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range of 7 to 8, the actual measured optimum varying with the buffer used. Estimated Molecular Weight. The sedimentation coefficient as determined by sucrose density gradient centrifugation is about 4.0 S, suggesting a molecular weight of about 52,000. Stability. Partially purified enzyme kept at 4 ° retained 40% of its activity after 2 months. Note on Occurrence and Specificity. Phospho-fl-glucosidase aci~ivity occurs in several members of the Enterobacteriaceae. 8 Some organisms (A. aerogenes and Escherichia coli) are reported to possess two phosphofl-glucosidases which have different ratios of activity on various phosphofl-glucosides, 8,9 but the enzymes in those investigations were not tested for activity on cellobiose monophosphate. Although the phosphocellobiase reported here has not been purified highly, several lines of evidence are consistent with the view t h a t the activity of the partially purified preparation on various phospho-fl-glucosides is the result of a single enzyme2 '~S. Schaefler and I. Schenkein, Proc. Nat. Acad. Sci. U.S. 59, 285 (1968). 9C. F. Fox and G. Wilson, Proc. Nat. Acad. Sci. U.S. 59, 988 (1968).

[75] ~-Galactosidases from N e u r o s p o r a crassa

By RICHARD STEPHENS and A. GIB DEBUSK (~)-R-O-D-galactopyranoside -~- H20 ~ ROH ~ D-galactose The occurrence of multiple forms of fl-galactosidase in Neurospora was first reported by Bates and Woodward, 1 who described an alkaline fl-galactosidase physically separable from the pH 4 optimum enzymes previously described by Landman and Bonner. ~ The acid fl-galactosidase has been further characterized by Johnson aDd De Busk, 3 who have described two stable forms of the enzyme with, in addition to other differences in physical properties, pH optima of 4.2 and 4.5. The fl-galactosidase system of Neurospora thus consists of the pH 7.5 enzyme and two activities at about pH 4, the 4. 2 fl-galactosidase and the 4.5 fl-galactosidase. The purification of the 4.2 enzyme is essentially that of Johnson and DeBusk. ~ The pH 7.5 fl-galactosidase has yet to be completely purified, and the properties given are those of partially purified extracts. i W. K. 20. E. 3H. N. 4H. N.

Bates and D. O. Woodward, Science 146, 777. Landman and D. M. Bonner, Arch. Biochem. Biophys. 41, 283 (1952). Johnson and A. G. DeBusk, Arch. Biochem. Biophys. 138, 412 (1970). Johnson and A. G. DeBusk, Arch. Biochem. Biophys. 138, 408 (1970).

498

GLYCOSIDASES

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I. pH 4.2 and 4.5 ~-Galactosidase

Assay Method Principle. fl-Galactosidase activity is assayed spectrophotometerically by measuring the release of o-nitrophenol from o-nitrophenyl-fl-Dgalactopyranose (ONPG). Absorbance is measured at 400 nm in alkaline solution. Reagents ONPG (Sigma), 10 mM in sodium phosphate-citrate buffer, 80 mM pH 4.3 Potassium carbonate, 0.5 M Enzyme. Clear supernatant of crude extract or product of subsequent steps. Dilute in 8 mM phosphate-citrate buffer, pH 4.3, so that 0.1 ml contains approximately 20 units as defined below. Procedure. All assays are at 37 ° . The reaction is initiated by the addition of 0.4 ml of ONPG to 0.1 ml of enzyme. At the end of 20 or 30 min, the reaction is terminated by the addition of 2.5 ml of 0.5 M potassium carbonate. The entire reaction mixture (3 ml) is placed in a silica cell, and absorbance is measured at 400 nm against a blank in which water replaces ONPG. Change in absorbance due to o-nitrophenol released is obtained by subtracting the absorbance of a control in which water replaced enzyme. Both the 42 and 4.5 fl-galactosidase occur in the culture medium of induced cells. The extracellular enzymes may be assayed by using 1.0 ml of the culture filtrate as enzyme, initiating the reaction by the addition of 1.0 ml of ONPG and terminating the reaction with 1.0 ml of 0.5 M potassium carbonate. Definition o] Enzyme Unit. One unit of activity is defined as that amount of enzyme required to release 1 nmole of o-nitrophenol per minute under the assay conditions. Specific activity is defined as units of activity per milligram of protein as measured by the method of Lowry et al. 5 with Armour Serum Albumin fraction V used as the reference standard. Purification Procedure Growth of Cultures. The strain of Neurospora crassa used is the Oak Ridge wild-type 74-0R23-1A obtained from the Fungal Genetic Stock Center, California State University, Humboldt, Arcata, California. 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

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~-GALACTOSIDASES FROM Neurospora crassa

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Stocks are maintained in continuous vegetative culture on Vogel's Medium N 6 supplemented with 2% commercial grade sucrose as a carbon source and 2% Difco Bacto-agar. Mycelia to be used for enzyme isolations are grown in 2800-ml Fernbach flasks containing 1500 ml of the same medium but without agar. These cultures are started from a heavy (approximately 50 mg) dry conidial inoculum and are grown for 60-72 hr at 25 ° on a gyrorotary shaker at approximately 100 rpm. At the end of this period the medium is removed by aspiration, 1500 ml of fresh Vogel's Medium N supplemented with B-lactose to 30 mM is added, and the cultures are left on the shaker for an additional 60 hr in order to induce the enzymes. Induced mycelia are harvested by vacuum filtration on a Biichner funnel lined with Whatman No. 1 filter paper, washed with 1 liter of cold water and lyophilized. All subsequent steps are carried out at 0-4 ° unless otherwise indicated. Step 1. Preparation o] Crude Extracts. Lyophilized mycelia are ground by hand under liquid nitrogen in a precooled porcelain mortar. The resulting fine powder is suspended in 10 mM phosphate buffer, pH 7.5, at a ratio of 15 ml of buffer per gram of mycelial powder. The suspension is homogenized in a VirTis homogenizer and sonicated in 100-ml batches in a Bronwill Biosonik II for 30 sec at full strength. This preparation is centrifuged at 15,000 g for 30 min. The clear supernatant contains virtually 100% of both the pH 4.2 and the pH 4.5 fl-galactosidase. Step 2. First Ammonium Sul]ate Fractionation. Solid ammonium sulfate (Mann, special enzyme grade) is added to the supernatant to 33% of saturation (196 g/100 ml) with continuous stirring. After 15 min the precipitate is removed by centrifugation at 27,000 g for 15 min, and the supernatant is taken to 75% of saturation (29.2 g/100 ml) with ammonium sulfate and stirred slowly for 4 hr. The precipitate, containing approximately 75% of the original 4.2 fl-galactosidase activity, is again collected by centrifugation and redissolved in one-tenth the original volume of 10 mM phosphate buffer, pH 7.5. The supernatant may be discarded. Step 3. Acid Precipitation. The redissolved ammonium sulfate precipitate is taken to pH 4.0 by the slow addition of 1 M citric acid. This is stirred 8-10 hr, at which time the copious precipitate is removed by centrifugation and discarded. Step 4. Dialysis. The acid supernatant is dialyzed 12 hr against at least 10 volumes of distilled water. The dialyzate should be changed once after about 4 hr. Step 5. Second Ammortium Sul]ate Fractionation. The dialyzed preparation is clarified by centrifugation at 15,000 g for 30 rain. The supernaH. J. Vogel, Microbial Genet. Bull. 13, 42 (1956).

500

GLYCOSIDASES

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rant is brought to 75% of saturation with solid ammonium sulfate and stirred for 8 hr. The precipitate is collected by centrifugation and redissolved in 1/100 the original volume of 8 mM sodium phosphate-citrate buffer, pH 4.3, made 10 mM with 2-mercaptoethanol. Step 6. CM-Sephadex Chromatography. The redissolved precipitate is chromatographed on CM-Sephadex, C-50, medium mesh column (Pharmacia of Uppsala, Sweden). The column is equilibrated with 8 mM phosphate-citrate buffer, pH 4.3, made 10 mM with 2-mercaptoethanol and eluted with a 500-ml linear 0.1 M to 1.0 M sodium chloride gradient. The 4.2 fl-galactosidase elutes at approximately 0.4 M NaC1. Peak fractions are homogeneous on polyacrylamide gel electrophoresis in both cathodic (pH 5.0) and anodie (pH 9.5) systems. A summary of the purification procedure is given in Table I. Since dialysis against distilled water causes a rapid conversion of the pH 4.5 fl-galactosidase to the 4.2 enzyme, the 4.5 enzyme may be isolated using steps 1 through 3 as above followed by steps 4 and 5 below. Step 4. Chromatography an Sephadex G-25. The supernatant from the acid precipitate is placed in dialysis tubing and packed in dry commercial sucrose. When the volume is reduced to approximately ~ o the starting volume, the preparation is layered onto a Sephadex G-25 column equilibrated with 8 mM phosphate-citrate buffer, pH 4.3, made 10 mM with 2-mercaptoethanol. Step 5. CM-Sephadex Chromatography. The active fractions from the Sephadex G-25 column are chromatographed on a CM-Sephadex column as described in step 6 above. The 4.5 fl-galactosidase elutes at approximately 0.6 M sodium chloride. Polyacrylamide gel electrophoresis of the

TABLE I SUMMARY OF PURIFICATION OF p H

4.2 ~-GALACTOSIDASEa Re-

Step 1. 2. 3. 4. 5.

Crude extract 75% (NH4)2SO~ ppt. p H 4.0 soluble After dialysis 75% (NH4)2SO4 ppt. after dialysis 6. CM eluent

Volume (ml)

Protein (mg)

Total activity b

Specific activity

covery (%)

200 100 100 110 20

3745 1075 178 38 6.6

20,255 15,333 15,333 15,000 13,775

5.4 14.1 86.1 392 2078

100 76 76 74 68

5

0.298

9,925

33,250

49

From H. N. Johnson and A. G. DeBusk, Arch. Biochem. Biophys. 138, 408 (1970). b Enzyme units.

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~-GALACTOSIDASES FROM Neurospora

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peak fractions results in two protein bands, both with fl-galactosidase activity. The major band corresponds to the 4.5 fi-galactosidase and a minor, faster moving, band having the properties of the 4.2 enzyme. It is felt that contamination by the 4.2 fl-galactosidase is due to conversion of the 4.5 enzyme to the 4.2 enzyme under conditions required for the electrophoresis rather than contamination of the CM-Sephadex eluate. Properties of the 4.5 fl-galactosidase were determined using the fractions of highest activity which were eluted from the CM-Sephadex column. That fraction of the initial activity of a crude extract attributable to the 4.5/?-galactosidase varies between 30 and 60% of the total depending on the age and condition of the culture.

Properties Stability. The purified 4.2 fl-galactosidase is stable at 4 ° with no significant loss of activity over a 30-day period. The 4.5 enzyme is slowly converted to the 4.2 form in solution. Both enzymes are considerably less stable in crude extracts. The 4.2 enzyme has a half-life of 28 rain at 52 °. The 4.5 enzyme has a half-life of 45 min under the same conditions. pH Optimum. The enzymes are designated according to their pH optima. The p H dependence curves are very similar, both enzymes retaining 18-20% activity at pH 7.5. Isoelectric Point. Using an LKB Ampholine column with pH 3-6 range ampholytes, the isoelectric point of the 4.2 fi-galactosidase is pH 4.82. The pI for the 4.5 enzyme is 5.83. Molecular Weight. Gel filtration on calibrated Sephadex G-200 columns gives a molecular weight estimate of 96,000. The enzymes are not separable by either gel filtration or centrifugation in sucrose gradients. Amino Acid Composition. An amino acid analysis of the 4.2/~-galactosidase is shown in Table II. The number of residues is based on a toolecular weight of 96,000. It is interesting that the enzyme contains no arginine. Carbohydrate Content. Assuming a molecular weight of 96,000, the purified 4.2 B-galactosidase is about 5% by weight carbohydrate. The enzyme contains 28 moles of covalently bound carbohydrate per mole protein. Carbohydrate was determined by the phenol-sulfuric acid method and compared to a glucose standard. A qualitative analysis of the carbohydrate portion of the enzyme has not been done. Substrate Specificity. Both forms of the enzyme are specific for /~galactosides. Thiogalactosides, a-galactosides, and fl-glucosides are not substrates. Galactosides substituted on the galactose ring have not been tested.

502

GLYCOSIDASES

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TABLE II AMINO ACID COMPOSITION Of TI-IE pH 4.2 ~-GALACTOSIDASEa

Residues/MW of 96,000 Amino acid

Found

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

80.77 14.48 0.0 84.58 43.18 202.06 140.84 26.04 258.57 92.33 -22.22 1.27 14.48 26.04 13.21 7.49

Nearest integer 81 15 0 85 43 202 141 26 259 92 -22

1 15 26 13 8 1029

a From H. N. Johnson and A. G. DeBusk, Arch. Biochem. Biophys. 138, 408 (1970).

Kinetics. The Km of the 4.2 fl-galactosidase for O N P G under the assay conditions is 0.45 mM. The Km of the 4.5 enzyme under the same conditions is 1.25 mM. II. p H 7.5 fl-Galactosidase

Assay Method Principle and Procedure. The assay for the 7.5 fl-galactosidase is identical to the assay for the 4.2 and 4.5 enzymes. .Reagents ONPG, 5.0 m M in potassium phosphate buffer, 10 mM, p H 7.5 Other reagents are as described for the pH 4.2 and 4.5 enzymes. Purification Procedure The procedure that follows, although not a complete purification, yields a stable preparation of the 7.5 fl-galactosidase which has been

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~-GALACTOSIDASES FROM Neurospora crassa

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used to determine the properties of the enzyme. The growth of cultures and preparation of crude extracts are the same as described for the 4.2 and 4.5 enzymes with the exception that the inducing medium should be supplemented with 30 mM D-galactose rather than fl-lactose. Solid ammonium sulfate is added to a crude extract to 40% of saturation and stirred for 5-10 min. This is immediately centrifuged at 27,000 g for 15 rain. The precipitate is redissolved in one-half the original volume of 10 mM potassium phosphate buffer, pH 7.5, and stirred for 2 hr. This preparation is rapidly heated to 50-54 ° and maintained there with continuous stirring for 15 min. This is cooled in an ice bath to 0-5 °, and the precipitate is removed by centrifugation. The clear supernatant contains approximately 20% of the 7.5 fl-galactosidase activity of the crude extract. This preparation is stable at 4 ° for several weeks and contains no fl-galaetesidase activity assayable at pH 4.3. Because of a tendency for the 7.5 enzyme to form enzymatically active but insoluble aggregates, additional fractionations with ammonium sulfate and further concentration of this preparation should be avoided. Further purification by a variety of ion exchange and affinity matrices has not been successful.

Properties Properties of the 7.5 fl-galactosidase were determined using the supernatant from the heat-treated preparation. The enzyme has a sharp pH optimum at pH 7.5. The isoelectric point is at pH 7.5. Gel filtration on calibrated Bio-Gel P-300 columns. (Bio-Rad Laboratories, Richmond, California) give a molecular weight estimate of 156,000. The enzyme is sensitive to disulfide reducing reagents, indicating the presence of at least one essential disulfide bond. Specificity. The preparation contains no detectable carbohydrase activity other than fl-galactosidase. The enzyme forms small amounts of an unidentified product different than the expected hydrolysis products with fl-lactose as the substrate. This "transferase" activity is a function of the 7.5 fl-galactosidase, not a contaminating protein because an electrophoretieally altered 7.5 fl-galactosidase which is isolated from a mutant strain of Neurospora forms only the expected hydrolysis products.

Beta-galactosidases from Neurospora crassa.

[75] f~-GALACTOSIDASES FROM Neurospora crassa 497 range of 7 to 8, the actual measured optimum varying with the buffer used. Estimated Molecular We...
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