[7] Nucleases of E. coli

44

ENZYMES OF NUCLEIC ACID METABOLISM

[7]

one-third to one-fifth maximal activity is observed. This residual activity is completely eliminated by the addition to the reduction mixture of Versene (0.02M), which suggests that the enzyme has an absolute requirement for some cation and that the residual activity is the result of traces of the metal ion present in the enzyme preparation. Mg ~ cannot be effectively replaced by a number of other divalent cations. Thus Mn ÷÷ and Ca +* each, at concentrations of 3 X 10-4 or 6 X 10-8 M, completely abolish enzymatic activity. Zn ÷÷ gives 64% of maximal activity at a concentration of 3 X 10-4 M, and less than 10% at 6 X 10-3 M. pH Optimum. The pH optimum for the purified enzyme is between pH 9.2 and 9.8 (0.07 M glycine buffer). At pH 10.7 (0.07 M glycine) about 30% of optimal activity is observed, and at pH 7.5 (0.07 M Tris buffer), about 20% of optimal activity. Enzymatic activity is enhanced by phosphate ion. Thus at pH 8.5 (0.07 M glycine), where the activityis 60% of optimal, the addition of 0.07 M K2HPO, (pH 8.5) increases the activity to the optimal level.

[ 7] N u c l e a s e s of E. coli II. An RNA-Inhibitable Endonucleaae

By I. R. L~.HMAN Assay Method

Principle. Assay of the E. coli endonuclease measures the conversion of PSi-labeled, native DNA to acid-soluble products. 1 An alternative assay for the more-purified enzyme fractions (CM-cellulose 2 or Amberlite XE-64) measures the increase in optical density at 260 m~ accompanying hydrolysis of the DNA. Reagents Pa2-1abeled E. call DNA, 1 mieromole of P per milliliter, 2 to 3 ~c./micromole2 Tris buffer, pH 7.5, 1 M. MgC12, 0.1 M. Crystalline pancreatic RNase (Armour Laboratories), 5 mg./ml. 1I. R. Lehman, G. G. Roussos, and E. A. Pratt, J. Biol. Chem. 237, 819 (1962). ' CM-cellulose-earboxymethylcellulose. DEAE-cellulose--diethylaminoethylcellulose. E. A. Peterson and It. A. Sober, J. Am. Chem. Soc. 78, 751 (1951). ' I . R. Lehman, J. Biol. Chem. 235, 1479 (1960); see Vol. VI [6].

[7]

NVCL~.^SES OF E. coli--II. ENDONUCLEASE

45

Crystalline cytochromc c (Sigma Chemical Company), 1 mg./ml. "Carrier" DNA. Calf thymus DNA, 2.5 mg./ml., isolated by the method of K a y et al. 4 Perchloric acid, 0.5 N. KOH, 1 N. Enzyme. Dilute the enzyme in a solution composed of 0.05 M Tris buffer, pH 7.5, 0.25 M ammonium sulfate, and crystalline bovine plasma albumin (Armour Laboratories), 1 mg./ml. Procedure. To tubes containing 0.02 ml. each of Tris buffer, MgC12, and DNA is added 0.01 to 0.07 unit of enzyme. In assays of the crude extract 0.01 ml. of RNase is added to destroy the inhibitory RNA present in this fraction (see below). All subsequent fractions are free of this inhibitor; however, 0.01 ml. of RNase or 0.04 ml. of cytochrome c is added to stabilize the enzyme during incubation. The reaction mixture (brought up to 0.3 ml. with water) is incubated for 30 minutes at 37°; then 0.2 ml. of "carrier" DNA and 0.5 ml. of cold perchloric acid are added. After 5 minutes at 0 ° the resulting precipitate is removed by centrifugation, and 0.2 ml. of the supernatant fluid is pipetted into a planchet. A drop of K 0 H is added directly to the aliquot on the planchet ; the solution is taken to dryness, and the radioactivity is determined. The supernatant fluids obtained from control incubations, with enzyme omitted, usually contain 0.05 to 0.1% of the added radioactivity. The spectrophotometric assay is performed with a Beckman Model DU or a Zeiss Model PMQ II spectrophotometer, equipped with a thermospacer through which water at 37 ° is circulated from a constanttemperature bath. The components of the reaction mixture are the same as those used in the isotopic assay with the exception that 0.03 ml. of DNA (unlabeled) and 0.1 to 0.5 unit of enzyme are added. A blank euvette of high absorbancy is used to permit direct measurements of optical density at the relatively high concentration of DNA used. Readings of optical density at 260 m~ are taken at 1-minute intervals. The increase in optical density is proportional to the amount of enzyme added. Thus with 0.01, 0.02, and 0.04 ml. of a 1:20 dilution of the CM-cellulose fraction, the rates of increase in optical density are 0.0034, 0.0066, and 0.0138 per minute, respectively. Defidition o/ Isotope Assay Unit and Specific Activity. A unit of enzyme is defined as the amount causing the production of 0.10 micromole of acid-soluble p~2 in 30 minutes. The specific activity is expressed 4E. R. M. Kay, N. S. Simmons, and A. L. Dounce, J. Am. Chem. Soc. 74, 1724 (1952).

46

ENZYMES OF NUCLEIC ACID METABOLISM

[7]

as units per milligram of protein. Protein is determined by the method of Lowry et al2 Application of the Isotope Assay Method to Crude Tissue Preparations. In crude extracts of E. coli the radioactivity made acid-soluble is proportional to the enzyme concentration at levels of 0.005 to 0.07 unit of enzyme. Thus, with the addition of 0.005, 0.01, 0.02, and 0.04 ml. of a 1:200 dilution of crude E. coli extract, values of 208, 229, 220, and 223 units of enzyme per milliliter of extract, respectively, are obtained. Purification Procedure Unless otherwise indicated, all operations are carried out at 0 ° to 4 °. All centrifugations are at 15,000 X g for 10 minutes. Preparation of Extracts. E. coli strain B is grown as described in Vol. VI [5]. The cells (450 g. wet weight) are mixed with 300 ml. of potassium phosphate buffer, 0.15 M, pH 7.0, in a large Waring blendor equipped with a cooling jacket and connected to a Variac. Stirring is begun at approximately one-third of maximal speed. To the suspension are gradually added 1350 g. of acid-washed glass beads (Superbrite, average diameter 200 ~, obtained from the Minnesota Mining and Manufacturing Company). When the mixture appears homogeneous, an additional 1500 ml. of buffer are added, and the homogenization is continued for 20 minutes at maximal speed. During this period, the temperature of the mixture should not rise above 12 ° . After the homogenization approximately 10 minutes are required for the beads to settle out. The broken cell suspension is then centrifuged, and the supernatant fluid (approximately 2000 ml.) is collected (fraction I). Protamine Precipitation and Elution. To 800 ml. of extract are added, with stirring, 400 ml. of a 1% protamine sulfate solution. After standing for 10 minutes, the suspension is centrifuged. The precipitate is transferred to a Waring blendor regulated by means of a Variac and homogenized with 800 ml. of potassium phosphate buffer, 0.2 M, pH 7.4, for 5 minutes at one-fourth maximal speed. The suspension is centrifuged, and the resulting precipitate is homogenized with 800 ml. of potassium phosphate buffer, 0.5 M, pH 7.4, for l0 minutes, then centrifuged as before. To the supernatant fluid are added an equal volume of distilled water and 5 ml. of 0.5 M MgCI2. The diluted solution is incubated at 37 ° for 2 hours. A heavy precipitate of inactive protein which forms during incubation is removed by centrifugation (fraction II). Ammonium Sulfate Fractionation. To 1600 ml. of protamine eluate are added, with stirring, 565 g. of solid ammonium sulfate. The suspen50. It. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, as cited in Vol. I I I [73].

[7]

NUCLEASE8

OF E. coli--II. ENDONUCLEASE

47

sion is allowed to stand for 10 minutes after the ammonium sulfate has dissolved and then is centrifuged. To the supernatant solution are added, with stirring, an additional 512 g. of ammonium sulfate. The suspension is allowed to stand and then is centrifuged as before. The resulting precipitate is dissolved in 80 mh of potassium phosphate buffer, 0.02 M, pH 7.4. This solution is dialyzed for 12 hours against 75 vol. of potassium phosphate buffer, 0.01 M, pH 7.4. An inactive precipitate which forms on dialysis is removed by centrifugation (fraction III). DEAE-cellulose Fractionation. A column of DEAE-cellulose 2 (16 X 3.3 cm.) is prepared and equilibrated with 2 1. of potassium phosphate buffer, 0.01 M, pH 7.4. The dialyzed ammonium sulfate fraction is applied to the column at the rate of 80 ml./hr. The adsorbent is then washed with 130 ml. of potassium phosphate buffer, 0.01 M, pH 7.4. Approximately 80% of the activity applied to the column is recovered in the wash (fraction IV). CM-cellulose Chromatography. A column of CM-cellulose (16 X 2.2 cm.) is equilibrated with 1 h of potassium phosphate buffer, 0.01 M, pH 6.5. DEAE-cellulose fraction (126 ml.), to which 1.26 ml. of 1 M potassium dihydrogen phosphate has been added, is applied to the column at the rate of 40 ml./hr., and the adsorbent is washed with 25 ml. of potassium phosphate buffer, 0.01 M, pH 6.5. A linear gradient of elution is applied with 0.04 M and 0.2 M potassium phosphate at pH 7.4 as limiting concentrations. The total volume of the gradient is 600 ml. The flow rate is 48 ml./hr., and fractions are collected at 10-minute intervals. Approximately 90% of the activity is eluted in a sharp peak between 4.4 and 6.4 resin-bed volumes of effluent. The peak fractions which contain enzyme of specific activity greater than 3500 (approximately 60% of the total) are pooled (fraction V). This fraction is purified 600-fold over the starting extract and contains 14% of the activity initially present. It can be concentrated by lyophilization. The pooled CMcellulose fraction (64 ml.) is first dialyzed for 5 hours against two 4-1. changes of Tris buffer, 0.01 M, pH 7.5, then lyophilized, and finally taken up in 4 ml. of distilled water. Approximately 80% of tile activity in tile CM-cellulose fraction is recovered after dialysis and lyophilization. Further purification of the enzyme can be achieved by chromatography on Amberlite XE-64 (IRC-50).6 A column of Amberlite XE-64 (20.5 X 0.7 cm.) is washed with 1 1. of potassium phosphate buffer, 0.1 M, pH 6.48. CM-cellulose fraction (34 mh), which has been dialyzed for a period of 5 hours against the equilibrating buffer, is added at the rate of 10 mh/hr, and washed into the column with 5 ml. of this same t-mffer. A linear gradient from 0.3 M KCI to 1 M KCI is apl)lied, t)oth limiting *C. H. W. Hits, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953).

48

ENZYMES OF NUCLEIC ACID METABOLISM

[7]

solutions containing potassium phosphate buffer, 0.1 M, pH 7.4. The total gradient volume is 100 ml. The flow rate is maintained at 12 ml./ hr., and 2-ml. fractions are collected. Approximately 8 0 ~ of the activity initially applied to the column appears in a sharp peak midway through the gradient. This procedure removes most of the contaminating ribonuclease activity present in the CM-cellulose fraction. Such preparations are not significantly different in their physical properties or in their action on DNA from the CM-cellulose fraction. The crude extract has been stored at --20 ° for as long as 1 year with no loss in activity. Fractions II, III, and IV have shown no significant decrease in activity on storage at --20 ° for 6 months. Fraction VI loses activity rapidly on storage at --20°; however, no loss in activity could be detected when this fraction was kept at 0 ~ for a 4-month period. The lyophilized CM-cellulose fraction showed no decrease in activity when stored at --20 ° for 2 months. An apparent loss in activity of the CM-cellulose fraction has been observed on prolonged dialysis at 0 ° for 24 hours or more. This loss is probably due to passage of enzyme protein through the cellophane dialysis bag. After dialysis of the CM-cellulose fraction (1 ml. containing 50 /~g. of protein) against 50 vol. of Tris buffer, 0.02 M, pH 7.5, containing 0.1 rag. of cytochrome c per milliliter, for a 72-hour period, approximately 5% of the activity initially present in the dialysis bag was detected in the surrounding fluid. A summary of the purification procedure is given in the table. PURIFICATION OF E. coli ENDONUCLEASE

Fraction

Step

Units/ml.

Total units, X 10-3

I II III IV V

Extract Protamine eluate Ammonium sulfate DEAE-cellulose CM-cellulose

177 42 444 264 318

141 67 42 33 20

Specific activity, Protein, units/mg. m g . / m L protein 17.4 0.30 1.70 0.50 0.05

10.2 140 260 528 6360

Properties

Specificity. The E. coli endonuclease degrades native DNA at a sevenfold greater rate than thermally denatured DNA. It carries out an endonucleolytic attack on DNA; that is, it produces scissions at many points along the DNA chain, yielding a limit digest whose oligonueleo-

[8]

PREPARATION OF LAMB BRAIN PHOSPHODIESTERASE

49

tides have an average chain length of approximately 7; these are terminated by a 5'-phosphoryl group. The isolated oligonucleotides are neither cleaved by high concentrations of the enzyme, nor can they, when added in equal concentrations, inhibit the hydrolysis of D:NA. The enzyme is highly specific for DNA. A ribonuelease present in the CM-cellulose fraction can be removed by chromatography on Amberlite XE-64 (IRC-50) (see above). The purified enzyme (CM-cellulose fraction) is free of any detectable acid or alkaline nucleotidase activity. The enzyme exists in the crude extract bound to an inhibitory RNA which is removed during purification. The purified enzyme can be inhibited by a variety of RNA's from E. cell, guinea pig liver, and tobacco mosaic virus. Of the RNA's tested, the amino acid-accepter RNA from E. cell is the most active. The kinetics of the enzyme inhibited by the amino acid-accepter RNA obey the equations for a competitive inhibition, and the calculated Ks is of the order of 1 X 10.8M (RNAnucleotide) .7 Requirement ]or Divalent Cations. The purified enzyme (CM-cellulose fraction) requires added magnesium ion for maximal activity. No activity is detectable in its absence. Under the conditions of the assay, maximal activity is observed at a Mg ÷÷ concentration of 7 X 10-3 M. At 1 X 10.3M and 3.4 X 10.2M, 66% and 50%, respectively, of maximal activity is observed. Magnesium ion can, to some extent, be replaced by manganous ion. Thus, at 7 X 10-3 M Mn ~, the activity is 57% of that observed with an equal concentration of Mg ÷÷. Ca ÷÷ and Zn ÷÷ are completely ineffective in replacing Mg ÷÷. No activity is observed when these ions are added at 3 X 10-4M, 1.5 X 10_3 M, or 7 X 10-3M. pH Optimum. The pH optimum for the purified enzyme with Tris buffer extends from 7.5 to 8.5. ;I. R. Lehman, G. G. Roussos, and E. A. Pralt, J. Bi~l Chem. 237, 829 (1962).

[8] P r e p a r a t i o n o f L a m b B r a i n P h o s p h o d i e s t e r a s e By J. W. HEALY, D. STOLLAR, and L. LEVINE

Assay Method Principle. The immunological assay method is based on the determination of denatured DNA serologically before and after treatment with the phosphodiesterase. Antibodies produced in rabbits to T-even