ZINC AND MAGNESIUM
IN E . COLI A L K A L I N E P H O S P H A T E S
Plant Physiol. 54, 678. Ludwig, L. J., and Canvin, D. T. (1971), Can. J . Bot. 49, 1299. Murai, T., and Akazawa, T. (1972), Biochem. Biophys. Res. Commun. 46, 2121. Ogren, W. L., and Bowes, G. (1971), Nature (London), New Biol. 230, 159. Packer, L. Murakami, S., and Mehard, C. W . (1970), Annu. Rev.Plant Physiol. 21, 27 1. Paulsen, J. M., and Lane, M. D. (1966), Biochemistry 5, 2350. Pedersen, T. A., Kirk, M., and Bassham, J. A. (1966), Physiol. Plant. 19, 219. Pon, N. G., Rabin, B. R., and Calvin, M. (1963), Biochem. Z . 338, 7. Sugiyama, T., Nakayama, N., and Akazawa, T. (1968a),
Biochem. Biophys. Res. Commun. 30, 118. Sugiyama, T., Nakayama, N., and Akazawa, T. (1968b), Arch. Biochem. Biophys. 126. 737. Vogels, G. D., and van der Drift, C. (1970), Anal. Biochem. 33, 143. Walker, D. A. (1971), in Proceedings of 2nd International Congress on Photosynthesis Research, Vol. 111, Forti, G . , Avron, M., and Melandri, A., Ed., W. Junk, The Hague, p 1773. Walker, D. A . (1973), New Phytol. 72, 209. Warburg, O., and Christian, W . (1942), Biochem. 2. 310, 384. Weissbach, A., Horecker, B. L., and Hurwitz, J. (1956), J . Biol. Chem. 218, 795. Werdan, K., and Heldt, H. W. (1972), Biochim. Biophys. Acta 283, 430.
Zinc and Magnesium Content of Alkaline Phosphatase from Escherichia coli f William F. Bosron,t F. Scott Kennedy,$ and Bert L. Vallee*
ABSTRACT: Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-atom of magnesium per molecular weight 80,000 (Plocke, D. J., Levinthal, C., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described
eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared. Collectively, the zinc content was found to be 4.0 f 0.3 g-atoms per molecular weight of 89,000 for enzyme purified and analyzed by procedures shown not to alter its intrinsic metal content adversely. Importantly, it is confirmed that the enzyme when isolated a t p H 7.2 and 7.5 contains an average of 1.3 f 0.2 g-atoms of magnesium/mol. This feature of the metal composition, hitherto largely unappreciated, bears significantly on the investigation of the structure of alkaline phosphatase as related to its chemical and functional properties.
T h e precision and sensitivity of methods applicable to the determination of the physical, chemical, and functional properties of enzymes have increased markedly in the last decade. Concurrently, there have been analogous advances in high resolution procedures for enzyme isolation. Purification of enzymes by means of such superior methodology may result in numerical values for the parameters characterizing their properties which differ from those obtained initially. Unfortunately, such determinations frequently do
not indicate definitively whether the numerical changes observed result from intrinsic differences in the enzyme being examined, variations in analytical methodology, or differing modes of calculation and representation of results. Escherichia coli alkaline phosphatase well illustrates these evolving problems in protein characterization. It was first isolated in 1960 (Garen and Levinthal, 1960) and soon thereafter shown to contain stoichiometric quantities of zinc and magnesium (Plocke et al., 1962). Since these early studies, native phosphatase, prepared from E. coli by a variety of methods, consistently has been shown to require minimally 2 g-atoms of zinc/mol of enzyme for catalytic activity (Simpson et al., 1968; Harris and Coleman, 1968; Reynolds and Schlesinger, 1969; Petitclerc et al., 1970; Trotman and Greenwood, 1971; Csopak and Szajn, 1973). Further, it was demonstrated that two additional zinc atoms stabilize secondary, tertiary, or quaternary structure and/or
~~~~~~
From the Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 021 15. Received March IO, 1975. This work was supported by Grant-in-Aid CM-15003 from the National Institutes of Health, Department of Health, Education and Welfare. *Recipient of traineeship under Grant GM-02123 from the National Institutes of Health, Department of Health. Education and Welfare.
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influence catalytic activity (Simpson et al., 1968; Reynolds and Schlesinger, 1969; Petitclerc et al., 1970; Trofman and Greenwood, 1971). However, the presence and the functional significance of these two additional zinc atoms have incurred considerable debate (Harris and Coleman, 1968; Applebury and Coleman, 1969; Csopak et ai., 1972a; Csopak and Szajn, 1973). Even though magnesium has been reported to enhance activity of the zinc enzyme (Plocke and Vallee, 1962), virtually all workers have examined only the stoichiometry of zinc in their preparations, while that of magnesium consistently has been, ignored, and systematic studies of its possible role in catalysis and/or structure stabilization are not on record. Furthermore, as a general rule phosphatase metal content has been expressed in terms of g-atom of metal per mole of protein rather than as the ratio of mass of metal per g of protein. Importantly, the molecular weight of the enzyme upon which such molar metal stoichiometry is based has varied significantly (Garen and Levinthal, 1960; Schlesinger and Barrett, 1965; Simpson et al., 1968; Applebury and Coleman, 1969; Trotman and Greenwood, 1971). Similarly, the absorbance a t 278 nm (Plocke et ai., 1962; Rothman and Byrne, 1963; Malamy and Horecker, 1964; Csopak et al., 1972b) and maximal specific activity (Plocke et al., 1962: Lazdunski and Lazdunski, 1967: Simpson et al., 1968; Harris and Coleman. 1968; Reynolds and Schlesinger, 1969; Csopak and Szajn, 1973) have differed widely. A number of factors may affect the value of these parameters: ( 1 ) the strain of E . coli employed, ( 2 ) the ambient conditions used in releasing phosphatase from the organism, (3) the procedures for subsequent purification, and (4) the analytical methodology and calculations used to define the characteristics of the enzyme. Initially, phosphatase was isolated from whole cell extracts and purified by precipitation at 80’ in high concentrations of magnesium followed by chromatography on DEAE-cellulose (Garen and Levinthal, 1960: Plocke et al., 1962). Since then, phosphatase purification procedures have been modified and improved repeatedly. Thus. following the finding that phosphatase is localized in the periplasmic space (Malamy and Horecker, 1961). the enzyme was released from E . coli by a variety of techniques, including lysozyme treatment in the presence of EDTA (Malamy and Horecker, I964), osmotic shock i n the presence of chelating agents (Neu and Heppel, 1965). and treatment of cells a t acid pH releasing metal-free subunits requiring activation (Schlesinger and Olsen. 1970). Following release, the enzyme generally n a s purified by means of DEAE-cellulose chromatography, gel filtration, and ammonium sulfate precipitation or crystallization. The purified enzyme. however, can be resolved into three forms by high resolution DEAE-cellulose chromatography, although the characteristics of these isozymes have not been examined in detail (Lazdunski and Lazdunski. 1967; Simpson et al., 1968). Several of the aforementioned procedures, viz., exposure to EDTA or ammonium sulfate and addition of high concentrations of magnesium and/or zinc, previously have been suggested as parameters responsible for altering the metal content of native enzyme (Simpson et al., 1968: Ulmer and Vallee, 1971: Csopak and Szajn, 1973). The present paper summarizes the metal content of the consecutively numbered alkaline phosphatase preparations since such studies were first initiated in this laboratory. They are compared with analogous data on preparations obtained elsewhere using various methods of purification.
2276
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The study confirms the presence of zinc and magnesium in alkaline phosphatase from E . coli, delineates their stoichiometry, and simultaneously identifies some of the factors which may affect the metal content of this enzyme. Further, a purification procedure is described which eliminates the features of all methods known to alter metal content of Phosphatase. In addition, it is emphasized that careful observance of metal-free technique (Thiers, 1957) and ashing of alkaline phosphatase samples prior to analysis by atomic absorption spectrometry are essential to accurate determination of the enzyme’s metal content. Collectively. the data demonstrate that the phosphatase contains 4.0 f 0.3 gatoms of zinc and 1.3 f 0.2 g-atoms of magnesium per molecular weight 89,000 when isolated and purified a t p H 7.2 and 7.5 by methods which do not affect its metal content adversely. Experimental Section
Purification of Alkaline Phosphatase. I n this laboratory preparations of alkaline phosphatase were initially obtained from cultures of E . coli K-12 either wild-type or constitutive for phosphatase, grown in a lactate-inorganic salts or tryptone-yeast extract medium (Table 11, no. 1-5) (Plocke et al.. 1962). Acetone powders from whole cell extracts w8ere then purified utilizing heat precipitation at 80’ in 0.01 M M g S 0 4 followed by chromatography on DEAE-cellulose. Preparations 6-26, Tables 11-IV, were obtained from periplasmic extracts of E. coli. Strain C-90 (Tables 11 and 111, no. 6-20) was grown to stationary phase at 37’ in 100or 14-1. cultures in a Tris-peptone medium (Simpson et al., 1968), and strains K-10, C4F1, and CW-3747’ (Table IV, no. 21-26) were grown similarly in a 3-1. culture using “ 1 21 Peptone” medium (Torriani, 1966). The cells were harvested by centrifugation and washed in 0.01 M Tris-C1 (pH 7.5). Periplasmic extracts were then prepared using osmotic shock either in sucrose and EDTA or sucrose and Chelex 100 (Neu and Heppel, 1965). When EDTA was utilized to release the enzyme (Tables 11-IV, no. 6-16 and 21-26) a 2-6% cell suspension was prepared a t room temperature in 20% sucrose, M EDTA, and 0.03 M Tris-CI (pH 7.5). and followed by centrifugation. The cells were shocked with the same volume of cold, distilled water. When Chelex 100 (Bio-Rad, 1-200 mesh) was used (Table 111, no. 17-20), a 2% cell suspension was prepared in 5% Chelex, 20% sucrose. and 0.03 M Tris-CI (pH 7.5). Under these circumstances, an additional centrifugation for 5 min at 600g was required to separate cells from Chelex, thereby assuring that the phosphatase released subsequently never comes into contact with any chelating agent and eliminating all effects in this step potentially attributable to EDTA (Ulmer and Vallee. 1971: Csopak and Szajn, 1973). The water extract from the osmotic shock medium was purified by batchwise absorption on DEAE-cellulose (Whatman DE52), equilibrated with 0.01 M imidazole-CI (pH 7.2), and eluted with 0.1 M NaC1. T h e protein was concentrated and dialyzed by ultrafiltration (Amicon PM-IO membrane). Preparations 6-26, Tables 11-IV, were purified by column chromatography on DEAE-cellulose in 0.01 M imidazole-CI (pH 7.2), using a linear gradient of NaCl to 0.1 M . The broad peak of activity, containing the isozymes. was
’
Kindlq provided by Dr. A . M . Torriani, Department of Biolog). V.1.T.
Z I N C A N D MAGNESIUM
IN
E . COLI
ALKALllU E
concentrated and dialyzed by ultrafiltration. In some cases rechromatography on DEAE-cellulose (Table 111, no. 15 and 19) or on Sephadex (3-150 (Pharmacia, 40-120 p ) in 0.01 M Tris-CI ( p H 7.5) (Table 111, 17 and 18) was employed to obtain enzyme of high specific activity. Preparations 6 and 7, Table 11, were prepared by Simpson et al. (1968) using crystallization from (NH4)2S04 and MgC12 according to the method of Malamy and Horecker (1964). Phosphatase Assays. Enzymatic activity was measured by following the hydrolysis of 4-nitrophenyl phosphate (Sigma 104) using a Gilford Model 222 spectrophotometer with a Heathkit Model IR-18M recorder. The release of 4nitrophenol in 1 M Tris-C1 ( p H 8.0) and 25' was determined a t 400 nm using a molar absorbance of 1.68 X lo4. A unit of activity is defined as the number of micromoles of substrate hydrolyzed per minute per milligram of protein under these conditions. Phosphatase concentration was determined spectrophotometrically using A27g( l%) 7.2 (Simpson et al., 1968). Electrophoresis. Polyacrylamide disc gel electrophoresis was performed in Tris-glycine buffer ( p H 9.4) (Gabriel, 1971) or in sodium dodecyl sulfate, as suggested by Weber and Osborn (1969). Protein was visualized on polyacrylamide gels using Coomassie Brilliant Blue stain (Smith, 1968) and phosphatase activity was determined using a-naphthyl phosphate and Fast Red T R (Gabriel, 1971). Metal Analysis. Two methods, spark emission spectrography and atomic absorption spectrometry, were employed for zinc and magnesium analysis. Primary working standards for metal analysis were prepared by dissolving weighed quantities of spectroscopically pure zinc rod and magnesium metal (Johnson, Matthey and Co., Ltd.) in metal-free HCI. To determine accuracy of the methods a zinc spelter, NBS sample 109; a copper-zinc-nickel alloy, N B S sample 157-A; a magnesium base alloy, N B S sample 171; and dolomite, N B S sample 88, all of certified metal content, were dissolved in metal-free HCI in the same manner as the aforementioned standards. All phosphatase samples were dialyzed for 4 days against multiple changes of a 100-fold excess of metal-free 0.01 M Tris-C1 ( p H 8.0) to remove unbound metals. For emission spectrography, 5-15 mg of protein was ashed in an electric muffle furnace at 450' for 24 hr (Thiers, 1957). The ash was dissolved in 6 N HCI containing 7 pg/ml of vanadium as an internal standard. Intensity ratio curves were constructed for each spectrographic plate by sparking, in triplicate, four standard concentrations of zinc, magnesium, and 15 other elements along with duplicate phosphatase samples (Vallee, 1955). For zinc concentrations between 1 and 30 pg/ml, the coefficient of variation for this method varies from 7 to 10% (Thiers and Vallee, 1957) and the accuracy is 100 f 7%. Atomic absorption spectrometry for zinc was performed with equipment previously described (Fuwa and Vallee, 1963). The coefficient of variation for this method is below 3% and the accuracy is 100 f 5% (Fuwa et al., 1964). Phosphatase samples were ashed, then dissolved in 0.1 N HCI, and diluted with metal-free water to a zinc concentration of between 0.01 and 0.1 g/ml. For comparative purposes samples were analyzed prior to ashing to determine the effect of the organic matrix on zinc determination (Table I). In every case the zinc content of an ashed sample was found to be higher than the identical nonashed sample. Furthermore, the average zinc content of ashed samples, 2900 f 180 pg/g or 4.0 f 0.3 g-atoms/89,000, was 12% higher than
PHOSPHATASE
Table I: Zinc Content of Alkaline Phosphatase as Determined by Atomic Absorption Analysis of Ashed and Nonashed Samples. Zinc Pretreatment
Number of Samplesa
Ashed Nonashed
14 14
g-atom/ 89,000 mol wt
wg/g
2900 2600
t i
180 190
4.0 + 0.3 3.6 i 0.3
a Preparation numbers 13-26.
that of the same nonashed samples, 2600 f 190 pg/g or 3.6 f 0.3 g-atoms/89,000. Atomic absorption spectrometry for magnesium was performed as described by Wacker et al. (1 965). Samples were initially diluted with metal-free water to a magnesium concentration ranging from 0.05 to 0.5 pg/ml and finally 1:l with 8-hydroxyquinoline (G. Fredrick Smith Chemical Co.) in 1 N HCI to obviate any interferences. The coefficient of variation for standards with this method is below 6% and the accuracy is 100 f 1% (Iida et a]., 1967). The metal content of phosphatase is expressed both in pg per g of protein and in g-atom of metal per molecular weight of 89,000 (Simpson et al., 1968) to facilitate comparsions with the results of these and other studies. Preparation of Reagents. Analytical grade chemicals were used throughout, and all solutions were prepared with deionized and distilled water. All buffers were extracted with 0.001% diphenyldithiocarbazone in carbon tetrachloride until free of transition metals. In order to remove magnesium, all solutions extracted in this manner were treated further with Chelex 100. Glassware and polyethylene containers were rendered metal-free by soaking overnight in a 1 : 1 mixture of concentrated nitric and sulfuric acids, followed by copious rinsing with metal-free water (Thiers, 1957). Results
Metal Content of Alkaline Phosphatase, 1962-1968. The earliest preparations of alkaline phosphatase in this laboratory (Plocke et al., 1962), obtained from whole cell extracts, contained zinc and magnesium as the major metal constituents, as measured by emission spectrography (Table 11, no. 1-5). The zinc content ranged from 1100 and 2300 pg/g of protein, averaging 1700 yg/g or 2.1 g-atoms of zinc per molecular weight of 80,000 as determined by Garen and Levinthal (1960) or 2.3 g-atoms/89,000 as determined later by Simpson et al. (1968). The concentration of magnesium ranged from 190 to 300 pg/g of protein, averaging 240 pg/g or 0.8 g-atom/80,000 or 0.9 g-atom/89,000. However, the sample containing the highest amount of zinc, 3.1 gatoms/89,000, also exhibited the highest activity, 41 units (Table 11, 2). Several preparations contained variable but stoichiometric quantities of iron and calcium. The purification scheme of Neu and Heppel (1965) releases alkaline phosphatase from the periplasmic space of E . coli using osmotic shock after treatment with EDTA and sucrose a t p H 7.5. This procedure was employed to obtain preparations 6-12, Table I (Simpson et al. 1968). Preparations 6 and 7, obtained by crystallization from (NH4)2S04 and MgC12, had activities of 36 and 40 units. They contained 2000 and 2200 pg of zinc/g or 2.8 and 3.0 g-atoms/ 89,000 in addition to 550 and 760 pg of magnesium/g or 2.0 and 2.8 g-atoms/89,000. Preparations 8-12, Table 11, BIOCHEMISTRY, VOL.
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Metal Content of Alkaline Phosphatase, Recent PrepaTable 11: Metal Content of Alkaline Phosphatase Measured by Emission Spectrogaphy (1962-1968). Specific Activity Sample (units) pg/g la 2 3 4 5 66 7 8C 9 10 11 12
24 41 24 35 39 36 40 39 43 40 39 42
1400 2300 1100 2000 1600 2200 2000 2500 3100 2800 2600 2700
Zinc
Magnesium
g-atom/ g-atom/ 80,000 89,000 1.8 2.8 1.4 2.4 2.0
1.9 3.1 1.5 2.7 2.2 3.0 2.8 3.5 4.2 3.9 3.7 3.8
pg/g 240 190 210 300 240 760 550 430
IN E . COLI A L K A L I N E P H O S P H A T E S
Plant Physiol. 54, 678. Ludwig, L. J., and Canvin, D. T. (1971), Can. J . Bot. 49, 1299. Murai, T., and Akazawa, T. (1972), Biochem. Biophys. Res. Commun. 46, 2121. Ogren, W. L., and Bowes, G. (1971), Nature (London), New Biol. 230, 159. Packer, L. Murakami, S., and Mehard, C. W . (1970), Annu. Rev.Plant Physiol. 21, 27 1. Paulsen, J. M., and Lane, M. D. (1966), Biochemistry 5, 2350. Pedersen, T. A., Kirk, M., and Bassham, J. A. (1966), Physiol. Plant. 19, 219. Pon, N. G., Rabin, B. R., and Calvin, M. (1963), Biochem. Z . 338, 7. Sugiyama, T., Nakayama, N., and Akazawa, T. (1968a),
Biochem. Biophys. Res. Commun. 30, 118. Sugiyama, T., Nakayama, N., and Akazawa, T. (1968b), Arch. Biochem. Biophys. 126. 737. Vogels, G. D., and van der Drift, C. (1970), Anal. Biochem. 33, 143. Walker, D. A. (1971), in Proceedings of 2nd International Congress on Photosynthesis Research, Vol. 111, Forti, G . , Avron, M., and Melandri, A., Ed., W. Junk, The Hague, p 1773. Walker, D. A . (1973), New Phytol. 72, 209. Warburg, O., and Christian, W . (1942), Biochem. 2. 310, 384. Weissbach, A., Horecker, B. L., and Hurwitz, J. (1956), J . Biol. Chem. 218, 795. Werdan, K., and Heldt, H. W. (1972), Biochim. Biophys. Acta 283, 430.
Zinc and Magnesium Content of Alkaline Phosphatase from Escherichia coli f William F. Bosron,t F. Scott Kennedy,$ and Bert L. Vallee*
ABSTRACT: Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-atom of magnesium per molecular weight 80,000 (Plocke, D. J., Levinthal, C., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described
eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared. Collectively, the zinc content was found to be 4.0 f 0.3 g-atoms per molecular weight of 89,000 for enzyme purified and analyzed by procedures shown not to alter its intrinsic metal content adversely. Importantly, it is confirmed that the enzyme when isolated a t p H 7.2 and 7.5 contains an average of 1.3 f 0.2 g-atoms of magnesium/mol. This feature of the metal composition, hitherto largely unappreciated, bears significantly on the investigation of the structure of alkaline phosphatase as related to its chemical and functional properties.
T h e precision and sensitivity of methods applicable to the determination of the physical, chemical, and functional properties of enzymes have increased markedly in the last decade. Concurrently, there have been analogous advances in high resolution procedures for enzyme isolation. Purification of enzymes by means of such superior methodology may result in numerical values for the parameters characterizing their properties which differ from those obtained initially. Unfortunately, such determinations frequently do
not indicate definitively whether the numerical changes observed result from intrinsic differences in the enzyme being examined, variations in analytical methodology, or differing modes of calculation and representation of results. Escherichia coli alkaline phosphatase well illustrates these evolving problems in protein characterization. It was first isolated in 1960 (Garen and Levinthal, 1960) and soon thereafter shown to contain stoichiometric quantities of zinc and magnesium (Plocke et al., 1962). Since these early studies, native phosphatase, prepared from E. coli by a variety of methods, consistently has been shown to require minimally 2 g-atoms of zinc/mol of enzyme for catalytic activity (Simpson et al., 1968; Harris and Coleman, 1968; Reynolds and Schlesinger, 1969; Petitclerc et al., 1970; Trotman and Greenwood, 1971; Csopak and Szajn, 1973). Further, it was demonstrated that two additional zinc atoms stabilize secondary, tertiary, or quaternary structure and/or
~~~~~~
From the Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 021 15. Received March IO, 1975. This work was supported by Grant-in-Aid CM-15003 from the National Institutes of Health, Department of Health, Education and Welfare. *Recipient of traineeship under Grant GM-02123 from the National Institutes of Health, Department of Health. Education and Welfare.
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influence catalytic activity (Simpson et al., 1968; Reynolds and Schlesinger, 1969; Petitclerc et al., 1970; Trofman and Greenwood, 1971). However, the presence and the functional significance of these two additional zinc atoms have incurred considerable debate (Harris and Coleman, 1968; Applebury and Coleman, 1969; Csopak et ai., 1972a; Csopak and Szajn, 1973). Even though magnesium has been reported to enhance activity of the zinc enzyme (Plocke and Vallee, 1962), virtually all workers have examined only the stoichiometry of zinc in their preparations, while that of magnesium consistently has been, ignored, and systematic studies of its possible role in catalysis and/or structure stabilization are not on record. Furthermore, as a general rule phosphatase metal content has been expressed in terms of g-atom of metal per mole of protein rather than as the ratio of mass of metal per g of protein. Importantly, the molecular weight of the enzyme upon which such molar metal stoichiometry is based has varied significantly (Garen and Levinthal, 1960; Schlesinger and Barrett, 1965; Simpson et al., 1968; Applebury and Coleman, 1969; Trotman and Greenwood, 1971). Similarly, the absorbance a t 278 nm (Plocke et ai., 1962; Rothman and Byrne, 1963; Malamy and Horecker, 1964; Csopak et al., 1972b) and maximal specific activity (Plocke et al., 1962: Lazdunski and Lazdunski, 1967: Simpson et al., 1968; Harris and Coleman. 1968; Reynolds and Schlesinger, 1969; Csopak and Szajn, 1973) have differed widely. A number of factors may affect the value of these parameters: ( 1 ) the strain of E . coli employed, ( 2 ) the ambient conditions used in releasing phosphatase from the organism, (3) the procedures for subsequent purification, and (4) the analytical methodology and calculations used to define the characteristics of the enzyme. Initially, phosphatase was isolated from whole cell extracts and purified by precipitation at 80’ in high concentrations of magnesium followed by chromatography on DEAE-cellulose (Garen and Levinthal, 1960: Plocke et al., 1962). Since then, phosphatase purification procedures have been modified and improved repeatedly. Thus. following the finding that phosphatase is localized in the periplasmic space (Malamy and Horecker, 1961). the enzyme was released from E . coli by a variety of techniques, including lysozyme treatment in the presence of EDTA (Malamy and Horecker, I964), osmotic shock i n the presence of chelating agents (Neu and Heppel, 1965). and treatment of cells a t acid pH releasing metal-free subunits requiring activation (Schlesinger and Olsen. 1970). Following release, the enzyme generally n a s purified by means of DEAE-cellulose chromatography, gel filtration, and ammonium sulfate precipitation or crystallization. The purified enzyme. however, can be resolved into three forms by high resolution DEAE-cellulose chromatography, although the characteristics of these isozymes have not been examined in detail (Lazdunski and Lazdunski. 1967; Simpson et al., 1968). Several of the aforementioned procedures, viz., exposure to EDTA or ammonium sulfate and addition of high concentrations of magnesium and/or zinc, previously have been suggested as parameters responsible for altering the metal content of native enzyme (Simpson et al., 1968: Ulmer and Vallee, 1971: Csopak and Szajn, 1973). The present paper summarizes the metal content of the consecutively numbered alkaline phosphatase preparations since such studies were first initiated in this laboratory. They are compared with analogous data on preparations obtained elsewhere using various methods of purification.
2276
BIOCHEMISTRY, VOL
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KEhNEDY. A U D \ A I
I Et.
The study confirms the presence of zinc and magnesium in alkaline phosphatase from E . coli, delineates their stoichiometry, and simultaneously identifies some of the factors which may affect the metal content of this enzyme. Further, a purification procedure is described which eliminates the features of all methods known to alter metal content of Phosphatase. In addition, it is emphasized that careful observance of metal-free technique (Thiers, 1957) and ashing of alkaline phosphatase samples prior to analysis by atomic absorption spectrometry are essential to accurate determination of the enzyme’s metal content. Collectively. the data demonstrate that the phosphatase contains 4.0 f 0.3 gatoms of zinc and 1.3 f 0.2 g-atoms of magnesium per molecular weight 89,000 when isolated and purified a t p H 7.2 and 7.5 by methods which do not affect its metal content adversely. Experimental Section
Purification of Alkaline Phosphatase. I n this laboratory preparations of alkaline phosphatase were initially obtained from cultures of E . coli K-12 either wild-type or constitutive for phosphatase, grown in a lactate-inorganic salts or tryptone-yeast extract medium (Table 11, no. 1-5) (Plocke et al.. 1962). Acetone powders from whole cell extracts w8ere then purified utilizing heat precipitation at 80’ in 0.01 M M g S 0 4 followed by chromatography on DEAE-cellulose. Preparations 6-26, Tables 11-IV, were obtained from periplasmic extracts of E. coli. Strain C-90 (Tables 11 and 111, no. 6-20) was grown to stationary phase at 37’ in 100or 14-1. cultures in a Tris-peptone medium (Simpson et al., 1968), and strains K-10, C4F1, and CW-3747’ (Table IV, no. 21-26) were grown similarly in a 3-1. culture using “ 1 21 Peptone” medium (Torriani, 1966). The cells were harvested by centrifugation and washed in 0.01 M Tris-C1 (pH 7.5). Periplasmic extracts were then prepared using osmotic shock either in sucrose and EDTA or sucrose and Chelex 100 (Neu and Heppel, 1965). When EDTA was utilized to release the enzyme (Tables 11-IV, no. 6-16 and 21-26) a 2-6% cell suspension was prepared a t room temperature in 20% sucrose, M EDTA, and 0.03 M Tris-CI (pH 7.5). and followed by centrifugation. The cells were shocked with the same volume of cold, distilled water. When Chelex 100 (Bio-Rad, 1-200 mesh) was used (Table 111, no. 17-20), a 2% cell suspension was prepared in 5% Chelex, 20% sucrose. and 0.03 M Tris-CI (pH 7.5). Under these circumstances, an additional centrifugation for 5 min at 600g was required to separate cells from Chelex, thereby assuring that the phosphatase released subsequently never comes into contact with any chelating agent and eliminating all effects in this step potentially attributable to EDTA (Ulmer and Vallee. 1971: Csopak and Szajn, 1973). The water extract from the osmotic shock medium was purified by batchwise absorption on DEAE-cellulose (Whatman DE52), equilibrated with 0.01 M imidazole-CI (pH 7.2), and eluted with 0.1 M NaC1. T h e protein was concentrated and dialyzed by ultrafiltration (Amicon PM-IO membrane). Preparations 6-26, Tables 11-IV, were purified by column chromatography on DEAE-cellulose in 0.01 M imidazole-CI (pH 7.2), using a linear gradient of NaCl to 0.1 M . The broad peak of activity, containing the isozymes. was
’
Kindlq provided by Dr. A . M . Torriani, Department of Biolog). V.1.T.
Z I N C A N D MAGNESIUM
IN
E . COLI
ALKALllU E
concentrated and dialyzed by ultrafiltration. In some cases rechromatography on DEAE-cellulose (Table 111, no. 15 and 19) or on Sephadex (3-150 (Pharmacia, 40-120 p ) in 0.01 M Tris-CI ( p H 7.5) (Table 111, 17 and 18) was employed to obtain enzyme of high specific activity. Preparations 6 and 7, Table 11, were prepared by Simpson et al. (1968) using crystallization from (NH4)2S04 and MgC12 according to the method of Malamy and Horecker (1964). Phosphatase Assays. Enzymatic activity was measured by following the hydrolysis of 4-nitrophenyl phosphate (Sigma 104) using a Gilford Model 222 spectrophotometer with a Heathkit Model IR-18M recorder. The release of 4nitrophenol in 1 M Tris-C1 ( p H 8.0) and 25' was determined a t 400 nm using a molar absorbance of 1.68 X lo4. A unit of activity is defined as the number of micromoles of substrate hydrolyzed per minute per milligram of protein under these conditions. Phosphatase concentration was determined spectrophotometrically using A27g( l%) 7.2 (Simpson et al., 1968). Electrophoresis. Polyacrylamide disc gel electrophoresis was performed in Tris-glycine buffer ( p H 9.4) (Gabriel, 1971) or in sodium dodecyl sulfate, as suggested by Weber and Osborn (1969). Protein was visualized on polyacrylamide gels using Coomassie Brilliant Blue stain (Smith, 1968) and phosphatase activity was determined using a-naphthyl phosphate and Fast Red T R (Gabriel, 1971). Metal Analysis. Two methods, spark emission spectrography and atomic absorption spectrometry, were employed for zinc and magnesium analysis. Primary working standards for metal analysis were prepared by dissolving weighed quantities of spectroscopically pure zinc rod and magnesium metal (Johnson, Matthey and Co., Ltd.) in metal-free HCI. To determine accuracy of the methods a zinc spelter, NBS sample 109; a copper-zinc-nickel alloy, N B S sample 157-A; a magnesium base alloy, N B S sample 171; and dolomite, N B S sample 88, all of certified metal content, were dissolved in metal-free HCI in the same manner as the aforementioned standards. All phosphatase samples were dialyzed for 4 days against multiple changes of a 100-fold excess of metal-free 0.01 M Tris-C1 ( p H 8.0) to remove unbound metals. For emission spectrography, 5-15 mg of protein was ashed in an electric muffle furnace at 450' for 24 hr (Thiers, 1957). The ash was dissolved in 6 N HCI containing 7 pg/ml of vanadium as an internal standard. Intensity ratio curves were constructed for each spectrographic plate by sparking, in triplicate, four standard concentrations of zinc, magnesium, and 15 other elements along with duplicate phosphatase samples (Vallee, 1955). For zinc concentrations between 1 and 30 pg/ml, the coefficient of variation for this method varies from 7 to 10% (Thiers and Vallee, 1957) and the accuracy is 100 f 7%. Atomic absorption spectrometry for zinc was performed with equipment previously described (Fuwa and Vallee, 1963). The coefficient of variation for this method is below 3% and the accuracy is 100 f 5% (Fuwa et al., 1964). Phosphatase samples were ashed, then dissolved in 0.1 N HCI, and diluted with metal-free water to a zinc concentration of between 0.01 and 0.1 g/ml. For comparative purposes samples were analyzed prior to ashing to determine the effect of the organic matrix on zinc determination (Table I). In every case the zinc content of an ashed sample was found to be higher than the identical nonashed sample. Furthermore, the average zinc content of ashed samples, 2900 f 180 pg/g or 4.0 f 0.3 g-atoms/89,000, was 12% higher than
PHOSPHATASE
Table I: Zinc Content of Alkaline Phosphatase as Determined by Atomic Absorption Analysis of Ashed and Nonashed Samples. Zinc Pretreatment
Number of Samplesa
Ashed Nonashed
14 14
g-atom/ 89,000 mol wt
wg/g
2900 2600
t i
180 190
4.0 + 0.3 3.6 i 0.3
a Preparation numbers 13-26.
that of the same nonashed samples, 2600 f 190 pg/g or 3.6 f 0.3 g-atoms/89,000. Atomic absorption spectrometry for magnesium was performed as described by Wacker et al. (1 965). Samples were initially diluted with metal-free water to a magnesium concentration ranging from 0.05 to 0.5 pg/ml and finally 1:l with 8-hydroxyquinoline (G. Fredrick Smith Chemical Co.) in 1 N HCI to obviate any interferences. The coefficient of variation for standards with this method is below 6% and the accuracy is 100 f 1% (Iida et a]., 1967). The metal content of phosphatase is expressed both in pg per g of protein and in g-atom of metal per molecular weight of 89,000 (Simpson et al., 1968) to facilitate comparsions with the results of these and other studies. Preparation of Reagents. Analytical grade chemicals were used throughout, and all solutions were prepared with deionized and distilled water. All buffers were extracted with 0.001% diphenyldithiocarbazone in carbon tetrachloride until free of transition metals. In order to remove magnesium, all solutions extracted in this manner were treated further with Chelex 100. Glassware and polyethylene containers were rendered metal-free by soaking overnight in a 1 : 1 mixture of concentrated nitric and sulfuric acids, followed by copious rinsing with metal-free water (Thiers, 1957). Results
Metal Content of Alkaline Phosphatase, 1962-1968. The earliest preparations of alkaline phosphatase in this laboratory (Plocke et al., 1962), obtained from whole cell extracts, contained zinc and magnesium as the major metal constituents, as measured by emission spectrography (Table 11, no. 1-5). The zinc content ranged from 1100 and 2300 pg/g of protein, averaging 1700 yg/g or 2.1 g-atoms of zinc per molecular weight of 80,000 as determined by Garen and Levinthal (1960) or 2.3 g-atoms/89,000 as determined later by Simpson et al. (1968). The concentration of magnesium ranged from 190 to 300 pg/g of protein, averaging 240 pg/g or 0.8 g-atom/80,000 or 0.9 g-atom/89,000. However, the sample containing the highest amount of zinc, 3.1 gatoms/89,000, also exhibited the highest activity, 41 units (Table 11, 2). Several preparations contained variable but stoichiometric quantities of iron and calcium. The purification scheme of Neu and Heppel (1965) releases alkaline phosphatase from the periplasmic space of E . coli using osmotic shock after treatment with EDTA and sucrose a t p H 7.5. This procedure was employed to obtain preparations 6-12, Table I (Simpson et al. 1968). Preparations 6 and 7, obtained by crystallization from (NH4)2S04 and MgC12, had activities of 36 and 40 units. They contained 2000 and 2200 pg of zinc/g or 2.8 and 3.0 g-atoms/ 89,000 in addition to 550 and 760 pg of magnesium/g or 2.0 and 2.8 g-atoms/89,000. Preparations 8-12, Table 11, BIOCHEMISTRY, VOL.
14, N O .
10, 1 9 1 5
2277
B O S R O N , K E N N E D Y , A Y D VAI.L.EE
Metal Content of Alkaline Phosphatase, Recent PrepaTable 11: Metal Content of Alkaline Phosphatase Measured by Emission Spectrogaphy (1962-1968). Specific Activity Sample (units) pg/g la 2 3 4 5 66 7 8C 9 10 11 12
24 41 24 35 39 36 40 39 43 40 39 42
1400 2300 1100 2000 1600 2200 2000 2500 3100 2800 2600 2700
Zinc
Magnesium
g-atom/ g-atom/ 80,000 89,000 1.8 2.8 1.4 2.4 2.0
1.9 3.1 1.5 2.7 2.2 3.0 2.8 3.5 4.2 3.9 3.7 3.8
pg/g 240 190 210 300 240 760 550 430
