JouRNAL oF BACRIoLOGY, May 1975, p. 599-606 Copyright ) 1975 American Society for Microbiology
Vol. 122, No. 2 Printed in U.S.A.
Purification and Properties of Streptococcal Nicotinamide Adenine Dinucleotide Glycohydrolase PHYLLIS S. GRUSHOFF, S. SHANY, I AND ALAN W. BERNHEIMER* Department of Microbiology, New York University School of Medicine, New York, New York 10016 Received for publication 13 November 1974
Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 x 10-4 mM. It was inhibited by a-iodoacetamide.
Many strains of streptococci belonging to groups A, C, and G produce an extracellular enzyme that catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD) at the linkage between nicotinamide and ribose (5). Earlier attempts to purify this enzyme resulted in preparations contaminated with other substances (18, 8). Recently, Shany et al. (20) described the separation of nicotinamide adenine dinucleotide glycohydrolase (NADase) (EC 3.2.2.5) and streptolysin 0 (SLO) in crude supernatant fluids, and they showed that hemolytic and NADase activities were two distinct activities attributable to two different proteins. In the present report an improved method for purification of streptococcal NADase is described. The relatively homogeneous preparations obtained were used for partial characterization of the enzyme. MATERIALS AND METHODS Production of streptococcal extracellular proteins. The C203U mutant strain of group A Strepto-
Bernheimer and Grushoff (2). One hemolytic unit is defmed as the smallest amount of lysin required to produce 50% lysis of 1 ml of a 0.7% erythrocyte suspension upon incubation at 37 C for 30 min. Prior to titration, SLO was activated by mixing an equal volume of 0.1 M dithiothreitol (Nutritional Biochemicals Corp., Cleveland, Ohio) dissolved in 0.067 M sodium phosphate, pH 7.4, and by allowing the mixture to stand for 15 min at room temperature. NADaw activity. The assay for NADase was performed by the cyanide method of Kaplan et al. (11) as modified by Carlson et al. (5). A unit of enzyme activity is defined as the amount of enzyme which destroys 1.0 jmol of NAD in 1.0 min at 37 C. The assay is linear for quantities of enzyme between 0.2 and 2 U/ml. The amount of NAD hydrolyzed was also found to increase linearly with time from 0 to 90 min.
Specific activity. The specific activity of NADase was expressed as units of NADase per mg of protein. Proteolytic activity. Streptococcal proteinase was
assayed by the method described by Kunitz (13) for the assay of trypsin with casein as a substrate. The proteinase was activated by the method of Liu et al. (15), but with dithiothreitol as the reducing agent. coccus pyogenes was grown by the method of Shany et One-half milliliter of test solution was incubated with al. (20). The supernatant fluid containing hemolytic 0.5 ml of 0.3 M dithiothreitol in 0.1 M phosphate activity (streptolysin 0), NADase activity, and pro- buffer, pH 7.6, for 30 min at 37 C. One milliliter of 1% teolytic activity was brought to 80% saturation by the casein (Fisher Scientific Co., Springfield, N.J.) in 0.1 addition of solid ammonium sulfate. The precipitate M phosphate buffer, pH 7.6, was added to 1-ml was recovered by centrifugation, collected in approxi- dilutions of activated enzyme. The reaction was mately 100 ml of 80% saturated ammonium sulfate, carried out at 37 C and stopped after 20 min by addition of 3 ml of 5% trichloroacetic acid. Appropriand stored at 4 C. Hemolytic activity. Hemolytic activity was as- ate blanks were included. After 1 h at room temperasayed with washed rabbit erythrocytes as described ture, the reaction mixtures were filtered and the by Bemheimer and Schwartz (3) and modified by absorbance of the filtrate was measured in a Beckman DU spectrophotometer. Proteolytic activity was ex'Present address: The Soroka Medical Center, Depart- pressed as units of absorbance at 280 nm. ment of Nephrology, Beer-Sheba, Israel. Protein estimation. Protein in the culture super599
600
GRUSHOFF, SHANY, AND BERNHEIMER
natant fluid and in subsequent stages of purification was estimated by the method of Lowry et al. (16), and also by determination of absorbance at 280 nm. Comparison was made with a standard curve with bovine plasma albumin (Armour Pharmaceutical Co., Chicago, Ill.). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Degree of purification was determined by the sodium dodecyl sulfate/polyacrylamide gel electrophoresis method of Weber and Osborn (23) under the conditions described by Shany et al. (20). Blocking of SLO and streptococcal proteinase by tetrathionate, and reactivation. The sulfhydryl groups of SLO and those of streptococcal proteinase were blocked by disodium tetrathionate by using a procedure based on the method described by Liu (14) and Fischetti et al. (9), as modified by Shany et al. (19) for blocking the sulfhydryl groups of cereolysin. Under these conditions nearly all the hemolytic and proteolytic activities were blocked, while the NADase activity was unaffected. Reactivation of streptococcal proteinase was achieved as described above for activation. SLO was reactivated similarly but by incubating at room temperature for 15 min. Gel filtration chromatography. NADase was filtered through beds of Sephadex G-100 superfine (Pharmacia Fine Chemicals, Piscataway, N.J.) equilibrated with 0.1 M sodium phosphate buffer, pH 7.0, containing 0.25 M sodium chloride in a watercooled K26/40 column (Pharmacia). Fractions were collected in a Buchler (Fort Lee, N.J.) refrigerated fraction collector. Molecular weight was estimated by the method of Andrews (1) with bovine plasma albumin, ovalbumin (Sigma Chemical Corp., St. Louis, Mo.), and soy bean trypsin inhibitor (Worthington Biochemical Corp., Freehold, N.J.) as standards. To determine elution volumes of enzymes, fractions were assayed for NADase activity and for absorbance at 280 nm. Amino acid analysis. The amino acid composition of purified NADase was determined by the method of Smith and Stockell (22). Samples of carboxymethylSephadex purified NADase were hydrolyzed for 20 h at 110 C with 6 N HCl in evacuated, sealed tubes, and amino acid analyses were performed with a Beckman Model 120 C amino acid analyzer. DL-P-2-Thienyl alanine was taken as the internal standard, as in the method of Siegel and Roach (21). Corrections for the values of serine, threonine, and cysteic acid were made by the method of Blackbum (4). Cysteine and methionine were determined as cysteic acid and methionine sulfone by the performic acid oxidation method described by Moore (17). The tryptophan content was determined by the spectrophotometric method of Edelhoch (6).
J. BACTEPIOL.
and at 37 C for 0.5, 1.0, 2.0, 4.0, and 24 h in the presence of 0.1 M dithiothreitol, and assayed for NADase, hemolytic, and proteolytic activities. The NADase and hemolytic activities decreased with time while the proteolytic activity increased (Fig. 1A). These effects were accelerated at 37 C (Fig.- 1B). Purification procedure. To prevent proteolysis of NADase and SLO by streptococcal proteinase, the crude streptococcal preparation was treated with disodium tetrathionate. Ten milliliters of ammonium sulfate precipitate (step 2), which represented 1,610 ml of streptococcal culture supernatant fluid (step 1; Table 1), was dialyzed against several changes of 0.2 M phosphate buffer, pH 7.4, for 24 h. The dialysate was reduced by adding an equal volume of 0.1 M dithiothreitol dissolved in the same buffer and allowed to stand for 15 min at 4 C. Blocking was carried out either by adding an equal volume of 0.2 M disodium tetrathionate (kindly supplied by V. Fischetti) dissolved in the same buffer or by adding solid disodium tetrathionate to the reduced preparation to a final concentration of 0.1 M. After 1 h of gentle stirring at room temperature, the mixture was concentrated to 21.3 ml by ultrafiltration with an Amicon PM 10 membrane (Amicon Corp., Lexington, Mass.) under nitrogen pressure (step 3). The excess disodium tetrathionate, which accounts for the low hemolytic and proteolytic activities of step 3, was removed by gel filtration with Sephadex G-25 (Pharmacia) equilibrated in 0.02 M phosphate buffer, pH 7.4, in a water-cooled K16/40 column (70-ml bed volume) (step 4). Whatman wet microgranular diethylaminoethyl (DEAE)-cellulose (DE 52) was suspended in 0.02 M tris(hydroxymethyl)aminomethanehydrochloride (Sigma 7-9, Sigma Chemical Co.), pH 7.4, packed in a water-cooled K26/40 column (180-ml bed volume) (Pharmacia) and equilibrated with the same buffer. After dialysis against the same buffer, the tetrathionate8
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002 RESULTS 01 40 40 10 20 Demonstration of proteolytic activity. A 10 20 TIME Hows) sample of extracellular streptococcal proteins that had been precipitated with ammonium FIG. 1. Proteolytic activity, NADase, and hemosulfate was dialyzed overnight against 0.02 M lytic activity as functions of time in the presence of phosphate, pH 6.8. Samples of the dialyzed dithiothreitol. Panels: (A) at room temperature; and solution were incubated at room temperature (B) at 37 C. 24
24
VOL. 122, 1975
STREPTOCOCCAL NADase 00
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treated streptococcal preparation eluted from gel filtration (36 ml) was added to the column and washed. Fractions (10 ml) were collected and assayed for absorbance at 280 nm, and NADase and hemolytic activities. The NADase peak (step 5, Table 1; Fig. 2) fractions were combined and assayed for NADase, hemolytic, and proteolytic activities; protein was determined by absorbance at 280 nm and by the Lowry method. Although the specific activity of NADase had increased some 700-fold, hemolytic and proteolytic activities could still be detected. The NADase peak eluted from the DEAE-cellulose column was dialyzed against 0.02 M phosphate buffer, pH 6.0 (step 6), and submitted to further purification on a carboxymethylSephadex (Pharmacia) K26/40 column equilibrated in the same buffer. After the column was washed with approximately 100 ml of equilibrating buffer, a gradient elution was carried out by use of an upper reservoir containing 0.02 M phosphate buffer, pH 6.0, and 0.6 M NaCl which fed into a constant mixing chamber containing 300 ml of 0.02 M phosphate buffer, pH 6.0. Fractions (5 ml) were collected and assayed for absorbance at 280 nm, and NADase and hemolytic activities. Fractions of each protein peak (Fig. 3) were combined and assayed for the above activities as well as for proteolytic activity and protein content by the Lowry method. From this last purification step NADase (peak III; step 7a) was obtained in highly purified form with a specific activity of 11,200 NADase units per mg of protein. No hemolytic or proteolytic activity could be detected. Peak II (step 7b) contained some hemolytic activity, proteolytic activity and traces of NADase activ-
J. BACTOL.
ity, whereas peak I (step 7c) showed only proteolytic activity. Assessment of purification on SDS/polyacrylamide gel electrophoresis. Increased purity of NADase was demonstrated at different stages by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Fig. 4A to 4E). A sample from crude ammonium sulfate dialysate gave rise to numerous bands (Fig. 4A), while the NADase peak obtained from DEAE-cellulose chromatography showed three major bands and several minor ones (4B). Figures 4C to 4E represent peaks I, II, and III from carboxymethylSephadex chromatography: proteinase zymogen (4C), active proteinase (4D), and purified NADase (4E). A single band was observed for NADase. Molecular weight estimation by gel filtra-
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VoL. 122, 1975
tion. The elution volume of the peak of NADase activity corresponded to a molecular weight of 50,000 4 2000 when determined by gel filtration by the method of Andrews ([1 ]; see Table 3). Amino acid composition. The results of the amino acid analysis are given in Table 2. The molecular weight, calculated by summation, assuming 10 histidine residues per mole, is 54,300. This is in agreement with the value obtained here by gel filtration and with the molecular weight obtained by sodium dodecyl sulfate/gel electrophoresis by Shany et al. ( [19]; Table 3). Effect of temperature on NADase activity. To determine the temperature at which NADase was most active, a sample of purified NADase (peak III from carboxymethylSephadex) was assayed as described under Materials and Methods but at different temperatures ranging from 0 to 60 C. NADase was active between 35 and 45 C with maximum enzyme activity at 40 C. TABLE 2. Amino acid composition of streptococcal NADase Amino acid
603
STREPTOCOCCAL NADase
number Bestofwhole residues
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
48 10 14 62 29 48 53 12 45 34 4 31 11 20 27 11 15 10
Total amino acids (% by weight)
11.33 2.52 4.02 13.14 5.40 7.70 12.60 2.14 4.73 4.45 0.82 5.65 2.65 6.04 5.63 3.30 4.06 3.72
Effect of pH on NADase activity. Purified NADase was assayed at different pH levels from 5 to 8.5 to determine the optimum pH for NADase activity. In most instances the assays were run in phosphate buffer (pH 6.0 to 7.5); for alkaline pH tris(hydroxymethyl)aminomethane-hydrochloride buffer was used, and for the lower pH assays acetate buffer was used. The enzyme was active from pH 6.5 to 8.0; the optimum pH was 7.3. Effect of substrate concentration on rate of NAD breakdown. The effect of various concentrations of NAD on the catalytic activity of streptococcal NADase was studied. The reaction was carried out with a constant concentration of purified NADase (1.9 units/ml) under the same conditions as for NADase determination. A Lineweaver-Burk plot is shown in Fig. 5. The Km calculated from these results is 5.1 x 10-4 mM. Inhibition of NADase by iodoacetamide. Twenty microliters of diluted purified NADase (4.64 units/ml) was incubated with 10, 20, 40, and 80 Asl of 0.6 M a-iodoacetamide. Volumes were made to 0.1 ml with 0.1 M potassium phosphate, pH 7.3. Incubation was carried out for 10 min at room temperature, followed by assay for NADase activity. The effect of various concentrations of a-iodoacetamide on NADase activity is shown in Fig. 6. (IO4 UNTS/MpUML.)
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TABLE 3. Molecular weight of streptococcal NADase Method
Sodium dodecyl sulfate/gel electrophoresis Summation of amino acid residues Gel filtration See reference 20. b See reference 8.
a
Molecular weight estimations
55,000a 54,300
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604
GRUSHOFF, SHANY, AND BERNHEIMER
DISCUSSION Enzymes similar to the NADase studied here have been found in both animal (10) and plant (12) tissues as well as in microorganisms. The microbial enzymes that have been investigated in some detail include that from Neurospora crassa (11) as well as that from the C203S strain of group A S. pyogenes (5), from the C203U mutant of the same strain (19) and from group C streptococci (7, 8). In the present study, pure NADase devoid of hemolytic activity was obtained. It had a specific activity of 11,200 NADase units per mg of protein, which is comparable with that (5,330 NADase units per mg of protein) of the most active preparation described by Pace and Pappenheimer (18), in their purification of NADase from group C streptococci. Although extensive purification was achieved by these authors it is not clear whether other streptococcal proteins were still detectable in the final .preparation. Fehrenbach (8) obtained crystalline material having a specific activity of 2,400 units per mg of protein and possessing hemolytic activity. The procedure described in this paper for the purification of NADase utilizes a methodology similar to that used by Fischetti et al. (9) for the stabilization and purification of the phageassociated lysin of group C streptococci. In our preparation, the blocking of sulfhydryl groups by disodium tetrathionate renders the streptococcal proteinase inactive, thus preventing proteolysis during the purification procedure. The subsequent use of two-step ion-exchange chromatography yields a sharp, pure peak of NADase activity. The reproducibility of this procedure is excellent, and yields of approximately 44% have been consistently achieved in several experiments. Purification steps monitored by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicate that NADase is restricted to one protein band. Several properties of purified NADase were studied. The molecular weight of NADase, determined in an earlier work by Shany et al. (21) using sodium dodecyl sulfate/gel electrophoresis and in the present work from calculation of amino acid residues and gel filtration studies (Table 3), was in agreement with the value obtained by Fehrenbach (7) using gel filtration (56,000 4 5000). Carlson et al. (5) described streptococcal NADase as a nondialyzable, heat-labile protein having optimal activity between pH 7.2 to 7.8. With purified enzyme, the optimum pH for NADase activity is 7.3 and the enzyme is active from pH 6.5 to 8.0. This is in agreement with the narrow range obtained by the above authors,
J. BACTERIOL.
but contrasts with the broad range of activity with respect to pH obtained by N. crassa (11). The temperature at which the purified enzyme appears to be most active is 40 C, but substantial activity is demonstrable also at 35 C and 45 C. The value of the Km found in the present study, 5.1 x 10-4 mM, is identical to the Km of 5 x 10-4 mM reported for Neurospora NADase (11). In view of this and of the fact that neither the streptococcal nor the Neurospora enzyme hydrolyzes the reduced form of NAD, it would appear that the two enzymes are rather similar. Analysis of the streptococcal enzyme revealed four half-cystines (Table 2), and since the enzyme is inhibited by a-iodoacetamide it is possible that at least two, perhaps four, halfcysteines are present in reduced form, that is, as cysteine which may be part of the active site. The availability of a method for obtaining streptococcal NADase in pure form may facilitate study of the biological activities of the enzyme, of its role in oxidative metabolism and of its possible significance in streptococcal infections. ACKNOWLEDGMENTS The authors are grateful to Maria Heincz and Charles Harman for assistance with the amino acid analyses. This investigation was supported by Public Health Service grant AI-02874 from the National Institute of Allergy and Infectious Diseases and by Public Health Career Program award 5K06-AI-14-198 to A.W.B. LITERATURE CITED 1. Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J.
91:222-233. 2. Bernheimer, A. W., and P. Grushoff. 1967. Cereolysin: 3. 4.
5.
6. 7.
8.
9.
production, purification and partial characterization. J. Gen. Microbiol. 46:143-150. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolation and composition of staphylococcal alpha toxin. J. Gen. Microbiol. 30:455-468. Blackburn, S. 1968. Amino acid determination, methods and techniques, p. 11-29. Marcel Dekker Inc., New York. Carlson, A. S., A. Kellner, A. W. Bernheimer, and E. B. Freeman. 1957. A streptococcal enzyme that acts specifically upon diphosphopyridine nucleotide: characterization of the enzyme and its separation from streptolysin 0. J. Exp. Med. 106:15-26. Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948-1954. Fehrenbach, F. J. 1969. Gel-filtration behavior and molecular weight of NAD-glycohydrolase (EC 3.2.2.5) from streptococci in column chromatography on Sephadex gels. J. Chromatogr. 41:43-52. Fehrenbach, F. J. 1971. Reinigung und kristallisation der NAD-glykohydrolase aus C-streptokokken. Eur. J. Biochem. 18:94-102. Fischetti, V. A., E. C. Gotschlich, and A. W. Bernheimer. 1971. Purification and physical properties of group C streptococcal phage-associated lysin. J. Exp. Med. 133:1105-1117.
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STREPITOCO(CCAL NADase
10. Handler, P., and J. R. Klein. 1942. The inactivation of pyridine nucleotides by animal tissues in vitro. J. Biol. Chem. 143:49-57. 11. Kaplan, N. O., S. P. Colowick, ard A. Nason. 1951. Neurospora diphosphopyridine nucleotidase. J. Biol. Chem. 191:473-483. 12. Kornberg, A., and W. E. Pricer, Jr. 1950. Nucleophosphatase. J. Biol. Chem. 182:763-778. 13. Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. 2. General properties. J. Gen. Physiol. 30:291-310. 14. Liu, T. Y. 1967. Demonstration of the presence of a histidine residue at the active site of streptococcal proteinase. J. Biol. Chem. 242:4020-4032. 15. Liu, T. Y., N. P. Neumann, S. D. Elliot, S. Moore, and W. H. Stein. 1963. Chemical properties of streptococcal proteinase and its zymogen. J. Biol. Chem. 238:251-256. 16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. 17. Moore, S. 1963. On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235-237.
605
18. Pace, M. G., and A. M. Pappenheimer, Jr. 1959. An immunochemical study of streptococcal diphosphopyridine nucleotidase. J. Immunol. 83:83-86. 19. Shany, S., A. W. Bernheimer, P. S. Grushoff, and Kwang-Shin Kim. 1974. Evidence for membrane cholesterol as the common binding site for cereolysin, streptolysin 0 and saponin. Mol. Cell. Biochem. 3:179-186. 20. Shany, S., P. S. Grushoff, and A. W. Bernheimer. 1973. Physical separation of streptococcal nicotinamide adenine dinucleotide glycohydrolase from streptolysin 0. Infect. Immun. 7:731-734. 21. Siegel, F. L., and M. K. Roach. 1961. fl-2-Thienyl-DLalanine, internal standard for automatic determination of amino acids. Anal. Chem. 33:1628. 22. Smith, E. L., and A. Stockell. 1954. Amino acid composition of crystalline carboxypeptidase. J. Biol. Chem. 207:501-514. 23. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl-sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
Vol. 122, No. 2 Printed in U.S.A.
Purification and Properties of Streptococcal Nicotinamide Adenine Dinucleotide Glycohydrolase PHYLLIS S. GRUSHOFF, S. SHANY, I AND ALAN W. BERNHEIMER* Department of Microbiology, New York University School of Medicine, New York, New York 10016 Received for publication 13 November 1974
Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 x 10-4 mM. It was inhibited by a-iodoacetamide.
Many strains of streptococci belonging to groups A, C, and G produce an extracellular enzyme that catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD) at the linkage between nicotinamide and ribose (5). Earlier attempts to purify this enzyme resulted in preparations contaminated with other substances (18, 8). Recently, Shany et al. (20) described the separation of nicotinamide adenine dinucleotide glycohydrolase (NADase) (EC 3.2.2.5) and streptolysin 0 (SLO) in crude supernatant fluids, and they showed that hemolytic and NADase activities were two distinct activities attributable to two different proteins. In the present report an improved method for purification of streptococcal NADase is described. The relatively homogeneous preparations obtained were used for partial characterization of the enzyme. MATERIALS AND METHODS Production of streptococcal extracellular proteins. The C203U mutant strain of group A Strepto-
Bernheimer and Grushoff (2). One hemolytic unit is defmed as the smallest amount of lysin required to produce 50% lysis of 1 ml of a 0.7% erythrocyte suspension upon incubation at 37 C for 30 min. Prior to titration, SLO was activated by mixing an equal volume of 0.1 M dithiothreitol (Nutritional Biochemicals Corp., Cleveland, Ohio) dissolved in 0.067 M sodium phosphate, pH 7.4, and by allowing the mixture to stand for 15 min at room temperature. NADaw activity. The assay for NADase was performed by the cyanide method of Kaplan et al. (11) as modified by Carlson et al. (5). A unit of enzyme activity is defined as the amount of enzyme which destroys 1.0 jmol of NAD in 1.0 min at 37 C. The assay is linear for quantities of enzyme between 0.2 and 2 U/ml. The amount of NAD hydrolyzed was also found to increase linearly with time from 0 to 90 min.
Specific activity. The specific activity of NADase was expressed as units of NADase per mg of protein. Proteolytic activity. Streptococcal proteinase was
assayed by the method described by Kunitz (13) for the assay of trypsin with casein as a substrate. The proteinase was activated by the method of Liu et al. (15), but with dithiothreitol as the reducing agent. coccus pyogenes was grown by the method of Shany et One-half milliliter of test solution was incubated with al. (20). The supernatant fluid containing hemolytic 0.5 ml of 0.3 M dithiothreitol in 0.1 M phosphate activity (streptolysin 0), NADase activity, and pro- buffer, pH 7.6, for 30 min at 37 C. One milliliter of 1% teolytic activity was brought to 80% saturation by the casein (Fisher Scientific Co., Springfield, N.J.) in 0.1 addition of solid ammonium sulfate. The precipitate M phosphate buffer, pH 7.6, was added to 1-ml was recovered by centrifugation, collected in approxi- dilutions of activated enzyme. The reaction was mately 100 ml of 80% saturated ammonium sulfate, carried out at 37 C and stopped after 20 min by addition of 3 ml of 5% trichloroacetic acid. Appropriand stored at 4 C. Hemolytic activity. Hemolytic activity was as- ate blanks were included. After 1 h at room temperasayed with washed rabbit erythrocytes as described ture, the reaction mixtures were filtered and the by Bemheimer and Schwartz (3) and modified by absorbance of the filtrate was measured in a Beckman DU spectrophotometer. Proteolytic activity was ex'Present address: The Soroka Medical Center, Depart- pressed as units of absorbance at 280 nm. ment of Nephrology, Beer-Sheba, Israel. Protein estimation. Protein in the culture super599
600
GRUSHOFF, SHANY, AND BERNHEIMER
natant fluid and in subsequent stages of purification was estimated by the method of Lowry et al. (16), and also by determination of absorbance at 280 nm. Comparison was made with a standard curve with bovine plasma albumin (Armour Pharmaceutical Co., Chicago, Ill.). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Degree of purification was determined by the sodium dodecyl sulfate/polyacrylamide gel electrophoresis method of Weber and Osborn (23) under the conditions described by Shany et al. (20). Blocking of SLO and streptococcal proteinase by tetrathionate, and reactivation. The sulfhydryl groups of SLO and those of streptococcal proteinase were blocked by disodium tetrathionate by using a procedure based on the method described by Liu (14) and Fischetti et al. (9), as modified by Shany et al. (19) for blocking the sulfhydryl groups of cereolysin. Under these conditions nearly all the hemolytic and proteolytic activities were blocked, while the NADase activity was unaffected. Reactivation of streptococcal proteinase was achieved as described above for activation. SLO was reactivated similarly but by incubating at room temperature for 15 min. Gel filtration chromatography. NADase was filtered through beds of Sephadex G-100 superfine (Pharmacia Fine Chemicals, Piscataway, N.J.) equilibrated with 0.1 M sodium phosphate buffer, pH 7.0, containing 0.25 M sodium chloride in a watercooled K26/40 column (Pharmacia). Fractions were collected in a Buchler (Fort Lee, N.J.) refrigerated fraction collector. Molecular weight was estimated by the method of Andrews (1) with bovine plasma albumin, ovalbumin (Sigma Chemical Corp., St. Louis, Mo.), and soy bean trypsin inhibitor (Worthington Biochemical Corp., Freehold, N.J.) as standards. To determine elution volumes of enzymes, fractions were assayed for NADase activity and for absorbance at 280 nm. Amino acid analysis. The amino acid composition of purified NADase was determined by the method of Smith and Stockell (22). Samples of carboxymethylSephadex purified NADase were hydrolyzed for 20 h at 110 C with 6 N HCl in evacuated, sealed tubes, and amino acid analyses were performed with a Beckman Model 120 C amino acid analyzer. DL-P-2-Thienyl alanine was taken as the internal standard, as in the method of Siegel and Roach (21). Corrections for the values of serine, threonine, and cysteic acid were made by the method of Blackbum (4). Cysteine and methionine were determined as cysteic acid and methionine sulfone by the performic acid oxidation method described by Moore (17). The tryptophan content was determined by the spectrophotometric method of Edelhoch (6).
J. BACTEPIOL.
and at 37 C for 0.5, 1.0, 2.0, 4.0, and 24 h in the presence of 0.1 M dithiothreitol, and assayed for NADase, hemolytic, and proteolytic activities. The NADase and hemolytic activities decreased with time while the proteolytic activity increased (Fig. 1A). These effects were accelerated at 37 C (Fig.- 1B). Purification procedure. To prevent proteolysis of NADase and SLO by streptococcal proteinase, the crude streptococcal preparation was treated with disodium tetrathionate. Ten milliliters of ammonium sulfate precipitate (step 2), which represented 1,610 ml of streptococcal culture supernatant fluid (step 1; Table 1), was dialyzed against several changes of 0.2 M phosphate buffer, pH 7.4, for 24 h. The dialysate was reduced by adding an equal volume of 0.1 M dithiothreitol dissolved in the same buffer and allowed to stand for 15 min at 4 C. Blocking was carried out either by adding an equal volume of 0.2 M disodium tetrathionate (kindly supplied by V. Fischetti) dissolved in the same buffer or by adding solid disodium tetrathionate to the reduced preparation to a final concentration of 0.1 M. After 1 h of gentle stirring at room temperature, the mixture was concentrated to 21.3 ml by ultrafiltration with an Amicon PM 10 membrane (Amicon Corp., Lexington, Mass.) under nitrogen pressure (step 3). The excess disodium tetrathionate, which accounts for the low hemolytic and proteolytic activities of step 3, was removed by gel filtration with Sephadex G-25 (Pharmacia) equilibrated in 0.02 M phosphate buffer, pH 7.4, in a water-cooled K16/40 column (70-ml bed volume) (step 4). Whatman wet microgranular diethylaminoethyl (DEAE)-cellulose (DE 52) was suspended in 0.02 M tris(hydroxymethyl)aminomethanehydrochloride (Sigma 7-9, Sigma Chemical Co.), pH 7.4, packed in a water-cooled K26/40 column (180-ml bed volume) (Pharmacia) and equilibrated with the same buffer. After dialysis against the same buffer, the tetrathionate8
A
09
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002 RESULTS 01 40 40 10 20 Demonstration of proteolytic activity. A 10 20 TIME Hows) sample of extracellular streptococcal proteins that had been precipitated with ammonium FIG. 1. Proteolytic activity, NADase, and hemosulfate was dialyzed overnight against 0.02 M lytic activity as functions of time in the presence of phosphate, pH 6.8. Samples of the dialyzed dithiothreitol. Panels: (A) at room temperature; and solution were incubated at room temperature (B) at 37 C. 24
24
VOL. 122, 1975
STREPTOCOCCAL NADase 00
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602
GRUSHOFF, SHANY, AND BERNHEIMER
treated streptococcal preparation eluted from gel filtration (36 ml) was added to the column and washed. Fractions (10 ml) were collected and assayed for absorbance at 280 nm, and NADase and hemolytic activities. The NADase peak (step 5, Table 1; Fig. 2) fractions were combined and assayed for NADase, hemolytic, and proteolytic activities; protein was determined by absorbance at 280 nm and by the Lowry method. Although the specific activity of NADase had increased some 700-fold, hemolytic and proteolytic activities could still be detected. The NADase peak eluted from the DEAE-cellulose column was dialyzed against 0.02 M phosphate buffer, pH 6.0 (step 6), and submitted to further purification on a carboxymethylSephadex (Pharmacia) K26/40 column equilibrated in the same buffer. After the column was washed with approximately 100 ml of equilibrating buffer, a gradient elution was carried out by use of an upper reservoir containing 0.02 M phosphate buffer, pH 6.0, and 0.6 M NaCl which fed into a constant mixing chamber containing 300 ml of 0.02 M phosphate buffer, pH 6.0. Fractions (5 ml) were collected and assayed for absorbance at 280 nm, and NADase and hemolytic activities. Fractions of each protein peak (Fig. 3) were combined and assayed for the above activities as well as for proteolytic activity and protein content by the Lowry method. From this last purification step NADase (peak III; step 7a) was obtained in highly purified form with a specific activity of 11,200 NADase units per mg of protein. No hemolytic or proteolytic activity could be detected. Peak II (step 7b) contained some hemolytic activity, proteolytic activity and traces of NADase activ-
J. BACTOL.
ity, whereas peak I (step 7c) showed only proteolytic activity. Assessment of purification on SDS/polyacrylamide gel electrophoresis. Increased purity of NADase was demonstrated at different stages by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Fig. 4A to 4E). A sample from crude ammonium sulfate dialysate gave rise to numerous bands (Fig. 4A), while the NADase peak obtained from DEAE-cellulose chromatography showed three major bands and several minor ones (4B). Figures 4C to 4E represent peaks I, II, and III from carboxymethylSephadex chromatography: proteinase zymogen (4C), active proteinase (4D), and purified NADase (4E). A single band was observed for NADase. Molecular weight estimation by gel filtra-
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FIG. 3. Carboxymethyl-Sephadex chromatography of DEAE cellulose peak I. Fractions (5 ml) were assayed for NADase activity (0), hemolytic activity (0), and absorbance at 280 nm (A). i
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FIG. 4. SDS/gel electrophoresis of NADase at variof purification. Crude ammonium sulfate dialysate (A), DEAE-cellulose peak I (B), carboxymethyl-Sephadex peak I (C), carboxymethylSephadex peak H (D), carboxymethyl-Sephadex peak III, purified NADase (E). ous stages
FIG.
2. DEAE-cellulose
chromatography of
tetra-
thionate-treated dialysate. Fractions (10 ml) were assayed for NADase activity (0), hemolytic activity (O), and absorbance at 280 nm (A).
VoL. 122, 1975
tion. The elution volume of the peak of NADase activity corresponded to a molecular weight of 50,000 4 2000 when determined by gel filtration by the method of Andrews ([1 ]; see Table 3). Amino acid composition. The results of the amino acid analysis are given in Table 2. The molecular weight, calculated by summation, assuming 10 histidine residues per mole, is 54,300. This is in agreement with the value obtained here by gel filtration and with the molecular weight obtained by sodium dodecyl sulfate/gel electrophoresis by Shany et al. ( [19]; Table 3). Effect of temperature on NADase activity. To determine the temperature at which NADase was most active, a sample of purified NADase (peak III from carboxymethylSephadex) was assayed as described under Materials and Methods but at different temperatures ranging from 0 to 60 C. NADase was active between 35 and 45 C with maximum enzyme activity at 40 C. TABLE 2. Amino acid composition of streptococcal NADase Amino acid
603
STREPTOCOCCAL NADase
number Bestofwhole residues
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
48 10 14 62 29 48 53 12 45 34 4 31 11 20 27 11 15 10
Total amino acids (% by weight)
11.33 2.52 4.02 13.14 5.40 7.70 12.60 2.14 4.73 4.45 0.82 5.65 2.65 6.04 5.63 3.30 4.06 3.72
Effect of pH on NADase activity. Purified NADase was assayed at different pH levels from 5 to 8.5 to determine the optimum pH for NADase activity. In most instances the assays were run in phosphate buffer (pH 6.0 to 7.5); for alkaline pH tris(hydroxymethyl)aminomethane-hydrochloride buffer was used, and for the lower pH assays acetate buffer was used. The enzyme was active from pH 6.5 to 8.0; the optimum pH was 7.3. Effect of substrate concentration on rate of NAD breakdown. The effect of various concentrations of NAD on the catalytic activity of streptococcal NADase was studied. The reaction was carried out with a constant concentration of purified NADase (1.9 units/ml) under the same conditions as for NADase determination. A Lineweaver-Burk plot is shown in Fig. 5. The Km calculated from these results is 5.1 x 10-4 mM. Inhibition of NADase by iodoacetamide. Twenty microliters of diluted purified NADase (4.64 units/ml) was incubated with 10, 20, 40, and 80 Asl of 0.6 M a-iodoacetamide. Volumes were made to 0.1 ml with 0.1 M potassium phosphate, pH 7.3. Incubation was carried out for 10 min at room temperature, followed by assay for NADase activity. The effect of various concentrations of a-iodoacetamide on NADase activity is shown in Fig. 6. (IO4 UNTS/MpUML.)
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TABLE 3. Molecular weight of streptococcal NADase Method
Sodium dodecyl sulfate/gel electrophoresis Summation of amino acid residues Gel filtration See reference 20. b See reference 8.
a
Molecular weight estimations
55,000a 54,300
50,000 + 2000 569000 + 5000jJ
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604
GRUSHOFF, SHANY, AND BERNHEIMER
DISCUSSION Enzymes similar to the NADase studied here have been found in both animal (10) and plant (12) tissues as well as in microorganisms. The microbial enzymes that have been investigated in some detail include that from Neurospora crassa (11) as well as that from the C203S strain of group A S. pyogenes (5), from the C203U mutant of the same strain (19) and from group C streptococci (7, 8). In the present study, pure NADase devoid of hemolytic activity was obtained. It had a specific activity of 11,200 NADase units per mg of protein, which is comparable with that (5,330 NADase units per mg of protein) of the most active preparation described by Pace and Pappenheimer (18), in their purification of NADase from group C streptococci. Although extensive purification was achieved by these authors it is not clear whether other streptococcal proteins were still detectable in the final .preparation. Fehrenbach (8) obtained crystalline material having a specific activity of 2,400 units per mg of protein and possessing hemolytic activity. The procedure described in this paper for the purification of NADase utilizes a methodology similar to that used by Fischetti et al. (9) for the stabilization and purification of the phageassociated lysin of group C streptococci. In our preparation, the blocking of sulfhydryl groups by disodium tetrathionate renders the streptococcal proteinase inactive, thus preventing proteolysis during the purification procedure. The subsequent use of two-step ion-exchange chromatography yields a sharp, pure peak of NADase activity. The reproducibility of this procedure is excellent, and yields of approximately 44% have been consistently achieved in several experiments. Purification steps monitored by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicate that NADase is restricted to one protein band. Several properties of purified NADase were studied. The molecular weight of NADase, determined in an earlier work by Shany et al. (21) using sodium dodecyl sulfate/gel electrophoresis and in the present work from calculation of amino acid residues and gel filtration studies (Table 3), was in agreement with the value obtained by Fehrenbach (7) using gel filtration (56,000 4 5000). Carlson et al. (5) described streptococcal NADase as a nondialyzable, heat-labile protein having optimal activity between pH 7.2 to 7.8. With purified enzyme, the optimum pH for NADase activity is 7.3 and the enzyme is active from pH 6.5 to 8.0. This is in agreement with the narrow range obtained by the above authors,
J. BACTERIOL.
but contrasts with the broad range of activity with respect to pH obtained by N. crassa (11). The temperature at which the purified enzyme appears to be most active is 40 C, but substantial activity is demonstrable also at 35 C and 45 C. The value of the Km found in the present study, 5.1 x 10-4 mM, is identical to the Km of 5 x 10-4 mM reported for Neurospora NADase (11). In view of this and of the fact that neither the streptococcal nor the Neurospora enzyme hydrolyzes the reduced form of NAD, it would appear that the two enzymes are rather similar. Analysis of the streptococcal enzyme revealed four half-cystines (Table 2), and since the enzyme is inhibited by a-iodoacetamide it is possible that at least two, perhaps four, halfcysteines are present in reduced form, that is, as cysteine which may be part of the active site. The availability of a method for obtaining streptococcal NADase in pure form may facilitate study of the biological activities of the enzyme, of its role in oxidative metabolism and of its possible significance in streptococcal infections. ACKNOWLEDGMENTS The authors are grateful to Maria Heincz and Charles Harman for assistance with the amino acid analyses. This investigation was supported by Public Health Service grant AI-02874 from the National Institute of Allergy and Infectious Diseases and by Public Health Career Program award 5K06-AI-14-198 to A.W.B. LITERATURE CITED 1. Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J.
91:222-233. 2. Bernheimer, A. W., and P. Grushoff. 1967. Cereolysin: 3. 4.
5.
6. 7.
8.
9.
production, purification and partial characterization. J. Gen. Microbiol. 46:143-150. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolation and composition of staphylococcal alpha toxin. J. Gen. Microbiol. 30:455-468. Blackburn, S. 1968. Amino acid determination, methods and techniques, p. 11-29. Marcel Dekker Inc., New York. Carlson, A. S., A. Kellner, A. W. Bernheimer, and E. B. Freeman. 1957. A streptococcal enzyme that acts specifically upon diphosphopyridine nucleotide: characterization of the enzyme and its separation from streptolysin 0. J. Exp. Med. 106:15-26. Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948-1954. Fehrenbach, F. J. 1969. Gel-filtration behavior and molecular weight of NAD-glycohydrolase (EC 3.2.2.5) from streptococci in column chromatography on Sephadex gels. J. Chromatogr. 41:43-52. Fehrenbach, F. J. 1971. Reinigung und kristallisation der NAD-glykohydrolase aus C-streptokokken. Eur. J. Biochem. 18:94-102. Fischetti, V. A., E. C. Gotschlich, and A. W. Bernheimer. 1971. Purification and physical properties of group C streptococcal phage-associated lysin. J. Exp. Med. 133:1105-1117.
VOL. 122, 1975
STREPITOCO(CCAL NADase
10. Handler, P., and J. R. Klein. 1942. The inactivation of pyridine nucleotides by animal tissues in vitro. J. Biol. Chem. 143:49-57. 11. Kaplan, N. O., S. P. Colowick, ard A. Nason. 1951. Neurospora diphosphopyridine nucleotidase. J. Biol. Chem. 191:473-483. 12. Kornberg, A., and W. E. Pricer, Jr. 1950. Nucleophosphatase. J. Biol. Chem. 182:763-778. 13. Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. 2. General properties. J. Gen. Physiol. 30:291-310. 14. Liu, T. Y. 1967. Demonstration of the presence of a histidine residue at the active site of streptococcal proteinase. J. Biol. Chem. 242:4020-4032. 15. Liu, T. Y., N. P. Neumann, S. D. Elliot, S. Moore, and W. H. Stein. 1963. Chemical properties of streptococcal proteinase and its zymogen. J. Biol. Chem. 238:251-256. 16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. 17. Moore, S. 1963. On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235-237.
605
18. Pace, M. G., and A. M. Pappenheimer, Jr. 1959. An immunochemical study of streptococcal diphosphopyridine nucleotidase. J. Immunol. 83:83-86. 19. Shany, S., A. W. Bernheimer, P. S. Grushoff, and Kwang-Shin Kim. 1974. Evidence for membrane cholesterol as the common binding site for cereolysin, streptolysin 0 and saponin. Mol. Cell. Biochem. 3:179-186. 20. Shany, S., P. S. Grushoff, and A. W. Bernheimer. 1973. Physical separation of streptococcal nicotinamide adenine dinucleotide glycohydrolase from streptolysin 0. Infect. Immun. 7:731-734. 21. Siegel, F. L., and M. K. Roach. 1961. fl-2-Thienyl-DLalanine, internal standard for automatic determination of amino acids. Anal. Chem. 33:1628. 22. Smith, E. L., and A. Stockell. 1954. Amino acid composition of crystalline carboxypeptidase. J. Biol. Chem. 207:501-514. 23. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl-sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
