Staphylococcal Acid Phosphatase: Preliminary Physical and Chemical Characterization

Transcription

1 JOURNAL OF BACTERIOLOGY, Mar. 1969, p American Society for Microbiology Vol. 97, No. 3 Printed In U.S.A. Staphylococcal Acid Phosphatase: Preliminary Physical and Chemical Characterization of the Loosely Bound Enzyme1 F. J. MALVEAUX2 AND C. L. SAN CLEMENTE Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication 14 December 1968 At temperatures between 45 and 5 C, staphylococcal acid phosphatase purified 44-fold had maximal activity at ph 5.2 to 5.3. However, the enzyme was most stable in the alkaline range (ph 8.5 to 9.5) at temperatures below 5 C. Iodoacetate and ethylenediamine-tetraacetic acid were effective inhibitors, whereas mercaptoethanol and Cu2+ acted as stimulators. The energy of activation for hydrolytic cleavage of the synthetic substrate, p-nitrophenyl phosphate, was 19.5 Kcal/mole. Km for the same substrate was 4.5 X 1-4 M. The purified enzyme was most active against the substrates p-nitrophenyl phosphate and glyceraldehyde 3-phosphate. Staphylococcal phosphatase, which has sometimes been regarded a virulence factor for the organism (1, 6, 8, 13), has recently been purified (16). Barnes and Morris (2) have partially characterized staphylococcal acid phosphatase present in a cell suspension. They found the enzyme maximally active at ph 5.6 and slightly inhibited by inorganic phosphate. The acid phosphatase of Escherichia coli has been studied in considerable detail (5, 11, 12, 18, 19, 23). This phosphatase can be released from the cells by an osmotic shock procedure (18, 19) and is presumed to be located near the surface of the cell. In fact, Dvorak et al. (5) caused the release of three separate E. coli acid phosphatase fractions by this same procedure. They successfully purified the acid hexose phosphatase 124- fold, but a nonspecific acid phosphatase resisted all purification attempts. In a previous report (15), we noted that a portion of the total staphylococcal acid phosphatase was found free in the culture medium, but the rest of the enzymatic activity was associated with the cells. Most of this latter activity was eluted from the surface of the cells with 1. M KCl under alkaline conditions. This eluted fraction which we called "loosely bound" was then purified 44-fold and partially characterized (16). The enzyme is maximally active at ph 5.2 to 5.3 and has a molecular weight of 58,. This paper describes further characterization of X Published with the approval of the Director of the Michigan Agricultural Station as Journal Article no 'Present address: Department of Microbiology, College of Medicine Howard University, Washington, D.C. 21. the activity, stability, and substrate specificity of staphylococcal acid phosphatase. MATERIALS AND METHODS Culture. Staphylococcus aureus PS 55 of the International Blair series (3) of phage-propagating strains was the source of enzyme in this study. Stock cultures were maintained at 4 C on Trypticase Soy Agar (BBL) and were transferred every 6 weeks. Source of purified enzyme. All characterizations of staphylococcal acid phosphatase were performed on loosely bound (eluted) enzyme that had been purified 44-fold (16). The purified enzyme had appeared homogeneous after gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. Acid phosphatase assay. Enzymatic activity was measured as already described (16) by a method modified from Barnes and Morris (2) using.8% p-nitrophenyl phosphate, disodium salt (Mann Research Laboratories, Inc., New York) as substrate. The unit of enzyme activity was expressed as micromoles of p-nitrophenol liberated per minute at 37 C. In initial velocity studies and comparison of the relative activity of acid phosphatase against different phosphate esters, the reaction was stopped with 1 ml of 1% trichloroacetic acid, and the liberated orthophosphate was measured according to the method of Fiske and SubbaRow (7). Intial velocity of acid phosphatase. Initial velocities were determined by stopping the reaction with trichloroacetic acid at certain intervals of time and assaying for liberated orthophosphate. Each set of assay tubes contained a different concentration of substrate, ranging from 5 X 1-4 M to 1 X 1 M. Three separate sets of controls were employed. In the first control, enzymatic activity was stopped with trichloroacetic acid at zero-time. In another, substrate was deleted from the assay system; in the third control, 1215

2 1216 MALVEAUX AND SAN CLEMENTE J. BACTERIOL. the enzyme was omitted. The data were plotted by the method of Lineweaver and Burk (14) and determination of Km was made as suggested by Dixon and Webb (4). Effect of temperature on the activity of acid phosphatase. Employing a uniform substrate concentration (5 g/liter), the initial velocity of equal amounts of enzyme was measured at 25, 3, 37, 45, 52, and 6 C. At 4 and 15 C, enzyme was more concentrated, but the final readings were multiplied by appropriate dilution factors so that all results were comparable. Effect of different compounds on the activity of acid phosphatase. At uniform substrate concentration (5 g/liter), the activity of acid phosphatase was determined in the presence of different compounds. Buffer concentration in all cases was 11 M acetate. In some cases different concentrations of the compounds were employed. In all assay systems, two controls were employed: in one, the reaction was stopped at zerotime; in the other, potential inhibitor was omitted. Effect of different divalent cations on the activity of acid phosphatase. Activity of the enzyme which had been dialyzed against water was determined in the presence of various divalent cations. Final concentration of metal ions (used as the chloride form) in the assay system was 1- M. Controls consisted of assay tubes devoid of metal and those in which the reaction was terminated at zero-time. Heat stability of the enzyme. Enzyme stability was observed in the presence and absence of Cu++ (I-3 M). In either case, the enzyme in 1- M tris(hydroxymethyl)aminomethane (tris) hydrochloride (ph 8.5) was heated to the desired temperature, kept at this temperature for 5 min, and quickly cooled in ice water. Treated samples were then assayed at 37 C and ph 5.2. An unheated control permitted calculation of per cent residual activity. Stability of the enzyme under different conditions. Dialyzed enzyme was stored at 25 C in water, 1. M acetic acid, 1. N NaOH, and in certain buffer solutions at ph 5.2, 7.5, 8.5, and 9.5. At the end of 1, 3, and 6 days, the samples were assayed for acid phosphatase activity at ph 5.2 and 37 C. Activity of acid phosphatase against various substrates. Hydrolytic activity of acid phosphatase against various phosphorylated esters was determined at ph 5.2 and 37 C. Final concentration of each substrate (dissolved in water) in the assay tubes was 1 M. Three separate sets of controls were employed. In the first case, enzymatic activity against each substrate was stopped at zero-time by adding trichloroacetic acid. In the second set of controls, substrate was deleted from the assay system; in the third set, enzyme was omitted. A value of 1 was arbitrarily assigned to the activity of acid phosphatase against p-nitrophenyl phosphate; relative enzymatic activity against other substrates was designated accordingly. RESULTS Initial velocity of acid phosphatase. Figure 1 is a double reciprocal plot of initial velocity of enzymatic activity. The Km for the substrate, p-nitrophenyl phosphate, was 4.5 x 1-' M. Effect of temperature on the activity of acid phosphatase. Maximal enzymatic activity (Fig. 2) occurred at approximately 49 C. Between 15 and 37 C there was a linear increase in initial activity, but below and above this range the same relationship was not applicable. The slope of the solid portion (between 15 and 37 C) of the plot was used to determine the Arrhenius function A (energy of activation). The value for A was 19.5 Kcal/mole. Effect of different compounds on the activity of acid phosphatase. Of the compounds tested, mer o 4 12 'DIs 2 FIG. 1. Double reciprocal plot of the relationship between initial velocity of acid phosphatase and p- nitrophenyl phosphate concentration. The reactions were run atph 5.2 and 37 C. 1.2 *Q. 1..\.8'..6 A ;3 3U FIG. 2. Arrhenius plot of the effect of temperature upon hydrolysis of p-nitrophenyl phosphate by acid phosphatase. The slope of the solid portion (between 15 and 37 C) of the plot was used to determine the Arrhenius function A (energy ofactivation).

3 VOL. 97, 1969 CHARACTERIZATION OF THE ACID PHOSPHATASE 1217 captoethanol was the only agent that stimulated enzymatic activity (Table 1). Ethylenediaminetetraacetic acid (EDTA), inorganic phosphate, sodium fluoride, sodium molybdate, iodoacetate, and urea inhibited enzymatic activity, but tartaric acid had no apparent effect. Effect of divalent cations on the activity of acid phosphatase. The cations Co2+, Mn2+, Mg2+, Ca2+, Zn2+, and Ba2+ had little or no effect on acid phosphatase activity (Table 2). Hg2+ and Pb2+, on the other hand, reduced enzymatic activity significantly. Cu2+ accelerated acid phosphatase activity twofold at its given concentration. Heat stability of the enzyme. In the absence of Cu2+, stability of purified acid phosphatase, under the conditions employed, decreased rapidly between 4 and 7 C. At this latter temperature, the enzyme was completely inactivated (Fig. 3). With Cu2+, the minimum temperature for complete inactivation was 8 C. Stability of the enzyme under different conditions. Purified acid phosphatase was completely inactivated in 1. M acetic acid and 1. N NaOH (Table 3). When suspended in water for 6 days, 55% of the initial activity was lost. Although maximal activity was in the acid range (16), the enzyme appeared most stable in moderately alkaline conditions (ph 8.5 and 9.5). Activity of acid phosphatase with various substrates. Of the substrates tested, acid phosphatase was most active against p-nitrophenyl phosphate (Table 4). Substantial activity against glyceralde- TABLE 1. Effect ofdifferent compounds on staphylococcal acid phosphatase Compounda None (no compound added) KH2PO4 (.3 M) KH2PO4 (.2 M) KH2PO4 (.34 M) Mercaptoethanol (.32 M) Mercaptoethanol (.64 M) Mercaptoethanol (.128 M) Cysteine (.5 M) EDTA (.5 M) Sodium fluoride (.5 M) Sodium molybdate (.5 M) Tartaric acid (.5 M) lodoacetate (.5 M) Urea (8 M) Units of enzymatic activity1b Relative activity afinal concentrations are given in parentheses. Buffer concentration in each case was.1 M acetate, ph 5.2. bexpressed as micromoles of p-nitrophenol per minute at 37 C. TABLE 2. Effect of different divalent cations on the activity of acid phosphatase Me tal1a Metal5 UUnits of Relative ~~~activity1' activity None (no metal added) Co, Mn Mg Ca Zn Cu Hg Pb Ba a Final concentration of the metal ions (used as chloride form) was 1-' M. b Expressed as micromoles of p-nitrophenol per minute at 37 C. 1-2 o-without Cu u 4 U 2- \ A-With Cu++ Oi.. n Temperature (-C) A FIG. 3. Heat stability of staphylococcal acid phosphatase with and without Cu+ (1-' m). The enzyme was heated at each temperature for S min in.1 M Tris-hydrochloride (ph 8.5) and cooled quickly in ice water. Assays were carried out at 37 C andph 5.2. hyde 3-phosphate, and moderate activity against a-glycerophosphate, fructose 6-phosphate, and phenolphthalein diphosphate were also observed. Acid phosphatase exerted little or no hydrolytic activity against the remaining substrates. DISCUSSION Determined from the double-reciprocal plot, Km for the substrate p-nitrophenyl phosphate was 4.5 X 1-1 M (Fig. 1). Barnes and Morris (2) reported the Km for p-nitrophenyl phosphate was 2. x 1-1 M. However, the enzyme preparation used in their studies was a suspension of whole cells. 1

4 1218 MALVEAUX AND SAN CLEMENTE J. BAcTERIoL. TABLE 3. Conditions Stability of staphylococcal acid phosphatasea Relative activityb 1 day 3 days 6 days Water (no buffer) M Acetate, ph M Tris-hydrochlo ride, ph M Tris-hydrochloride, ph M Glycine-NaOH, ph M KCl, ph O MKCl, ph O MKCl, ph O MKCl, ph O M Acetic acid 1.O N NaOH a Samples were stored at room temperature (25 C) for 6 days. b The activities are relative to zero-time. A value of 1 was assigned to the activity of the acid phosphatase which liberated.58 pmoles of inorganic phosphate from p-nitrophenyl phosphate in 3 min at 37 C. As noted by Dixon and Webb (4), the effects of temperature on enzyme reactions are very complex. Discontinuity of the slope of an Arrhenius plot (Fig. 2) suggests irreversible inactivation of staphylococcal acid phosphatase above 48 C. The Arrhenius function A (energy of activation) for the enzyme was 19.5 Kcal/mole. This value is comparable to that for a 3', 5'-cyclic nucleotide phosphodiesterase which was isolated and purified 173-fold from dog heart (17), but differs from that of alkaline phosphatase from E. coli (6.9 Kcal/mole; reference 1). The divalent cation Cu2+ stimulated the activity of staphylococcal acid phosphatase (Table 2). The activity of nonspecific acid phosphatase of E. coli was stimulated 1% by the combined effect of Mg2+ and Co2+, but was inhibited by Zn2+ and Ca2+ (5); hydrolysis of p-nitrophenyl phosphate by a cyclic phosphodiesterase from the same microorganism was stimulated 6-fold by Co2+. The phosphomonoesterase activity of Bacillus subtilis was stimulated at least threefold by Co2+ and twofold by Ni2+ (21). On the other hand, alkaline phosphatase of E. coli was inhibited by Cd2+, Co2+, and Cu2+, which displaced the native Zn2+ in this enzyme (2). Staphylococcal acid phosphatase probably requires a free sulfhydryl group for maximal activity. lodoacetate, which usually reacts with thiol groups to give alkylated derivatives (4), proved TABLE 4. Relative activities of staphylococcal acid phosphatase against different phosphate esters (1-2 M)a Inor- Relative Compound ganic phos- activ- ityb phate umolc p-nitrophenyl phosphate.29 1 Glucose 6-phosphate a-glycerophosphate a-glycerophosphate Dihydroxyacetone phosphate Fructose 6-phosphate Ribose 5-phosphate Phosphoglyceric acid Glyceraldehyde 3-phosphate Fructose 1,6-diphosphate Guanosine diphosphate Cytidine 3'-(2')phosphoric acid Adenosine triphosphate Adenosine diphosphate Adenosine monophosphate Deoxyribonucleic acid Sodium pyrophosphate Phenolphthalein diphosphate Casein a-casein Lecithin a Samples were incubated at 37 C for 3 min. b A value of 1 was assigned to the activity of acid phosphatase against p-nitrophenyl phosphate. to be the most effective inhibitor (Table 1); mercaptoethanol, which usually preserves sulfhydryl groups, had a stimulatory effect. Since EDTA also inhibited enzymatic activity, the enzyme may be more active in the presence of certain metals. As noted above, acid phosphatase was stimulated twofold by Cu2+. Dvorak et al. (5) noted that activity of nonspecific acid phosphatase of E. coli was reduced by 18% with EDTA; in the case of acid hexose phosphatase no such effect was noted. Inorganic phosphate also slightly inhibited the activity of staphylococcal acid phosphatase. Barnes and Morris (2) made the same observation and also noted that phosphate ions skewed the ph activity curve towards the acid side. Instability of an enzyme can be studied by first exposing the enzyme to various temperatures for a definite period of time and then measuring its activity at a temperature where it is stable (4). Above 4 C, stability of staphylococcal acid phosphatase was lost rapidly (Fig. 3). Our studies indicate that Cu2+ may not only stimulate enzymatic activity but also may give greater sta-

5 VOL. 97, 1969 CHARACrERIZATION OF THE ACID PHOSPHATASE 1219 bility to the enzyme between 5 and 8 C. On the other hand, Terac and Ukita (22) noted that a purified, bovine pancreatic phosphodiesterase retained its full activity if heated at any temperature below 75 C for 5 min at ph 7.. The acid phosphatase purified by Hofsten and Porath (12) from E. coli was unstable in dilute solutions, stable in 1. M acetic acid, and denatured by neutral salts. Staphylococcal acid phosphatase was relatively stable in water and 1. M KCl, but was inactivated by 1. M acetic acid (Table 3). The enzyme was more stable in a slightly alkaline menstruum, although optimal activity occurred at low ph values. However, at both extremes of the ph scale, staphylococcal acid phosphatase was completely inactivated. Relative activity of the enzyme with the different substrates tested (Table 4) gives little insight into the natural substrate and role of the enzyme in vivo. Acid phosphatase was most reactive against p-nitrophenyl phosphate with significant activity for glyceraldehyde 3-phosphate. The nonspecific acid phosphatase of E. coil was also most active against the same synthetic substrate, but the acid hexose phosphatase was most active against glucose 6-phosphate, glucose 1-phosphate, and galactose 1-phosphate (5). The latter enzyme also had considerable hydrolytic activity on fructose 1, 6-diphosphate. Alkaline phosphatase of E. coli acted more like a general phosphomonoesterase, since the organic moieties of certain phosphoester substrates had little effect on the rate of enzymatic activity (1). The pattern of substrates hydrolyzed by alkaline phosphatase from Pseudomonas fluorescens was similar to that of compounds hydrolyzed by the same enzyme from E. coli (9). The natural substrate and the role of staphylococcal acid phosphatase must await further kinetic and other critical characterizations of the enzyme. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-8121 from the National Institute of Allergy and Infectious Diseases. We are grateful for the assistance of Carl L. Winely in the preparation of this paper. LITERATURE CITED 1. Barber, M., and S. W. A. Kuper Identification of Staphylococcus pyogenes by the phosphatase reaction. J Pathol. Bacteriol. 63: Barnes, E. H., and J. F. Morris A quantitative study of the phosphatase activity of Micrococcus pyogenes. J. Bacteriol. 73: Blair, J. E., and M. Carr The techniques and interpretation of phage typing of staphylococci. J. Lab. Clin. Med. 55: Dixon, M., and E. C. Webb Enzymes. Academic Press Inc., New York. 5. Dvorak, H. F., R. W. Brockman, and L. A. Heppel Purification and properties of two acid phosphatase fractions isolated from osmotic shock fluid of Escherichia coli. Biochemistry 6: Elek, S. D Staphylococcus pyogenes and its relation to disease. E. and S. Livingstone, Ltd., London, England. 7. 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Pniewska Enzymatic activity of coagulase-positive Staphylococcus aureus strains isolated from patients and healthy carriers. Pathol. Microbiol. 29: Lineweaver, H., and D. Burk The determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56: Malveaux, F. J., and C. L. San Clemente Elution of loosely bound acid phosphatase from Staphylococcus aureus. Appl. Microbiol. 15: Malveaux, F. J., and C. L. San Clemente Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme. J. Bacteriol. 97: Nair, K. G Purification and properties of 3', 5'-cyclic nucleotide phosphodiesterase from dog heart. Biochemistry 5: Neu, H. C., and L. A. Heppel On the surface localization of enzymes in E. coli. Biochem. Biophys. Res. Commun. 17: Neu, H. C., and L. A. Heppel The release of enzymes from Escherichai colt by osmotic shock and during the formation of spheroplasts. J. Biol. Chem. 24: Plocke, D. J., and B. L. Vallee Interaction of alkaline phosphatase of E. colt with metal ions and chelating agents. Biochemistry 1: Takeda, K., and A. Tsugita Phosphoesterases of Bacillus subtilis. II. Crystallization and properties of alkaline phosphatase. J. Biochem. 6: Teroa, T., and T. Ukita Purification and properties of phosphodiesterase from bovine pancreas. J. Biochem. 58: Torriani, A Influence of inorganic phosphate in the formation of phosphatases by Escherichia coli. Biochim. Biophys. Acta 38: