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A low molecular weight tyrosinase isoenzyme
Life nt~edi n
reat
Vol. 9, Part H,
1970 . pp . 553-559,
Pergamon Press
A LOW MOLECULAR WEIGHT TYROSINASE ISOENZYME Clarence Madhosingh Cell Biology Research Institute', Research Branch Canada Department of Agriculture, Ottawa
(Received 18 November 1989; in final form 23 March 1970) o-Diphenol :oxygen oxidoreductase (E .C . 1 .10 .3 .1 .), commonly called tyrosinase, occurs widespread in plant and animal tissues .
Signs of the activity of
the enzyme indicated by darkening of tissues, become conspicuous during morphogenesis, senescence, disease and injury . A review of the pertinent literature (1, 2, 3) of the past two decades reveal that the enzyme exists in multiple forms which interconvert relatively easily .
This inherent characteristic has given rise to varying reports re
garding the number of catalytic forms which exist in different species, substrate specificities . catecholase/cresolase ratios and molecular weights (2, 3) . The isoenzymes of mushroom trrosinase have been described as polymeric associations of units of 32,400 M .W . (4) . a molecular weight of 123,800 (5) .
The largest isoenzyme isolated had
This range of molecular weights does not
accommodate the five isoenzymes demonstrated previously (3) nor the multiplicity of forms observed by other workers (1, 6) .
This multiplicity of forms
suggests the possible existence of even lower molecular weight entities in the tryosinase complex .
A previous report (7) mentioned preliminary studies on
subunits of about 17,000 M .W . in mouse melanoma tryosinase . In order to understand better certain aspects of the structural and catalytic variations observed in this enzyme system, the approach has been to attempt to obtain a basic catalytic unit and examine the parameters which alter its structure and catalytic properties . ' Contribution No . 661 . 553
554
TYROSINASE ISOENZYMES
Vol . 9, No. 10
Methods Acetone powder was prepared from A ag ricus hortensis sporophore tissues as Initially, the freeze dried
described by Bouchilloux, McMahill and Mason (2) .
protein powder was dissolved in 0 .01 M phosphate buffer, pH 7 .2, and filtered under nitrogen and pressure (40 psi) in the cold (2°-4° C) successively through a 50,000, 10,000 and finally 1,000 molecular weight limits ultrafilters (phenolphthalein-free, Diaflo-Ultrafilters .
Amicon Corp ., Mass .) .
The
movement of protein through the ultrafilters was examined with the following standards :
albumen (67,000 M.W .), rtyoglobin (17,800 M .W .), cytochrome C
(12,400 M .W .), protein molecular weight markers obtained from Mann Research Laboratories, New York, and glucagon (3,485 M.W .) obtained from Eli Lilly and Company, Indianapolis, U.S .A .
Subsequently, the technique of electrodialysis
with ultrafiltration was adopted producing higher yields and better delineated fractions .
This procedure was as follows (Fig . 1) :
200 mg . acetone powder
was dissolved in Tris-Glycine buffer solution, pH 8 .9 (8), sealed in a dialysing membrane about 17,000 M.W . cut-off, (Fisher Scientific Co ., Montreal) container (A) and placed in an electrolyte cell (B) which is connected to another cell (C) by a 1,000 M .W . limit ultrafilter .
Cell B is connected by a paper
electrolyte bridge to a third cell (D) which contains the anode . contained thd same Tris-Glycine buffer (8) . 25°C at 200 volts for 5 hours .
All cells
The electrodialysis was run at
Fractions in cells B and C were then freeze
dried and labelled B and C respectively . The initial ultra-filtered fraction between 1,000 M .W . and 10,000 M .W . and the subsequent electrodialysed fraction B were applied to Sephadex G-50 superfine columns (2 .5 x 80 cm) . buffer, pH 7.2 .
The eluent in each case was 0 .01 M phosphate
The protein standards listed above were used also as standards
in the columns for estimating molecular weights . Protein fractions were identified by the Lowry method (9) .
DOPA-catalytic
fractions were located by mixing and incubating at 37°C for 3 min 0 .60 ml sample and 2 .9 ml substrate (0 .15% DL-DOPA in 0 .01 M phosphate buffer, pH 7 .2)
Vol . 9, No. 10
TYROSINASE ISOENZYMES
555
DIALYSIS MEMBRANE CONTAINER MOLECULAR FILTER (1000 M.W. LIMIT) PAPER ELECRTOLYTE BRIDGE
FIG . 1 Electrodialysis apparatus used for obtaining low molecular weight DL-DOPA-catalytic protein . All cells contained TrisGlycine buffer, pH 8 .9 (8) . Cell A contained, in addition, acetone powder obtained from Agaricus hortensis .
and measuring colorimetrically dopachrome fromation at 470 mu .
Both protein
and DOPA-catalytic estimations were performed on the Technicon autoanalyser or the Gilford spectrophotometer .
Samples of acetone powder (600 mg in 200 ml
phosphate buffer 0 .01 M, pH 7 .2) were treated with 3M and 6M urea .
The ultra-
filtrates (less than 10,000 M .W .) were then passed through Sephadex G-50 columns, equilibrated with buffer and 3 M and 6 M urea respectively . Results The Sephadex fractionation of the electrodialysate B (Fig . 2) produced two DL-DOPA-catalytic fractions .
One fraction, consistently emerging with
the void volume suggests a conversion of lower molecular weight entities (less than 10,000 M .W .) to larger catalytic molecules (25,000 M .W .-30,000 M .W .) .
The preponderant catalytic activity was obtained in an elution fraction
emerging between glucagon (3,485 M .W .) and ribonuclease (13,000 M .W .) and had
558
TYROSINASE ISOENZYMES
a molecular weight estimated as 8,000 to 10,000 .
Vol. 9, No. 10
The catalytic fraction also
coincided with the major protein fraction in this sample .
Another non-cata-
lytic protein of lower molecular weight was also separated on the column . The fractionation patterns of protein and catalytic entities of the electrodialysed fraction B (shown in Fig. 2) and that obtained for the ultrafiltered undialysed sample (1,000 M.W . to 10,000 M.W .) were the same . The urea treated samples are compared in Fig. 3 with the fractions obtained from a similar non-treated sample .
The data suggested a reduction in
the 3 M urea treatment and elimination in the 6 M urea treatment of the higher molecular weight catalytic peak .
There was however, a distinct increase in
the lower molecular weight major catalytic unit (8,000-10,000 M .W .) in the 3 M urea treatment but no further increase in this fraction in the 6 M treatment .
There was also an increase in non-catalytic lower molecular weight
protein entities in the acetone extracts generally as a result of the increasing concentrations of the urea treatments as would be expected .
This is indi-
cated by the shift in the major protein peak to the right in the diagram . Discussion The assay for tryos1nase using DL-DOPA as a substrate and measuring dopachrone formation has been discussed (10) .
The technique is simple and
sensitive and useful for locating catalytic entities in large numbers of fractions .
Catalytic protein fractions which are not detectable by absorbance
at 280 mu or by the Lowry method are detectable by the catalysed dopachrome formation .
These observations suggest that the analysis for enzyme, only in
those fractions which have demonstrable levels of proteins, as has been done previously (2,5), is inadequate and may serve to eliminate certain isoforms . The data presented therefore suggest that the low molecular weight DLDOPA-catalytic protein demonstrated could be a basic unit in some species which have polymerized isoenzymes of tryosinase .
The existence of such a unit would
accommodate the multiple forms of the enzyme within the range of molecular
Vol. 9, No. 10
TYROBINASE ISOENZYMES
60-
.11
Z W 0
PROTEIN
I I II I 1
50
I
40 ~
I R
30-
_j
I f
1 1 I 1\
G
I I 1
a 20 ~
U
a O
557
iW
10-
01
ENZYME
60
1\
\
1--'
f
11 11 f 1 1 I 1 I 1 1 1
80
120 140 100 160 ELUTION VOLUME ML . FIG . 2
--I
Sephadex G-50 superfine column fractionation of electrodialysate B (Fig . 1) showi n g two protein peaks (- and two DOPA-catalytic peaks ( ) . The blank control is also shown . Rate : 12 samples/hr ., each 1 .9 ml . R and G indicate the relative positions of ribonuclease and glucagon respectively .
weights reported for the isoenzymes in different species .
Further, the vari-
ations in the catalytic activities reported (3) for certain enzyme forms suggest the hybridization of at least two dissimilar structural ànits . recent study (5) substantiates this experimentally .
A
558
TYROSINASE ISOENZYMES
Vol. 9, No . 10
O.DL
100 O
m
50
Z
FIG. 3 Sephadex G-50 superfine column fractionation of equivalent volumes of ultrafiltrates (10,000 M .W .) of treated (3 M and 6 M urea) and non-treated acetone powder preparations from A aricus~ hortensis . The columns for the treated samples were equilibrated wwRY -TIT and 6 M urea in phosphate buffer, pH 7 .2, before fractionation of the respective samples . Base line was corrected with the blank-controls . Rate : 12 samples/hr ., each 2.5 ml .
Summary A low molecular weight DL-dihydroxypbenylalanine (DOPA)-catalytic protein 8,000-10,000 M .W ., has been obtained from an extract of the mushroom Agaricus hortensis .
The protein was isolated by electrodialysis, ultrafiltration and
finally by passage through a Sephadex G-50 superfine column .
The partial
conversion to a higher molecular weight catalytic entity was prevented by treatment with 6 M urea . Acknowledgements The technical assistance of Mr . Ian Wood is appreciated with thanks .
Vol. 9, No . 10
TYROSINASE LSOENZYMES
559
References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 .
Constantides, S.M ., Bedford, C.L ., J . Food Science 32, 446 (1967) . hem. 238 Bouchilloux, S ., McMahill, P ., Mason, H .S ., J . Biol . 1699 (1963) . Jolley Jr ., R.L ., Mason, H.S ., J . Biol . Chem . 240, PC 1498 (1965) . Jolley Jr ., R.L ., Robb, D .A ., Mason, H.S ., J . M. Chem . 244, 1593 (1969) . Jolley Jr ., R.L ., Nelson, R .M ., Robb, D .A ., J . Biol . Chem . 244 , 3251 (1969) . Madhosingh, C ., Un ubllshed data . Burnett, J .B ., Sei Rer, H., Brown, I .V ., Cancer Research 27, 880 (1967) . Davis, B .J ., Ann. N.Y . Acad . Sci . 121, 404 (1964) . Lowry, O .H ., Rosebrough, N .J ., Farr,A .L ., Randall, R .J ., J . Biol . Chem . Chem . 193, 265 (1951) . Flin , M .,l~orowitz, N.H ., Heinemann, S.F ., J . Biol . Chem . 238, 2045 1963) .
reat
Vol. 9, Part H,
1970 . pp . 553-559,
Pergamon Press
A LOW MOLECULAR WEIGHT TYROSINASE ISOENZYME Clarence Madhosingh Cell Biology Research Institute', Research Branch Canada Department of Agriculture, Ottawa
(Received 18 November 1989; in final form 23 March 1970) o-Diphenol :oxygen oxidoreductase (E .C . 1 .10 .3 .1 .), commonly called tyrosinase, occurs widespread in plant and animal tissues .
Signs of the activity of
the enzyme indicated by darkening of tissues, become conspicuous during morphogenesis, senescence, disease and injury . A review of the pertinent literature (1, 2, 3) of the past two decades reveal that the enzyme exists in multiple forms which interconvert relatively easily .
This inherent characteristic has given rise to varying reports re
garding the number of catalytic forms which exist in different species, substrate specificities . catecholase/cresolase ratios and molecular weights (2, 3) . The isoenzymes of mushroom trrosinase have been described as polymeric associations of units of 32,400 M .W . (4) . a molecular weight of 123,800 (5) .
The largest isoenzyme isolated had
This range of molecular weights does not
accommodate the five isoenzymes demonstrated previously (3) nor the multiplicity of forms observed by other workers (1, 6) .
This multiplicity of forms
suggests the possible existence of even lower molecular weight entities in the tryosinase complex .
A previous report (7) mentioned preliminary studies on
subunits of about 17,000 M .W . in mouse melanoma tryosinase . In order to understand better certain aspects of the structural and catalytic variations observed in this enzyme system, the approach has been to attempt to obtain a basic catalytic unit and examine the parameters which alter its structure and catalytic properties . ' Contribution No . 661 . 553
554
TYROSINASE ISOENZYMES
Vol . 9, No. 10
Methods Acetone powder was prepared from A ag ricus hortensis sporophore tissues as Initially, the freeze dried
described by Bouchilloux, McMahill and Mason (2) .
protein powder was dissolved in 0 .01 M phosphate buffer, pH 7 .2, and filtered under nitrogen and pressure (40 psi) in the cold (2°-4° C) successively through a 50,000, 10,000 and finally 1,000 molecular weight limits ultrafilters (phenolphthalein-free, Diaflo-Ultrafilters .
Amicon Corp ., Mass .) .
The
movement of protein through the ultrafilters was examined with the following standards :
albumen (67,000 M.W .), rtyoglobin (17,800 M .W .), cytochrome C
(12,400 M .W .), protein molecular weight markers obtained from Mann Research Laboratories, New York, and glucagon (3,485 M.W .) obtained from Eli Lilly and Company, Indianapolis, U.S .A .
Subsequently, the technique of electrodialysis
with ultrafiltration was adopted producing higher yields and better delineated fractions .
This procedure was as follows (Fig . 1) :
200 mg . acetone powder
was dissolved in Tris-Glycine buffer solution, pH 8 .9 (8), sealed in a dialysing membrane about 17,000 M.W . cut-off, (Fisher Scientific Co ., Montreal) container (A) and placed in an electrolyte cell (B) which is connected to another cell (C) by a 1,000 M .W . limit ultrafilter .
Cell B is connected by a paper
electrolyte bridge to a third cell (D) which contains the anode . contained thd same Tris-Glycine buffer (8) . 25°C at 200 volts for 5 hours .
All cells
The electrodialysis was run at
Fractions in cells B and C were then freeze
dried and labelled B and C respectively . The initial ultra-filtered fraction between 1,000 M .W . and 10,000 M .W . and the subsequent electrodialysed fraction B were applied to Sephadex G-50 superfine columns (2 .5 x 80 cm) . buffer, pH 7.2 .
The eluent in each case was 0 .01 M phosphate
The protein standards listed above were used also as standards
in the columns for estimating molecular weights . Protein fractions were identified by the Lowry method (9) .
DOPA-catalytic
fractions were located by mixing and incubating at 37°C for 3 min 0 .60 ml sample and 2 .9 ml substrate (0 .15% DL-DOPA in 0 .01 M phosphate buffer, pH 7 .2)
Vol . 9, No. 10
TYROSINASE ISOENZYMES
555
DIALYSIS MEMBRANE CONTAINER MOLECULAR FILTER (1000 M.W. LIMIT) PAPER ELECRTOLYTE BRIDGE
FIG . 1 Electrodialysis apparatus used for obtaining low molecular weight DL-DOPA-catalytic protein . All cells contained TrisGlycine buffer, pH 8 .9 (8) . Cell A contained, in addition, acetone powder obtained from Agaricus hortensis .
and measuring colorimetrically dopachrome fromation at 470 mu .
Both protein
and DOPA-catalytic estimations were performed on the Technicon autoanalyser or the Gilford spectrophotometer .
Samples of acetone powder (600 mg in 200 ml
phosphate buffer 0 .01 M, pH 7 .2) were treated with 3M and 6M urea .
The ultra-
filtrates (less than 10,000 M .W .) were then passed through Sephadex G-50 columns, equilibrated with buffer and 3 M and 6 M urea respectively . Results The Sephadex fractionation of the electrodialysate B (Fig . 2) produced two DL-DOPA-catalytic fractions .
One fraction, consistently emerging with
the void volume suggests a conversion of lower molecular weight entities (less than 10,000 M .W .) to larger catalytic molecules (25,000 M .W .-30,000 M .W .) .
The preponderant catalytic activity was obtained in an elution fraction
emerging between glucagon (3,485 M .W .) and ribonuclease (13,000 M .W .) and had
558
TYROSINASE ISOENZYMES
a molecular weight estimated as 8,000 to 10,000 .
Vol. 9, No. 10
The catalytic fraction also
coincided with the major protein fraction in this sample .
Another non-cata-
lytic protein of lower molecular weight was also separated on the column . The fractionation patterns of protein and catalytic entities of the electrodialysed fraction B (shown in Fig. 2) and that obtained for the ultrafiltered undialysed sample (1,000 M.W . to 10,000 M.W .) were the same . The urea treated samples are compared in Fig. 3 with the fractions obtained from a similar non-treated sample .
The data suggested a reduction in
the 3 M urea treatment and elimination in the 6 M urea treatment of the higher molecular weight catalytic peak .
There was however, a distinct increase in
the lower molecular weight major catalytic unit (8,000-10,000 M .W .) in the 3 M urea treatment but no further increase in this fraction in the 6 M treatment .
There was also an increase in non-catalytic lower molecular weight
protein entities in the acetone extracts generally as a result of the increasing concentrations of the urea treatments as would be expected .
This is indi-
cated by the shift in the major protein peak to the right in the diagram . Discussion The assay for tryos1nase using DL-DOPA as a substrate and measuring dopachrone formation has been discussed (10) .
The technique is simple and
sensitive and useful for locating catalytic entities in large numbers of fractions .
Catalytic protein fractions which are not detectable by absorbance
at 280 mu or by the Lowry method are detectable by the catalysed dopachrome formation .
These observations suggest that the analysis for enzyme, only in
those fractions which have demonstrable levels of proteins, as has been done previously (2,5), is inadequate and may serve to eliminate certain isoforms . The data presented therefore suggest that the low molecular weight DLDOPA-catalytic protein demonstrated could be a basic unit in some species which have polymerized isoenzymes of tryosinase .
The existence of such a unit would
accommodate the multiple forms of the enzyme within the range of molecular
Vol. 9, No. 10
TYROBINASE ISOENZYMES
60-
.11
Z W 0
PROTEIN
I I II I 1
50
I
40 ~
I R
30-
_j
I f
1 1 I 1\
G
I I 1
a 20 ~
U
a O
557
iW
10-
01
ENZYME
60
1\
\
1--'
f
11 11 f 1 1 I 1 I 1 1 1
80
120 140 100 160 ELUTION VOLUME ML . FIG . 2
--I
Sephadex G-50 superfine column fractionation of electrodialysate B (Fig . 1) showi n g two protein peaks (- and two DOPA-catalytic peaks ( ) . The blank control is also shown . Rate : 12 samples/hr ., each 1 .9 ml . R and G indicate the relative positions of ribonuclease and glucagon respectively .
weights reported for the isoenzymes in different species .
Further, the vari-
ations in the catalytic activities reported (3) for certain enzyme forms suggest the hybridization of at least two dissimilar structural ànits . recent study (5) substantiates this experimentally .
A
558
TYROSINASE ISOENZYMES
Vol. 9, No . 10
O.DL
100 O
m
50
Z
FIG. 3 Sephadex G-50 superfine column fractionation of equivalent volumes of ultrafiltrates (10,000 M .W .) of treated (3 M and 6 M urea) and non-treated acetone powder preparations from A aricus~ hortensis . The columns for the treated samples were equilibrated wwRY -TIT and 6 M urea in phosphate buffer, pH 7 .2, before fractionation of the respective samples . Base line was corrected with the blank-controls . Rate : 12 samples/hr ., each 2.5 ml .
Summary A low molecular weight DL-dihydroxypbenylalanine (DOPA)-catalytic protein 8,000-10,000 M .W ., has been obtained from an extract of the mushroom Agaricus hortensis .
The protein was isolated by electrodialysis, ultrafiltration and
finally by passage through a Sephadex G-50 superfine column .
The partial
conversion to a higher molecular weight catalytic entity was prevented by treatment with 6 M urea . Acknowledgements The technical assistance of Mr . Ian Wood is appreciated with thanks .
Vol. 9, No . 10
TYROSINASE LSOENZYMES
559
References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 .
Constantides, S.M ., Bedford, C.L ., J . Food Science 32, 446 (1967) . hem. 238 Bouchilloux, S ., McMahill, P ., Mason, H .S ., J . Biol . 1699 (1963) . Jolley Jr ., R.L ., Mason, H.S ., J . Biol . Chem . 240, PC 1498 (1965) . Jolley Jr ., R.L ., Robb, D .A ., Mason, H.S ., J . M. Chem . 244, 1593 (1969) . Jolley Jr ., R.L ., Nelson, R .M ., Robb, D .A ., J . Biol . Chem . 244 , 3251 (1969) . Madhosingh, C ., Un ubllshed data . Burnett, J .B ., Sei Rer, H., Brown, I .V ., Cancer Research 27, 880 (1967) . Davis, B .J ., Ann. N.Y . Acad . Sci . 121, 404 (1964) . Lowry, O .H ., Rosebrough, N .J ., Farr,A .L ., Randall, R .J ., J . Biol . Chem . Chem . 193, 265 (1951) . Flin , M .,l~orowitz, N.H ., Heinemann, S.F ., J . Biol . Chem . 238, 2045 1963) .