts. The RNA strand of the hybrid substrate phage f l DNA * [3H]RNA was hydrolyzed rapidly by the Mn2+-activatedhybrid enzyme; the RNA strand in the complementary combination was not hydrolyzed significantly by linkage may not be the most practical RNase alone. Although an -S-Slinkage, the concept of the conjugation offers the possibility of delivering in vivo two enzymes that differ in size, charge, and biological function to the same site at the same time. V.
Actin as a n Inhibitor of DNase I
The presence in most mammalian cells of a protein that will inhibit pancreatic DNase has been known for over thirty years (I). Lazarides and Lindberg (S3)found in 1974 that the protein is cytoplasmic actin. Purified actin from chicken skeletal muscle inhibits DNase; the DNase inhibitor isolated from various tissues and cells is found to be closely similar to actin in its physical and chemical properties. Antibodies to the purified DNase inhibitor show reactivity toward actin-containing fibers in human skin fibroblasts. The inhibitory protein is thus a major cellular component that usually constitutes 5-10% of the soluble protein. The attribution of a 52. D. Wang, Biochemistry 18, 4449 (1979). 53. E. Lazarides and U . Lindberg, PNAS 71, 4742 (1974).
294
STANFORD MOORE
physiological role to the DNase-inhibitory action of actin is limited by the paucity of information on the existence of DNase I-type enzymes in nonpancreatic cells. The situation is very different from that which pertains to the highly specific cytoplasmic RNase inhibitor (54), K i= 3 X lo-’’’ M, which is present in trace amounts (about 0.01-0.02% of the soluble protein) along with a one-fifth to one-tenth molar quantity of a pancreatictype RNase, the activity of which is thus modulated. In line with Laskowski’s (I) suggestion, inhibition by actin can be a key criterion for characterization of intracellular nucleases of the DNase I type. Actin can be removed from cellular extracts by adsorption on a column of DNase coupled to agarose (55); so far, it has not been possible to elute the actin from the affinity column in a way that will preserve the inhibitory activity. Elution with 3 M guanidinium chloride, 1 M in sodium acetate (pH 6 3 , and 30% in glycerol yields the actin-like protein, but with more than 90% of the inhibitory activity lost. A pH 2.8 buffer did not release the actin from the adsorbent. Hitchcock et al. (56) and Mannherz et al. (57) have shown that DNase I causes depolymerization of filamentous actin to form a stable complex of 1 mole of DNase I with 1 mole of globular actin (MW -49,000). Wang and Goldberg (58) have utilized the affinity of DNase for actin-containing fibers to visualize microfilament bundles in nonmuscle cells: DNase I was added to formaldehyde-fixed and acetone-extracted chick or human fibroblasts followed by antibody to DNase for indirect immunofluorescence microscopy, or rhodamine-conjugated DNase was used for direct fluorescent microscopy. A selective assay for monomeric and filamentous actin in cell extracts has been developed by Blikstad et al. (59); the inhibition of DNase activity by G-actin is measured a few seconds after the addition of the enzyme and also after depolymerization of F-actin by 0.75 M guanidinium chloride, 0.5 M in sodium acetate, 0.5 mM in CaCl,, 0.5 mM in ATP, and 10 mM in Tris-HC1 (pH 7.5). Under this condition for 5 min at O”, the F-actin is converted to G-actin without loss of the DNase-inhibitory activity in the monomer. With fluorescently labeled DNase, Mannherz et al. (60) estimated the 54. P. Blackburn and S. Moore, “The Enzymes,” 3rd Ed., Vol. 15, in press. 55. U. Lindberg and S. Eriksson, EJB 18, 474 (1971). 56. S. E. Hitchock, L. Carlsson, and U . Lindberg, Cell 7, 531 (1976). 57. H. G. Mannherz, J. Barrington Leigh, R. Leberman, and H. Pfrang,FEBS (Fed.Eur. Biochem. S o c . ) Lett. 60, 34 (1975). 58. E. Wang and A. R. Goldberg, J . Histochem. Cytochem. 26,745 (1978). 59. I. Blikstad, F. Markey, L. Carlsson, T. Persson, and U. Lindberg, Cell 15,935 (1978). 60. H. G. Mannherz, R . S. Goody, M. Konrad, and E. Nowak, EJB 104, 367 (1980).
15. PANCREATIC DNase
295
binding constant for DNase I and monomeric rabbit skeletal muscle actin to be 5 x 10*M-' and the inhibition to be competitive. With fluorescently labeled actin, Ikkai et al. (61) obtained a K b value of 1 x lo6 M-I. A binding constant of 1.2 x 104 M-' was obtained with filamentous actin (60). Crystals of actin . DNase I complexes have been obtained for cystallographic studies (62, 6 3 ) . When Rohr and Mannherz (64)examined rat pancreatic juice by SDS gel electrophoresis for bands coinciding with actin and DNase actin complex, both were observed. When the juice was treated with 0.25 N H2S04, as in the first steps of the Kunitz (7) procedure for the isolation of pancreatic nucleases, the DNase activity doubled; the inhibitory action of actin is destroyed in this step but DNase is stable. The complex could also be dissociated under physiological conditions by rat or human bile; an activating component has been found to be 5'-nucleotidase (65). The enzyme from snake venom can produce the same results; the process is slow (14 hours at 23") with 10 mM DNase * actin complex and 2 mM nucleotidase. Since 5'-nucleotidase is a constituent of plasma membranes, they were tested (66) and found to effect a slow liberation of DNase from the complex; Grazi and Magri (67) suggest that phosphorylation of actin may have a role in the process. It has been proposed (60) that the interaction of DNase and actin may be a physiological process in the extracellular space of the gastrointestinal tract. VI.
Research Applications
DNase I has received wide use as a probe in the study of the structure of chromatin. Active genes are expected to be more readily accessible to digestion by DNase than the transcriptionally inert segments. Experiments in several laboratories (68-71 ) have demonstrated that limited 61. T. Ikkai, K . Mihashi, andT. Kouyarna,FEBS (Fed. Eur.Eiochem. S o c . ) L e t t . 109,216 (1980). 62. H. G. Mannherz, W. Kabsch, and R . Leberman, FEBS (Fed. E w . Biochem. S o c . ) Lett. 73, 141 (1977). 63. H . Sugino, N. Sakabe, K. Sakabe, S. Hatano, F. Oosawa, T. Mikawa, and S. Ebashi, J . Biochem. (Tokyo) 86, 257 (1979). 64. G. Rohr and H. G. Mannherz, EJB 89, 151 (1978). 65. H. G. Mannherz and G . Rohr, FEBS (Fed. E r r . Eiochem. Soc.) Lett. 95, 284 (1978). 66. G . Rohr and H . G . Mannherz, FEBS (Fed. Eur. Eiochem. Soc.) Lett. 99, 351 (1979). 67. E. Grazi and E. Magri, FEES (Fed. Eur. Eiochem. S o c . ) Lett. 104, 284 (1979). 68. H. Weintraub and M. Groudine, Stience 193, 848 (1976). 69. A. Garel and R. Axel, PNAS 73, 3966 (1976). 70. B . Sollner-Webb and G. Felsenfeld, Cell 10, 537 (1977). 71. S. Weisbrod and H. Weintraub, PNAS 76, 630 (1979).
296
STANFORD MOORE
digestion by DNase can provide information on the structure of the nucleosome. High resolution electrophoretic separation of solubilized oligonucleotides has provided evidence (72-74) for a repetitive internal structure of the chromatin subunit. The periodicity in the fragment lengths can be correlated with helical and super helical orientation of DNA in the nucleosome core (75). In a study of the role of postsynthetic modification of histones in gene activation, Vidali et NI. (76) have used DNase I digestion to show that DNA is more readily liberated from chromatin that contains an increased quantity of acetylated histones; Kastern et al. (77) have used DNase coupled to Sepharose in the preparation of DNA-dependent RNA polymerase 11; the use of an immobilized enzyme makes it possible to release RNA polymerase from actively transcribing genes without having the product contaminated by DNase. The interaction of DNase and actin has formed the basis of a procedure for visualizing cellular microfilaments by fluorescent microscopy (58) and for a differential assay for monomeric and filamentous actin in cell extracts (59).
Thoroughly RNase-free DNase (13) has proved useful (78, 79) to digest chromatin in the preparation of nuclear RNA by the procedure of Penman (80).The protease-free nature of the same DNase preparation is of potential value in the isolation of nuclear proteins. On the basis that the process of replication in virus-infected cells is preceded by liberation of the viral nucleic acid from its protective protein coating, Trukhachev and Salganik (81) studied the inhibition of viral synthesis by DNase in cell cultures. In a clinical study, favorable results (82) have been reported from the local application of DNase to patients suffering from herpes infections of the eye. M . NOH, Nitcleic Acids Res. 1, 1573 (1974). L. C. Lutter, J M B 117, 53 (1977). L. C. Lutter, Nircleic Acids Res. 6, 41 (1979). A. Prunell, R. D. Kornberg, L. Lutter, A. Klug, M. Levitt, and F. H. C. Crick, Science 204, 855 (1979). 76. G. Vidali, L. C. Boffa. E. M . Bradbury, and V. G . Allfrey, /“AS 75, 2239 (1978). 77. W. H. Kastern, J. D. Eldridge, and K. P. Mullinix, JBC 254, 7368 (1979). 78. I. Tamm and T. Kikuchi, P N A S 76, 5750 (1979). 79. I. Tamm, T. Kikuchi, J. E. Darnell, Jr., and M. Salditt-Georgieff, Biockemisrry 19, 2743 (1980). 80. S. Penman, in “Fundamental Techniques in Virology” ( K. Habel and N. P. Salzman, eds.), p. 35. Academic Press, New York, 1969. 81. A . A. Trukhachev and R. 1. Salganik, Virology 33, 552 (1967). 82. A . A. Colain, R . 1. Salganik, I . E . Mikhailovskaya, and I . M. Gorban,Ann. h i / . 203, 371 (1970). 72. 73. 74. 75.