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Purification and characterization of intracellular galactose oxidase from Dactylium dendroides
ARCHIVESOFBIOCHEMISTRY ANDBIOPHYSICS February 1, pp. 507-514,1987
Vol. 252, No. 2,
Purification
and Characterization of Intracellular Oxidase from Dactylium dendroides
MARCIA Departamento
de B&&mica, Received
H. MENDONCA Universidade May
12,1986,
Federal
AND do Parana,
and in revised
GLACI Caixa form,
Galactose
T. ZANCAN’ Postal
9S9,8O.o00
September
Curitiba,
PR., Brazil
5,1986
The intracellular galactose oxidase from Dactylium dendmides was purified to homogeneity with a 64% yield. The enzyme is a glycoprotein (7.7% neutral sugars, 1.7% aminosugars) with 72,000 Da of molecular mass. The enzyme showed nonlinear double reciprocal plots with O2 and D-galactose, suggesting cooperative binding for both substrates. The intracellular galactose oxidase catalyzes the oxidation of galactose derivatives and dihydroxyacetone but not of glycerol, glycolaldehyde, /3-hydroxipyruvate, and ally1 alcohol which are substrates for the extracellular enzyme. Compared with the extracellular galactose oxidase, the intracellular enzyme showed higher carbohydrate content and sensitivity to diethyldithiocarbamate. o 1981 Academic press, I~~.
Galactose oxidase (EC 1.1.3.9) is secreted into the medium during the exponential growth phase of Dactylium dendroides (1). The extracellular copper-containing enzyme (2) has a broad specificity (1, 3) and its properties have been extensively studied (4). Levels of extracellular galactose oxidase are enhanced by high oxygen tension whereas increases in the intracellular levels are much less significant (5), suggesting an efficient secretion mechanism. Secretion of enzymes in lower eukaryotes is associated with glycosylation (6,7). The extracellular galactose oxidase has a low (1% ) carbohydrate content (8) but the intracellular enzyme has not yet been studied. The present paper deals with the purification and characterization of intracellular galactose oxidase from D. dendroides and makes a study of its secretion possible. MATERIAL
AND
METHODS
Enzyme purification. Dactylium (formerly Polypwus circinatus) from (9) was grown as described by Markus
dendroides our collection et al. (10) with
1 To whom
correspondence
should
be addressed. 507
1% glucose as the sole carbon source. The mycelia were harvested by filtration after 36 h of growth, washed with distilled water, and kept frozen (-17°C). Frozen mycelia (120 g) were suspended in 240 ml of 10 mM phosphate buffer, pH 7.0, containing 0.5 mM EDTA. The suspension was submitted twice to treatment at 20,000 psi in a French Press (Amincon Instruments), centrifuged at 17,600g for 15 min, and the supernatant used as enzyme source. Fractions of 50 ml were heated for 5 min at 6O”C, then cooled in an ice-salt bath (-5°C). The precipitate was removed by centrifugation at 17,SOOg for 15 min, the supernatant was adjusted to 45% saturation with ammonium sulfate, and after standing for 10 min the mixture was centrifuged at 17,600 for 15 min. The ammonium sulfate content in the supernatant was increased to 65% saturation and the pellet which formed after centrifugation was dissolved in 10 ml of 10 mM phosphate buffer, pH 7.0. The preparation was dialyzed against 100 mM ammonium acetate buffer, pH 7.2, containing 0.5 mM CuSOl. After dialysis, the preparation was chromatographed on a Sepharose 6B column (1.0 X 18 cm) previously equilibrated with the same solution. The column was washed with 100 mM ammonium acetate, pH 7.2, until the protein concentration was less than 0.0025 mg ml-‘. The enzyme was eluted with 100 mM D-fUCOSe in 100 mM ammonium acetate, pH 7.2, and the enzymically active fractions (20-80 IJ) were pooled and frozen at -17’C. For kinetic experiments, the enzyme was dialyzed for 4 h in 100 mM ammonium sulfate, pH 7.2, to eliminate D-fUCOSe but the enzyme lost 35% of its activity with this procedure. No modification 0003-9861/W Copyright All rights
$3.00
0 1987 hy Academic Press, Inc. of reproduction in any form reserved.
508 in the kinetic of D-fucose.
MENDONCA parameters
was observed
in the presence
Enzyme assays. The catalytic activity during the enzyme purification was monitored by determining the hydrogen peroxide produced, according to the method of Aisaka and Terada (11). In most kinetic experiments, the amount of oxygen consumed was measured polarographically. Assay A. The standard assay mixture (1.0 ml) contained 15 pmol phosphate buffer, pH ‘7.0, ‘7.0 rmol phenol, ‘75 rmol D-galactose, 0.48 nmol of 4-aminoantipyrine, 6.7 U peroxidase, and enzyme. The reaction was initiated by the additions of galactose oxidase and the rate of HpOz formation was measured at 500 nm in a DU-2 Beckman spectrophotometer. To eliminate interference in the reaction, controls without enzyme or substrate were carried out. One enzyme unit was defined as the amount that catalyzed the formation of 1 rmol of HzOz per minute. Specific activities were defined as units per milligram of protein. Assay B. The reaction mixture (1.4 ml) contained 20 pmol phosphate buffer, pH 7.0,lOO nmol D-galactose, and the enzyme. The oxygen consumption was monitored using a Gilson oxygraph Model KIC, equipped with a Clark-type electrode and a water-bath assembly to maintain the temperature at 30°C. All solutions were previously equilibrated at 30°C for 15 min. When oxygen concentrations were higher than 20% a stream of pure OX was passed through the solutions, and for concentrations below 20% a stream of argon was passed through the solutions to obtain the appropriate oxygen concentrations. Enzyme inhibition studies. To determine the inhibition of galactose oxidase by several agents, these were preincubated with the enzyme for 10 min at 30°C in an air-saturated buffer solution before starting the reaction by 300 mM of D-galactose. Results are expressed as percentages of the noninhibited activity. Protein determination Protein concentrations were determined by the method of Lowry et al. (12) using serum albumin as standard, and by the method of Warburg and Christian (13). Purification of extracellular gabctose oxidase. The purification of extracellular galactose oxidase was performed according to Amaral et al. (14) until the DEAE-cellulose step. The DEAE fraction was chromatographed on Sepharose 6B column as previously described by Hatton (15). The final preparation (74 U/mg of protein) was homogenous on examination by polyacrylamide gel electrophoresis at pH 8.6 and 3.6 and had a molecular weight similar to that found by Kosman et al. (8). Polyacrylamide gel electrqphoresis The enzyme preparation was concentrated in a rotatory evaporator and subjected to electrophoresis in 8% polyacrylamide gel using 65 mM Tris-borate buffer, pH 8.9, or 100 mM glycine-HCI buffer, pH 3.6. Electrophoresis was performed at 2 mA per tube until the indicator dye reached the end of the gel.
AND
ZANCAN
Electrophoresis was carried out in an Ortee pulse power apparatus. The gels were stained for protein with Coomasie blue (16) and for enzyme activity visualization, gels run in parallel were incubated in 100 mM galactose and revealed by the peroxidase-dianisidine reagent. Molecular weight detevknation The molecular weight was determined by gel permeation chromatography using Bio-Gel P-150, according to Andrews (17). The following protein molecular weight standards were used to calibrate the column: la&albumin (14,200), carbonic anidrase (20,000), egg albumin (45,000), serum albumin monomer (66,000), and serum albumin dimer (132,000). The elution of protein standards was monitored by light absorption at 230 nm and that of galactose oxidase by enzymic activity using Assay A. Electrophoresis under denaturing conditions was carried out using a modification of the procedure of Fairbanks (18). Molecular weight standards used were crosslinked albumin (66,000 and 132,000) and hemoglobin (16,000, 32,000, 48,000, and 64,000). Analysis of carbohydrate composition. The homogenous preparations of intra- and extracellular galactose oxidase were exhaustively (76 h) dialyzed against distilled water to eliminate free sugars. The solutions were concentrated at reduced pressure at 20°C. The total neutral sugar concentration was determined by the phenol-sulfuric method (19). A sample (3 mg) of homogenous enzyme was hydrolyzed in 2 ml of trifluoracetic acid (TFA)2 for 15 h at 100°C in a sealed tube. The hydrolysates were concentrated in a rotatory evaporator, distilled water was added to facilitate removal of trace residues of TFA, and evaporation was carried out again. This procedure was repeated five times. Neutral sugars were converted into alditol acetates and analyzed by gas chromatography as previously described (9). Aminosugars were determined according to Boas (20) after hydrolysis by the method of Maxhimex (21). Neutral sugars were fractionated by descending paper chromatography on Whatman No. 1 paper using benzene:butanol:pyridine:water (1: 5:3:3) as eluant and visualized with silver nitrate (22). Reagents. All reagents were of analytical grade and were obtained from Sigma Chemical Company. The lactose, D-galactose, melibiose, and raffinose were from Calbiochem, 2-deoxy-D-galactose was from Nutritional Biochemical Company, and D-galactosamine, Dglucosamine, D-ghCOSe were from Merck-Darmstadt. RESULTS
PurQicatim and properties. The results of a typical purification of intracellular galactose oxidase are presented in Table I. Similar purification patterns were obtained in eight independent runs. The purification a Abbreviation
used: TFA,
trifluoracetic
acid.
INTRACELLULAR
GALACTOSE
OXIDASE TABLE
PURIFICATION
-
OF INTRACELLULAR Total volume (ml)
Fraction
Cell extract Thermal inactivation Ammonium sulfate, 45-65% Sepharose 6B column
390 370 9.5 10
OXIDASE Units two1 HA min-’ * min-‘)
7.68 1.90 18.00 0.175
0.810
1.900 20.760
FROM
D. de&-tides
(U/mg
Sp act protein)
Recovery (%I
0.105 0.387
100 86
1.153
20.300
procedure gave approximately 1104-fold purified enzyme with a 64% overall yield. The enzyme was eluted as a single peak from a Sepharose 6B gel column using Dfucose as eluent (Fig. 1). The pooled activity fraction showed a single homogenous band on polyacrylamide disc electrophoresis at pH 8.6 and 3.6 (Fig. 2) and this fraction was used for further characterization of
509
D. dendroides
I
GALACTOSE
Protein (mg * ml-‘)
FROM
63
116.000
64
the enzyme. This enzyme was stable for at least 6 months at -17°C. In all purification steps, the enzymatic activity was monitored by polyacrylamide gel electrophoresis and only one band of activity was detected throughout. Molecular weight. The molecular weight of intracellular galactose oxidase was estimated from its chromatographic mobility
1% 0 ELUTION
FIG. 1. Affinity chromatography of intracellular monium sulfate fraction (171 mg) was percolated washed with the equilibration buffer and eluted assayed for protein and enzyme activity.
WIldi
(ml)
galactose oxidase on Sepharose 4B column. using a flow rate of 0.5 ml min-‘. The column with 0.1 M D-fUCOSe. Fractions were collected
Amwas and
510
MENDONCA
AND
ZANCAN
also a substrate. Compounds such as glycerol, /I-hydroxypyruvate, glycolaldehyde, and ally1 alcohol were not oxidized. (Table III)
FIG. 2. Homogeneity of intracellular galactose oxidase. 100 gg of protein was subjected to analytical polyacrylamide gel electrophoresis, as described under Materials and Methods. Gels were stained (bands A and C) for protein and for activity (bands B and D). Migration was from the bottom to the top.
on gel by comparison with those of standard proteins. The molecular weight calculated from the plot of V,/V, versus log molecular weight was 72,000 (Fig. 3A). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a single Coomasie blue positive band with a molecular weight of 72,000 (Fig. 3B). Carbohydrate ccmpositim Analysis of intracellular galactose oxidase by the phenol-sulfuric acid assay for total neutral sugars gave a value of 7.7% by weight. The extracellular enzyme on submission to the same conditions, gave a value of 1.7% (Table II). The Elson-Morgan method, for analysis of amino sugars, gave a value of 1.5% for the intracellular enzyme and 0.40% for the extracellular form. Hydrolysis of the enzyme with 2 M TFA followed by paper chromatography revealed the presence of bound glucose, arabinose, galactose, and mannose. The identity of monosaccharides was confirmed by GLC analysis of derived hexitol acetates. Quantitative values for the monosaccharide composition are shown in Table II. The relative proportion of sugars was different for each enzymic form. Substrate specz&itg. Like the extracellular galactose oxidase (1, 13), the intracellular enzyme catalyzes the oxidation of the primary hydroxyl -._group _ of galactose and its derivatives. Dihydroxyacetone is
6
5 MXECULAFt
IO WEIGHT
IO4
FIG. 3. Determination of molecular weight of intracellular galactose oxidase. (A) The calibration curve for gel filtration column (1 X 41 cm) of Biogel P150. Each protein (150 fig) in 0.3 ml of 50 mM phosphate buffer, pH 7.2, was applied to the column previously equilibrated with the same buffer. Fractions of 1 ml were collected at a flow rate of 0.4 ml min-‘. Void volume (V,) was calculated from the elution volume (V,) of Dextran Blue. Standards used were (A) carbonic anidrase, (A) egg albumin, (0) serum albumin monomere, (Cl) serum albumin dimere, (0) intracellular galactose oxidase. The continuous trace in the insert shows the elution profile of enzyme. (B) The calibration curve for SDS electrophoresis. The electrophoresis conditions were described in the text using the following standards (molecular weights in parentheses): (0) hemoglobin (l-16,ooO, 2-32,000,3-48,000; 464,000); (0) bovine serum albumin (5-66,W 6132.000): ~~~,~ ,, (0) \ - , _palactose oxidase intracellular.
INTRACELLULAR TABLE
GALACTOSE
II
SUGAR COMPOSITION OF GALACTOSE OXIDASE FROM D. devuirodes Enzyme Intracellular Ilarbobydrate Total neutral sugars (%) Total amino sugars (%)
7.7 1.5
Neutral sugars Mannose Galactose Glucose Arabinose
Molar 1.0 0.98 4.8 5.8
Extracellular
1.7 0.4 proportion 1.0 1.2 3.8 1.9
Kinetic properties. Intracellular galactose oxidase activity gave rise to a sigmoida1 relation between velocity and galactose concentration, when measuring oxygen
TABLE
III
SUBSTRATE SPECFICITY OF INTRA- AND EXTRACELLULAR GALACTOSE OXIDASE Relative Substrate (100 mM) D-Galactose Dihydroxyacetone 2-Deoxy-D-galactose D-Galactosamine Methyl-a-D-galactopyranoside O-Nitrophenyl-P-Dgalactopyranoside Lactose Melibiose Raffinose Stachyose Guaran Ally1 alcohol” Glycerol Glycoaldehyde” /&Hydroxypiruvate”
Intracellular
velocity
(% )
FROM
511
D. clewhides
consumption or H202 formation (Fig. 4). Initial rate measurements with dihydroxyacetone gave a linear double plot with a Km value of 20 mM. The Oz saturation curve in presence of saturating galactose concentration has also shown a sigmoidal pattern (Fig. 5). The So,s value for Oz was 0.29 mM. The Hill coefficients were 1.36 for galactose and 1.66 for oxygen. Effect of inhibitors. The intracellular galactose oxidase activity was inhibited by metal ligands such as cyanide, sodium azide, and hydroxylamine (Table IV). The intracellular form is probably a copper enzyme, as its activity was affected by 5 pM diethyldithiocarbamate, a known copper chelator. (Fig. 6). DISCUSSION
The purification of intracellular galactose oxidase by affinity chromatography on Sepharose (polymer of D-galactose and 3,6 anidro-L-galactose) was only achieved when D-fucose (6-deoxi-D-galactose) was used as eluent. When galactose was used the enzyme lost its activity, probably due to H202 formation. The extracellular enzyme form was eluted using a column buffer without sugar additions (15), sug-
Extracellular
100 179 52 60
100 263 65 39
170
106
40 11 68 144 161 47 0.0 0.0 0.0 0.0
OXIDASE
21 13 83 96 3.5 1.3 13.5 15.0
Note. Assays performed by method A using and 0.25 pg of intraand extracellular enzyme, spectively. Guaran concentration was estimated described by Avigad et aL (1). a Assays performed using 0.1 pg of each enzyme 300 mM of substrates.
0.13 reas and
D-GALKTCSE
(mM)
FIG. 4. Dependence of reaction rate on galactose concentration. Assays were performed as described under Materials and Methods using method B except that the concentration of substrate was changed: (0) 20% oxygen; (0) 60% oxygen.
512
MENDONCA
AND
I /iSI 0
03
LO OXYGEN
CmM)
FIG. 5. Effect of oxygen on the rate of galactose oxidase oxidation. Assays were performed measuring the oxygen consumption (Assay B) using 1.0 pg of protein. Oxygen concentration was adjusted as described under Materials and Methods. Galactose concentration was 300 InM.
gesting that the intracellular form was more tightly bound to Sepharose than the extracellular enzyme. Only a single band of galactose oxidase activity was observed in our crude preparations of mycelial extracts of D. dendroides, although Tressel and Kosman (23) have described isoenzymic forms in their extracellular preparations. It seems probable that the isoenzymic forms are postsecretional products since it is known that proteases are present in crude extracellular enzyme preparations (15). The substrate specificity of the intracellular galactose oxidase for galactose derivatives was found to be similar to that of the extracellular enzyme (1,3,24). The extracellular galactose oxidase catalyzes the oxidation of dihydroxiacetone (25) and nearly all primary alcohols (4) while the intracellular form had a more restricted specificity (Table III). Intra- and extracellular galactose oxidase behave differently with respect to the kinetics of galactose oxidation. Under the same assay conditions, the extracellular form behaved as a Michaelian enzyme
ZANCAN
while the intracellular form gave a sigmoidal pattern (using air or 60% of 02 as gas phase). Sigmoidal behavior has also been described for the extracellular galactose oxidase from Gibberella fujikumi (26). The suggestion originally made by Hamilton et al. (27) that the extracellular enzyme could exist as inactive and active forms, and recently stressed by Johnson et al. (28), who proposed a turnover between inactive Cu,,-enzyme to active Cut-enzyme, could explain the kinetic cooperativity we observed for the intracellular homogenous galactose oxidase, in the presence of oxygen or galactose. However no catalytic lags or bursts could be detected as would be expected if low molecular transitions similar to those observed for hysteretic enzymes (29) had occurred. Galactose and Oz apparently induce a conformational modification promoting the conversion of a less active form to a more active enzyme form. Another significant difference observed between the intra- and extracellular enzymic forms was their sensibility to metal ligands and copper chelators, suggesting that the copper in the active center is more accessible in the intracellular form than in the extracellular one. (Fig. 6). No activity of superoxide dismutase could be detected in the homogenous intracellular enzyme, as has been described for the extracellular form (30). The intracellular galactose oxidase is a single polypeptide chain with a molecular weight of 72,000, larger than that of 68,000 described for the extracellular form (8).
TABLE
IV Inhibition
Inhibitors Sodim azide” Diethyldithiocarbamate” Hydroxylamim? Ethylenediamine Cyanide* Iodoacetamideb HIOPa a Assay ‘Assay
Concentration 0.1 mM
51rM 0.1 mM tetracetate”
by method by method
0.5 mM 10 pM 0.1 mM 2mM
B (V,, A (V,,
97 nmol 102 nmol
On min-I). HeOz min-I).
(9) 54 87 47 0
37 22 42
INTRACELLULAR
GALACTOSE
OXIDASE
513
D. dendmides
FROM
sitive to the presence of ligands and has a more limited specificity. The secretion of enzymes through membranes involves glycosylation of the enzyme (6, ‘7) so that the extracellular forms have higher carbohydrate contents (31,34, 43). Higher carbohydrate contents in the intracellular form of the enzyme were also observed with glucosidases in A. niger (44), which coincidently had a higher proportion of glucose. It would, therefore, be interesting to look for the differences in carbohydrate structures of intra- and extracellular galactose oxidase forms as the first approach in studying its secretion mechanism. Diethyldithiocarbamate FIG.
6. Effect
oxidase forms. Each enzyme di.fferent fore the cellular
diethyldithiocarbamate Assays (0.5 pg)
were performed was preincubated
diethylditbiocarbamate addition of 300 enzyme;
(M)
(0)
concentrations mM D-g&?.CtOSe. (0)
extracellular
ACKNOWLEDGMENTS
on galactose by method A. 10 min with beIntra-
This
research
was
do Desenvolvimento The authors thank and
supported
by Conselho
Cientifico Dr. Fabio
e Tecnologico 0. Pedrosa
National for
(CNPq). his help
criticism.
enzyme. REFERENCES
Both enzymes are glycoproteins containing glucose, arabinose, mannose, galactose, and hexosamines. The intracellular enzyme has a higher content of carbohydrates than the extracellular form, which could explain the higher overall molecular weight of the intracellular galactose oxidase. Most extracellular fungal glycoprotein enzymes have mannose as the major carbohydrate component (31-36). The neutral sugar composition of both forms of galactose oxidase showed a different pattern, with glucose and arabinose as major components. The proportions of glucose remained the same even after treatment with chloroform methanol to eliminate the glucose contaminants (3’7). Arabinose was also detected as a component of the a-amylase of Aspergillus niger (38). The higher carbohydrate contents in the intracellular galactose oxidase, compared to the extracellular one, could be a means of protection against proteolytic degradation, similar to that described for other enzymes in yeast (39,40). Another possibility is that the higher carbohydrate content promotes the stabilization of the enzymes conformation (41, 42) which is more sen-
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Biochem.
Vol. 252, No. 2,
Purification
and Characterization of Intracellular Oxidase from Dactylium dendroides
MARCIA Departamento
de B&&mica, Received
H. MENDONCA Universidade May
12,1986,
Federal
AND do Parana,
and in revised
GLACI Caixa form,
Galactose
T. ZANCAN’ Postal
9S9,8O.o00
September
Curitiba,
PR., Brazil
5,1986
The intracellular galactose oxidase from Dactylium dendmides was purified to homogeneity with a 64% yield. The enzyme is a glycoprotein (7.7% neutral sugars, 1.7% aminosugars) with 72,000 Da of molecular mass. The enzyme showed nonlinear double reciprocal plots with O2 and D-galactose, suggesting cooperative binding for both substrates. The intracellular galactose oxidase catalyzes the oxidation of galactose derivatives and dihydroxyacetone but not of glycerol, glycolaldehyde, /3-hydroxipyruvate, and ally1 alcohol which are substrates for the extracellular enzyme. Compared with the extracellular galactose oxidase, the intracellular enzyme showed higher carbohydrate content and sensitivity to diethyldithiocarbamate. o 1981 Academic press, I~~.
Galactose oxidase (EC 1.1.3.9) is secreted into the medium during the exponential growth phase of Dactylium dendroides (1). The extracellular copper-containing enzyme (2) has a broad specificity (1, 3) and its properties have been extensively studied (4). Levels of extracellular galactose oxidase are enhanced by high oxygen tension whereas increases in the intracellular levels are much less significant (5), suggesting an efficient secretion mechanism. Secretion of enzymes in lower eukaryotes is associated with glycosylation (6,7). The extracellular galactose oxidase has a low (1% ) carbohydrate content (8) but the intracellular enzyme has not yet been studied. The present paper deals with the purification and characterization of intracellular galactose oxidase from D. dendroides and makes a study of its secretion possible. MATERIAL
AND
METHODS
Enzyme purification. Dactylium (formerly Polypwus circinatus) from (9) was grown as described by Markus
dendroides our collection et al. (10) with
1 To whom
correspondence
should
be addressed. 507
1% glucose as the sole carbon source. The mycelia were harvested by filtration after 36 h of growth, washed with distilled water, and kept frozen (-17°C). Frozen mycelia (120 g) were suspended in 240 ml of 10 mM phosphate buffer, pH 7.0, containing 0.5 mM EDTA. The suspension was submitted twice to treatment at 20,000 psi in a French Press (Amincon Instruments), centrifuged at 17,600g for 15 min, and the supernatant used as enzyme source. Fractions of 50 ml were heated for 5 min at 6O”C, then cooled in an ice-salt bath (-5°C). The precipitate was removed by centrifugation at 17,SOOg for 15 min, the supernatant was adjusted to 45% saturation with ammonium sulfate, and after standing for 10 min the mixture was centrifuged at 17,600 for 15 min. The ammonium sulfate content in the supernatant was increased to 65% saturation and the pellet which formed after centrifugation was dissolved in 10 ml of 10 mM phosphate buffer, pH 7.0. The preparation was dialyzed against 100 mM ammonium acetate buffer, pH 7.2, containing 0.5 mM CuSOl. After dialysis, the preparation was chromatographed on a Sepharose 6B column (1.0 X 18 cm) previously equilibrated with the same solution. The column was washed with 100 mM ammonium acetate, pH 7.2, until the protein concentration was less than 0.0025 mg ml-‘. The enzyme was eluted with 100 mM D-fUCOSe in 100 mM ammonium acetate, pH 7.2, and the enzymically active fractions (20-80 IJ) were pooled and frozen at -17’C. For kinetic experiments, the enzyme was dialyzed for 4 h in 100 mM ammonium sulfate, pH 7.2, to eliminate D-fUCOSe but the enzyme lost 35% of its activity with this procedure. No modification 0003-9861/W Copyright All rights
$3.00
0 1987 hy Academic Press, Inc. of reproduction in any form reserved.
508 in the kinetic of D-fucose.
MENDONCA parameters
was observed
in the presence
Enzyme assays. The catalytic activity during the enzyme purification was monitored by determining the hydrogen peroxide produced, according to the method of Aisaka and Terada (11). In most kinetic experiments, the amount of oxygen consumed was measured polarographically. Assay A. The standard assay mixture (1.0 ml) contained 15 pmol phosphate buffer, pH ‘7.0, ‘7.0 rmol phenol, ‘75 rmol D-galactose, 0.48 nmol of 4-aminoantipyrine, 6.7 U peroxidase, and enzyme. The reaction was initiated by the additions of galactose oxidase and the rate of HpOz formation was measured at 500 nm in a DU-2 Beckman spectrophotometer. To eliminate interference in the reaction, controls without enzyme or substrate were carried out. One enzyme unit was defined as the amount that catalyzed the formation of 1 rmol of HzOz per minute. Specific activities were defined as units per milligram of protein. Assay B. The reaction mixture (1.4 ml) contained 20 pmol phosphate buffer, pH 7.0,lOO nmol D-galactose, and the enzyme. The oxygen consumption was monitored using a Gilson oxygraph Model KIC, equipped with a Clark-type electrode and a water-bath assembly to maintain the temperature at 30°C. All solutions were previously equilibrated at 30°C for 15 min. When oxygen concentrations were higher than 20% a stream of pure OX was passed through the solutions, and for concentrations below 20% a stream of argon was passed through the solutions to obtain the appropriate oxygen concentrations. Enzyme inhibition studies. To determine the inhibition of galactose oxidase by several agents, these were preincubated with the enzyme for 10 min at 30°C in an air-saturated buffer solution before starting the reaction by 300 mM of D-galactose. Results are expressed as percentages of the noninhibited activity. Protein determination Protein concentrations were determined by the method of Lowry et al. (12) using serum albumin as standard, and by the method of Warburg and Christian (13). Purification of extracellular gabctose oxidase. The purification of extracellular galactose oxidase was performed according to Amaral et al. (14) until the DEAE-cellulose step. The DEAE fraction was chromatographed on Sepharose 6B column as previously described by Hatton (15). The final preparation (74 U/mg of protein) was homogenous on examination by polyacrylamide gel electrophoresis at pH 8.6 and 3.6 and had a molecular weight similar to that found by Kosman et al. (8). Polyacrylamide gel electrqphoresis The enzyme preparation was concentrated in a rotatory evaporator and subjected to electrophoresis in 8% polyacrylamide gel using 65 mM Tris-borate buffer, pH 8.9, or 100 mM glycine-HCI buffer, pH 3.6. Electrophoresis was performed at 2 mA per tube until the indicator dye reached the end of the gel.
AND
ZANCAN
Electrophoresis was carried out in an Ortee pulse power apparatus. The gels were stained for protein with Coomasie blue (16) and for enzyme activity visualization, gels run in parallel were incubated in 100 mM galactose and revealed by the peroxidase-dianisidine reagent. Molecular weight detevknation The molecular weight was determined by gel permeation chromatography using Bio-Gel P-150, according to Andrews (17). The following protein molecular weight standards were used to calibrate the column: la&albumin (14,200), carbonic anidrase (20,000), egg albumin (45,000), serum albumin monomer (66,000), and serum albumin dimer (132,000). The elution of protein standards was monitored by light absorption at 230 nm and that of galactose oxidase by enzymic activity using Assay A. Electrophoresis under denaturing conditions was carried out using a modification of the procedure of Fairbanks (18). Molecular weight standards used were crosslinked albumin (66,000 and 132,000) and hemoglobin (16,000, 32,000, 48,000, and 64,000). Analysis of carbohydrate composition. The homogenous preparations of intra- and extracellular galactose oxidase were exhaustively (76 h) dialyzed against distilled water to eliminate free sugars. The solutions were concentrated at reduced pressure at 20°C. The total neutral sugar concentration was determined by the phenol-sulfuric method (19). A sample (3 mg) of homogenous enzyme was hydrolyzed in 2 ml of trifluoracetic acid (TFA)2 for 15 h at 100°C in a sealed tube. The hydrolysates were concentrated in a rotatory evaporator, distilled water was added to facilitate removal of trace residues of TFA, and evaporation was carried out again. This procedure was repeated five times. Neutral sugars were converted into alditol acetates and analyzed by gas chromatography as previously described (9). Aminosugars were determined according to Boas (20) after hydrolysis by the method of Maxhimex (21). Neutral sugars were fractionated by descending paper chromatography on Whatman No. 1 paper using benzene:butanol:pyridine:water (1: 5:3:3) as eluant and visualized with silver nitrate (22). Reagents. All reagents were of analytical grade and were obtained from Sigma Chemical Company. The lactose, D-galactose, melibiose, and raffinose were from Calbiochem, 2-deoxy-D-galactose was from Nutritional Biochemical Company, and D-galactosamine, Dglucosamine, D-ghCOSe were from Merck-Darmstadt. RESULTS
PurQicatim and properties. The results of a typical purification of intracellular galactose oxidase are presented in Table I. Similar purification patterns were obtained in eight independent runs. The purification a Abbreviation
used: TFA,
trifluoracetic
acid.
INTRACELLULAR
GALACTOSE
OXIDASE TABLE
PURIFICATION
-
OF INTRACELLULAR Total volume (ml)
Fraction
Cell extract Thermal inactivation Ammonium sulfate, 45-65% Sepharose 6B column
390 370 9.5 10
OXIDASE Units two1 HA min-’ * min-‘)
7.68 1.90 18.00 0.175
0.810
1.900 20.760
FROM
D. de&-tides
(U/mg
Sp act protein)
Recovery (%I
0.105 0.387
100 86
1.153
20.300
procedure gave approximately 1104-fold purified enzyme with a 64% overall yield. The enzyme was eluted as a single peak from a Sepharose 6B gel column using Dfucose as eluent (Fig. 1). The pooled activity fraction showed a single homogenous band on polyacrylamide disc electrophoresis at pH 8.6 and 3.6 (Fig. 2) and this fraction was used for further characterization of
509
D. dendroides
I
GALACTOSE
Protein (mg * ml-‘)
FROM
63
116.000
64
the enzyme. This enzyme was stable for at least 6 months at -17°C. In all purification steps, the enzymatic activity was monitored by polyacrylamide gel electrophoresis and only one band of activity was detected throughout. Molecular weight. The molecular weight of intracellular galactose oxidase was estimated from its chromatographic mobility
1% 0 ELUTION
FIG. 1. Affinity chromatography of intracellular monium sulfate fraction (171 mg) was percolated washed with the equilibration buffer and eluted assayed for protein and enzyme activity.
WIldi
(ml)
galactose oxidase on Sepharose 4B column. using a flow rate of 0.5 ml min-‘. The column with 0.1 M D-fUCOSe. Fractions were collected
Amwas and
510
MENDONCA
AND
ZANCAN
also a substrate. Compounds such as glycerol, /I-hydroxypyruvate, glycolaldehyde, and ally1 alcohol were not oxidized. (Table III)
FIG. 2. Homogeneity of intracellular galactose oxidase. 100 gg of protein was subjected to analytical polyacrylamide gel electrophoresis, as described under Materials and Methods. Gels were stained (bands A and C) for protein and for activity (bands B and D). Migration was from the bottom to the top.
on gel by comparison with those of standard proteins. The molecular weight calculated from the plot of V,/V, versus log molecular weight was 72,000 (Fig. 3A). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a single Coomasie blue positive band with a molecular weight of 72,000 (Fig. 3B). Carbohydrate ccmpositim Analysis of intracellular galactose oxidase by the phenol-sulfuric acid assay for total neutral sugars gave a value of 7.7% by weight. The extracellular enzyme on submission to the same conditions, gave a value of 1.7% (Table II). The Elson-Morgan method, for analysis of amino sugars, gave a value of 1.5% for the intracellular enzyme and 0.40% for the extracellular form. Hydrolysis of the enzyme with 2 M TFA followed by paper chromatography revealed the presence of bound glucose, arabinose, galactose, and mannose. The identity of monosaccharides was confirmed by GLC analysis of derived hexitol acetates. Quantitative values for the monosaccharide composition are shown in Table II. The relative proportion of sugars was different for each enzymic form. Substrate specz&itg. Like the extracellular galactose oxidase (1, 13), the intracellular enzyme catalyzes the oxidation of the primary hydroxyl -._group _ of galactose and its derivatives. Dihydroxyacetone is
6
5 MXECULAFt
IO WEIGHT
IO4
FIG. 3. Determination of molecular weight of intracellular galactose oxidase. (A) The calibration curve for gel filtration column (1 X 41 cm) of Biogel P150. Each protein (150 fig) in 0.3 ml of 50 mM phosphate buffer, pH 7.2, was applied to the column previously equilibrated with the same buffer. Fractions of 1 ml were collected at a flow rate of 0.4 ml min-‘. Void volume (V,) was calculated from the elution volume (V,) of Dextran Blue. Standards used were (A) carbonic anidrase, (A) egg albumin, (0) serum albumin monomere, (Cl) serum albumin dimere, (0) intracellular galactose oxidase. The continuous trace in the insert shows the elution profile of enzyme. (B) The calibration curve for SDS electrophoresis. The electrophoresis conditions were described in the text using the following standards (molecular weights in parentheses): (0) hemoglobin (l-16,ooO, 2-32,000,3-48,000; 464,000); (0) bovine serum albumin (5-66,W 6132.000): ~~~,~ ,, (0) \ - , _palactose oxidase intracellular.
INTRACELLULAR TABLE
GALACTOSE
II
SUGAR COMPOSITION OF GALACTOSE OXIDASE FROM D. devuirodes Enzyme Intracellular Ilarbobydrate Total neutral sugars (%) Total amino sugars (%)
7.7 1.5
Neutral sugars Mannose Galactose Glucose Arabinose
Molar 1.0 0.98 4.8 5.8
Extracellular
1.7 0.4 proportion 1.0 1.2 3.8 1.9
Kinetic properties. Intracellular galactose oxidase activity gave rise to a sigmoida1 relation between velocity and galactose concentration, when measuring oxygen
TABLE
III
SUBSTRATE SPECFICITY OF INTRA- AND EXTRACELLULAR GALACTOSE OXIDASE Relative Substrate (100 mM) D-Galactose Dihydroxyacetone 2-Deoxy-D-galactose D-Galactosamine Methyl-a-D-galactopyranoside O-Nitrophenyl-P-Dgalactopyranoside Lactose Melibiose Raffinose Stachyose Guaran Ally1 alcohol” Glycerol Glycoaldehyde” /&Hydroxypiruvate”
Intracellular
velocity
(% )
FROM
511
D. clewhides
consumption or H202 formation (Fig. 4). Initial rate measurements with dihydroxyacetone gave a linear double plot with a Km value of 20 mM. The Oz saturation curve in presence of saturating galactose concentration has also shown a sigmoidal pattern (Fig. 5). The So,s value for Oz was 0.29 mM. The Hill coefficients were 1.36 for galactose and 1.66 for oxygen. Effect of inhibitors. The intracellular galactose oxidase activity was inhibited by metal ligands such as cyanide, sodium azide, and hydroxylamine (Table IV). The intracellular form is probably a copper enzyme, as its activity was affected by 5 pM diethyldithiocarbamate, a known copper chelator. (Fig. 6). DISCUSSION
The purification of intracellular galactose oxidase by affinity chromatography on Sepharose (polymer of D-galactose and 3,6 anidro-L-galactose) was only achieved when D-fucose (6-deoxi-D-galactose) was used as eluent. When galactose was used the enzyme lost its activity, probably due to H202 formation. The extracellular enzyme form was eluted using a column buffer without sugar additions (15), sug-
Extracellular
100 179 52 60
100 263 65 39
170
106
40 11 68 144 161 47 0.0 0.0 0.0 0.0
OXIDASE
21 13 83 96 3.5 1.3 13.5 15.0
Note. Assays performed by method A using and 0.25 pg of intraand extracellular enzyme, spectively. Guaran concentration was estimated described by Avigad et aL (1). a Assays performed using 0.1 pg of each enzyme 300 mM of substrates.
0.13 reas and
D-GALKTCSE
(mM)
FIG. 4. Dependence of reaction rate on galactose concentration. Assays were performed as described under Materials and Methods using method B except that the concentration of substrate was changed: (0) 20% oxygen; (0) 60% oxygen.
512
MENDONCA
AND
I /iSI 0
03
LO OXYGEN
CmM)
FIG. 5. Effect of oxygen on the rate of galactose oxidase oxidation. Assays were performed measuring the oxygen consumption (Assay B) using 1.0 pg of protein. Oxygen concentration was adjusted as described under Materials and Methods. Galactose concentration was 300 InM.
gesting that the intracellular form was more tightly bound to Sepharose than the extracellular enzyme. Only a single band of galactose oxidase activity was observed in our crude preparations of mycelial extracts of D. dendroides, although Tressel and Kosman (23) have described isoenzymic forms in their extracellular preparations. It seems probable that the isoenzymic forms are postsecretional products since it is known that proteases are present in crude extracellular enzyme preparations (15). The substrate specificity of the intracellular galactose oxidase for galactose derivatives was found to be similar to that of the extracellular enzyme (1,3,24). The extracellular galactose oxidase catalyzes the oxidation of dihydroxiacetone (25) and nearly all primary alcohols (4) while the intracellular form had a more restricted specificity (Table III). Intra- and extracellular galactose oxidase behave differently with respect to the kinetics of galactose oxidation. Under the same assay conditions, the extracellular form behaved as a Michaelian enzyme
ZANCAN
while the intracellular form gave a sigmoidal pattern (using air or 60% of 02 as gas phase). Sigmoidal behavior has also been described for the extracellular galactose oxidase from Gibberella fujikumi (26). The suggestion originally made by Hamilton et al. (27) that the extracellular enzyme could exist as inactive and active forms, and recently stressed by Johnson et al. (28), who proposed a turnover between inactive Cu,,-enzyme to active Cut-enzyme, could explain the kinetic cooperativity we observed for the intracellular homogenous galactose oxidase, in the presence of oxygen or galactose. However no catalytic lags or bursts could be detected as would be expected if low molecular transitions similar to those observed for hysteretic enzymes (29) had occurred. Galactose and Oz apparently induce a conformational modification promoting the conversion of a less active form to a more active enzyme form. Another significant difference observed between the intra- and extracellular enzymic forms was their sensibility to metal ligands and copper chelators, suggesting that the copper in the active center is more accessible in the intracellular form than in the extracellular one. (Fig. 6). No activity of superoxide dismutase could be detected in the homogenous intracellular enzyme, as has been described for the extracellular form (30). The intracellular galactose oxidase is a single polypeptide chain with a molecular weight of 72,000, larger than that of 68,000 described for the extracellular form (8).
TABLE
IV Inhibition
Inhibitors Sodim azide” Diethyldithiocarbamate” Hydroxylamim? Ethylenediamine Cyanide* Iodoacetamideb HIOPa a Assay ‘Assay
Concentration 0.1 mM
51rM 0.1 mM tetracetate”
by method by method
0.5 mM 10 pM 0.1 mM 2mM
B (V,, A (V,,
97 nmol 102 nmol
On min-I). HeOz min-I).
(9) 54 87 47 0
37 22 42
INTRACELLULAR
GALACTOSE
OXIDASE
513
D. dendmides
FROM
sitive to the presence of ligands and has a more limited specificity. The secretion of enzymes through membranes involves glycosylation of the enzyme (6, ‘7) so that the extracellular forms have higher carbohydrate contents (31,34, 43). Higher carbohydrate contents in the intracellular form of the enzyme were also observed with glucosidases in A. niger (44), which coincidently had a higher proportion of glucose. It would, therefore, be interesting to look for the differences in carbohydrate structures of intra- and extracellular galactose oxidase forms as the first approach in studying its secretion mechanism. Diethyldithiocarbamate FIG.
6. Effect
oxidase forms. Each enzyme di.fferent fore the cellular
diethyldithiocarbamate Assays (0.5 pg)
were performed was preincubated
diethylditbiocarbamate addition of 300 enzyme;
(M)
(0)
concentrations mM D-g&?.CtOSe. (0)
extracellular
ACKNOWLEDGMENTS
on galactose by method A. 10 min with beIntra-
This
research
was
do Desenvolvimento The authors thank and
supported
by Conselho
Cientifico Dr. Fabio
e Tecnologico 0. Pedrosa
National for
(CNPq). his help
criticism.
enzyme. REFERENCES
Both enzymes are glycoproteins containing glucose, arabinose, mannose, galactose, and hexosamines. The intracellular enzyme has a higher content of carbohydrates than the extracellular form, which could explain the higher overall molecular weight of the intracellular galactose oxidase. Most extracellular fungal glycoprotein enzymes have mannose as the major carbohydrate component (31-36). The neutral sugar composition of both forms of galactose oxidase showed a different pattern, with glucose and arabinose as major components. The proportions of glucose remained the same even after treatment with chloroform methanol to eliminate the glucose contaminants (3’7). Arabinose was also detected as a component of the a-amylase of Aspergillus niger (38). The higher carbohydrate contents in the intracellular galactose oxidase, compared to the extracellular one, could be a means of protection against proteolytic degradation, similar to that described for other enzymes in yeast (39,40). Another possibility is that the higher carbohydrate content promotes the stabilization of the enzymes conformation (41, 42) which is more sen-
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