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Superoxide dismutase in the housefly Musca domestica (L.)

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Archives of Insect Biochemistry and Physiology 3:31-43 (1986)
Superoxide Dismutase in the Housefly,
Musca dornestica (L.)
Thomas G . Bird, Marvin L. Salin, JohnA. Boyle, and James R. Heitz
Department of Biockemisty, Mississippi State University, Mississippi State
Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present i n
whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese,
and the other three contained copper and zinc. All were observed in each of
the body tagma (head, abdomen, and thorax) and at each developmental
stage (egg t o adult).
The copper- and zinc-containing isozymes purified from newly emerged,
adult M. domestica had a relative molecular weight of 34,800 as determined
by gel filtration chromatography but consisted of t w o equal-size subunits of
16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three
copper, zinc isozymes were identified as charge variants. The amino acid
composition of the enzyme was similar to that of copper, zinc-containing
superoxide dismutases from other sources.
Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able t o protect lactic dehydrogenase against deactivation in the
presence of light and rose bengal, a known generator of singlet oxygen. The
role of SOD in the phototoxic reaction involving rose bengal is discussed.
Key words: superoxide dismutase, housefly, Musca domestica, enzyme purification and
characterization, rose bengal, singlet oxygen, enzyme deactivation
Acknowledgments: We thank Mr. William E. Poe, Department of Biochemistry, and Mr.
Michael Aide, Department of Agronomy, Mississippi State University, for able assistance with
the amino acid analysis and atomic absorption spectrophotometry, respectively. We also
thank Dr. Paul A. Hedin, USDA-ARS, Boll Weevil Research Laboratory, Mississippi State, Dr.
S.B. Ramaswamy, Department of Entomology, and Dr. Robert B. Koch, Department of Biochemistry, Mississippi State, for critical review of the manuscript. This work was supported
by funds from the Mississippi Agricultural and Forestry Experiment Station MAFES, publication No. 5879.
Received May 26,1985; accepted June 12,1985.
T.C.B.’s present address i s Center for Alluvial Plains Studies, Delta State University, Cleveland, MS 38732.
Address reprint requests to James R. Heitz, Department of Biochemistry, Drawer BB, Mississippi State, MS 39762.
@ 1986 Alan R. Liss, Inc.
32
Bird et al
INTRODUCTION
As a consequence of living in an atmosphere enriched in oxygen, aerobic
organisms have undergone a pressure to evolve enzymes capable of detoxifying deleterious by-products of oxygen metabolism. Among such enzymes
are the class of metalloproteins known as superoxide dismutases.These enzymes detoxify the 02-by catalyzing a disproportionation reaction between
two 02-molecules:
Several isozymes of SOD* have been characterized with differences in
amino acid sequence and metal content. CuZnSOD has been found in most
eucaryotic organisms and consists of a dimer with a molecular weight of
32,000 and has 2 Cu and 2 Zn atoms per molecule [l-51. MnSOD has been
found in bacteria as well as in the mitochondria1 matrix of plants and animals.
The enzyme has a molecular weight of 40,000-90,000 depending on source
and a variable metal content of 1to 4 atoms Mn per molecule. A third class,
FeSOD, has generally been found in procaryotes [l],though exceptions have
been reported [2,3].This isozyme has a molecular weight of about 40,000,
consists of two subunits, and has a metal content of 1to 2 atoms of Fe per
molecule.
SODs from insects had been largely ignored until Lee et a1 [4] purified and
characterized CuZnSOD from the fruit fly, Drosopkila rnelunoguster. As a class
of enzymes, the SODs are of particular interest to us owing to their postulated ability of interacting with '02and inhibiting its reaction [6,7] or by
acting as protectant against the effects of ionizing radiation [4]. Specifically,
xanthene dyes, known to generate ' 0 2 as part of their photooxidative reaction, are now in use as insecticides [8]. If SOD does afford a degree of
protection to the organism that has been exposed to '02, a possible source of
resistance to the pesticide may exist. Therefore, the intent of the current
study is to purify and characterize CuZnSOD from housefly prior to initiating
a study on '02and its effect on housefly SOD. We also report results of in
vitro studies into the interaction of '02and CuZnSOD from house fly.
MATERIALS AND METHODS
Newly emerged, adult houseflies from a colony initially established in 1977
with wild insects from a caged-layer poultry house in Pelahatchie, MS and
maintained in the Department of Biochemistry, Mississippi State University,
were used in the purification studies. Other stages used are noted with each
study. The adult fly rearing colony was maintained on dry milk, sugar, and
*Abbreviations: CM-Sepharose = carboxyrnethyl Sepharose; DEAE-Sephadex = diethylaminoethyl Sephadex; pl = isoelectric point; LDH = lactic dehydrogenase; M, = relative
molecular weight; M W = molecular weight; PAGE = polyacrylamide gel electrophoresis; RE3
= rose bengal; SDS-PACE = sodium dodecyl sulfate PAGE; lo2 = singlet oxygen; 0 2 -=
superoxide anion; SOD = superoxide dismutase.
Housefly Superoxide Dismutase
33
H20 with a 14L:lOD photoperiod at approximately 27°C. The standard larval
diet used throughout contained 7 ml of malt solution (50 ml of commercial
malt extract plus 100 ml H20), 27 ml of a solution of brewer’s yeast (66 g of
yeast in 540 ml H2O; ICN Nutritional Biochemicals; Cleveland, OH), and 190
ml H201100 g of wheat bran. The mixture was allowed to ferment 24 h prior
to introduction of eggs or larvae. Gravid females laid their eggs directly onto
the larval media. The eggs were allowed to develop, and first-instar larvae
were transferred to fresh media.
CuZnSOD from 1kg of houseflies was purified according to the procedure
of McCord and Fridovich [5] with two additional steps. The presence of
contaminants not resolved by the original procedure led to the inclusion of a
CM-Sepharose column following the usual DEAE-Sephadex chromatography. The sample was loaded onto the 2.5 x 20 cm CM-Sepharose column in
5 mM, pH 6.0, phosphate buffer and eluted without adhering to the matrix.
Following gel filtration on Sephadex G-75 ultrafine, the presence of impurities necessitated development of a method for preparative PAGE. A Canalco
PREP-DISC@unit, outfitted with a I’D-150 gel column, was employed for
preparative PAGE. The analytical electrophoresis method described below
was modified by increasing the polyacrylamide gel concentration to 15% in
the resolving gel. A 1.5-cm resolving gel and 3.0-cm concentrating gel were
supported by fine mesh nylon netting across the column end. Flow rate of
the elution buffer was controlled by a Gilson Minipuls 2 peristalic pump at
0.5 mllmin, and temperature was maintained at 4°C by circulating 50%
ethylene glycol through a refrigeration unit. A driving force of 4 mA was
applied until the sample entered the resolving gel and was then increased to
8 mA for the duration of the run.
The unit of SOD activity was initially defined by McCord and Fridovich
[5] in an indirect assay. Xanthine oxidase is added in sufficient quantity to a
reaction mixture containing xanthine (5 x lo-’ M) as an oxidizable substrate
and ferricytochrome c (1 x lo-’ M) as a reducible substrate in phosphate
buffer (pH 7.8, 50 mM). A rate of reduction measured at 550 nm of 0.025
absorbance unitslmin at 22.5
1°C is desired. Under these conditions,
McCord and Fridovich defined 1 unit of enzyme activity as the amount of
SOD required to inhibit the rate of ferricytochrome c reduction by 50% (0.0125
absorbance unitslmin). A drawback to expressing the unit of activity in this
manner is the lack of linearity over a wide range of SOD concentrations. By
using the equation of Asada et a1 [9]:
(Vlv) = 1 + K’ [SOD]
where V and v represent the reaction rate of the assay in the absence and
presence of SOD, Giannopolitis and Ries [lo] demonstrated linearity over a
wider range of SOD concentrations when the reaction rate constant, K’, is
set equal to 1.By derivation they arrived at the following expression:
SOD unitslml = [(Vlv) - 11 (dilution factor)
It is this expression of enzyme activity that the present paper uses.
34
Bird et al
Protein levels were estimated by either the modified Lowry method of
Schacterlee and Pollack [ll]or the differential absorbance method of Murphy
and Kies 1121 as indicated. The use of two methods for protein assay was
necessitated by numerous nonproteinaceous compounds which absorb at 215
nm and interfered with the latter method in the early stages of purification,
thereby giving highly erroneous results, and also by rapidly diminishing
enzyme levels in the latter stages of purification which made use of the
destructive Lowry method infeasible.
Electrophoresis on 7.5% polyacrylamide gels was performed according to
the procedures of Davis [13] and Ornstein 1141 and stained by the method of
Beauchamp and Fridovich [15]. CuZnSOD isozymes were identified by convention on polyacrylamide gels as bands sensitive to 1 mM KCN [15], whereas
bands identified as MnSOD were insensitive to both KCN and 1 mM H202
m1.
Development of SOD isozymes from egg to adult was followed by first
collecting a large number of freshly laid eggs. A representative sample was
frozen at -70°C until needed for the comparative study, but the remainder
were allowed to develop. Frozen samples did not differ from fresh ones
when compared electrophoretically. Larvae or pupae were taken every 24 h
until adult emergence and frozen. Upon adult emergence, accumulated samples were prepared for electrophoresis.
Distribution of the isozymes in the body tagma (head, abdomen, and
thorax) was determined by sectioning live insects, freezing immediately in
liquid N2, and electrophoresing as described above.
Molecular weights were measured by both gel filtration chromatography
and SDS-PAGE. For the former, a Sephadex G-200 column (40-120 mesh, 1.5
x 70 cm) was equilibrated with standard phosphate buffer (50 mM, pH 7.8)
and calibrated with ribonuclease A (MW 13,700), chymotrypsinogen A (MW
25,000), 6-lactoglobulin (MW 36,800), ovalbumin (MW 45,000), bovine serum
albumin (MW 66,000), and aldolase (MW 158,000, Sigma, St. Louis). SDSPAGE was performed by the method of Laemmli 1171. Standards used to
calibrate included egg white lysozyme (MW 14,300), 6-lactoglobulin (MW
36,800, subunits of 18,400), porcine stomach mucosa pepsin (MW 34,700),
ovalbumin (MW 45,000), and bovine serum albumin (MW 66,000).
Copper and zinc levels were determined on a Perkin-Elmer model 305B
atomic absorption spectrophotometer. Purified enzyme was dialyzed extensively against 0.1 mM ethylenediamine tetraacetic acid in deionized water
prior to the determination.
The amino acid composition of the three CuZnSOD isozymes in one
solution was determined after hydrolysis in vacuo in 6 M HC1 at 110°C for 24
h followed by analysis on a Beckman model 120C amino acid analyzer.
Tryptophan was measured spectrophotometrically using the N-bromosuccinimide method of Spande and Witkop 1181.
The PI of the housefly CuZnSOD was determined by the method of Salin
and Bridges 1191 using a DesagalBrinkman double-chamber gel isoelectric
focusing apparatus. The absorbance spectrum was determined on a PerkinElmer model 552 spectrophotometer. Purified, adult housefly CuZnSOD was
used in two in vitro experiments to determine the effects of RB-generated
Housefly Superoxide Dismutase
35
on the enzyme. In the first RB (1mM) and CuZnSOD were prepared in
1 ml phosphate buffer (50 mM, pH 7.8). The amount of CuZnSOD present
was sufficient to halve the reduction rate of cytochrome cox to cytochrome
C,,d when a 10-p1aliquot was used in the previously described enzyme assay.
After 30 min dark preincubation the test tube containing the solution was
placed into a cooling jacket constructed of a chromatography jar and a
recirculating water bath to maintain a constant temperature of 25°C for the
exposure period. The solution was then illuminated with a 650-W Sylvania
sun gun. Activity was measured at 3-min intervals for 60 min. In the second
experiment housefly CuZnSOD was tested as a protectant for a second
enzyme, LDH, against the effects of lo2.Five different assay tubes were
prepared for each of the four replicates: a dark control with LDH only, a dark
control with LDH and RB, an illuminated tube with LDH only, an illuminated
tube with LDH and RB, and an illuminated tube with LDH, RB, and 3 pM
housefly CuZnSOD. Tubes were maintained as before at 25°C. Each tube
contained 100 pl of bovine heart LDH (550 units; 1 unit converts 1 pM
pyruvate to L-lactate per min at pH 7.5, 37°C) in 15 ml phosphate buffer (50
mM, pH 7.5). RB was added to produce absorbance = 0.80 at 556 nm, which
previously was shown to produce an acceptable rate of LDH inactivation
when illuminated. LDH activity was measured at 2-min intervals using
previously published methods [20]. An aliquot of the illuminated LDH-RB
mixture was added to an enzyme assay mixture containing NADH (0.2 mM)
and pyruvate (0.5 mM) in phosphate buffer (50 mM, pH 7.5) at 37°C. The
conversion of NADH to NAD+ was measured at 340 nm until greater than
80% LDH deactivation was observed.
' 0 2
RESULTS
Electrophoresis and activity staining of various samples without inhibitors
always showed four achromatic bands. All four resolvable bands were present in each of the adult body tagma (Fig. l A , B, and C) and from egg to adult
(data not shown). In the presence of the inhibitors KCN or H202, the three
fastest migrating bands (bands 2, 3, and 4) were eliminated but the slowest
(band 1)was not affected (Fig. 1D and B). Based on the known sensitivities
of the various SOD isozymes [15, 161, it was concluded that the triplet peaks
were CuZnSOD isozymes and that the slowest band was MnSOD. There
was no evidence for FeSOD, in keeping with the general observation that
this isozyme is found predominantly in prokaryotes [l].
The crude homogenate had a specific activity of 7 units Img protein (Table
1).Treatment of the sample with organic solvents eliminated a large portion
of the contaminant while not affecting the total activity. Chromatography
over three columns resulted in a 132-fold increase in specific activity while
only half of the total activity was lost. Contaminating proteins were effectively eliminated by preparative electrophoresis, with the faster-migrating
contaminants eluting prior to most SOD activity. Other contaminants were
unable to enter the 15% PAGE resolving gel and were visible as a dark-brown
band at the concentrating ge1:resolving gel interface. The inclusion of this
step increased the specific activity approximately 2.9-fold over the Sephadex
36
x,-
Bird et al
D
W
0
t
0
I
?
0
In
n
a
c
2cm
Fig. 1. SOD activity from newly emerged, adult houseflies localized o n polyacrylamide
electrophoresis gels. Results from the body tagma (head, A; thorax, B; and abdomen, C) are
represented by enzyme stained without inhibitor present. Whole-body homogenates were
electrophoresed and stained in the presence of KCN (D) or H 2 0 2 (E) after the method of
Beauchamp and Fridovich [I51 and Asada et al [I61 to determine the presence of MnSOD,
peak 1; CuZnSOD, peaks 2 , 3 , and 4; and/or FeSOD.
TABLE 1. Purification of CuZnSOD From Newly Emerged, Adult Houseflies
Crude
homogenate
Acetone
precipitate
DEAE-Sephadex
CM-Sepharose
G-75 Sephadex
Preparative
electrophoresis
Specific
activity
(unitsimg
protein)
Volume
(mu
Total
protein
(mg)
Total
units
7,075
57,770a
382,100
7
100
1
690
3,367a
340,900
101
89
15
1,080
1,090
275
410
1,159a
672a
223b
56b
315,400
272,500
195,800
141,040
272
406
878
2,519
83
71
51
37
41
61
132
381
'Protein measured by method of Schacterlee and Pollack [ll].
%otein measured by method of Murphy and Kies [12].
Percent
recovery
Fold
purification
Housefly Superoxide Dismutase
37
G-75 step. Analytical electrophoresis indicated that the purified sample contained three CuZnSOD isozymes but no other bands, whereas only a single
peak of SOD activity was observed when chromatographed on Sephadex G200.
Analysis of PAGE gels according to the method of Hedrick and Smith [21]
indicated that the purified isozymes were charge isomers (data not shown).
An M, for these CuZnSOD isozymes as measured by Sephadex G-200 gel
filtration was approximately 34,800. The determination of M, by SDS-PAGE
[17] from a solution containing all of the housefly CuZnSOD isozymes produced a single band corresponding to approximately 16,000 when heated at
100°C for 5 min in both the presence and absence of 2-mercaptoethanol.
The UV-absorbance spectrum of a solution containing all three of the
housefly CuZnSOD isozymes (Fig. 2) was essentially the same as that of
CuZnSOD from other sources. Absorption shoulders were observed at 264
and 259 nm. Extinction coefficients corresponding to these peaks were 7,850
M-' cm-' and 7,890 M-' cm-' based on an M, of 32,000. Sufficient enzyme
was not available to generate the spectrum in the visible region. Analysis for
metal content of the enzyme showed the presence of 1.6 mol of copper and
1.7 mol of zinc per mol of dimer.
The amino acid composition of the three CuZnSOD isozymes based on a
dimeric molecular weight of 32,000 is shown in Table 2 along with that of
several other organisms. The most noteworthy features of the housefly
CUZIISOD amino acid profile are the absence of tryptophan and the pres-
WAVELENGTH, nm
Fig. 2. Absorbance spectrum of housefly CuZnSOD from newly emerged adults in the U V
region. A protein concentration of 0.6 m g h l was used to generate this spectrum. An M, of
32,000 was used in calculations. All three isozymes were present in solution.
38
Bird et al
TABLE 2. Comparison of the Amino Acid Composition of CuZnSOD
From Musca dornestica and Several Other Sources
Amino acid
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Cysteine
Valine
Methionine
Isoleucine
leucine
Tryptophan
Phenylalanine
Lysine
Histidine
Arginine
Tyrosine
Total residues
Chicken
livera
Spinachb
32
18
14
23
12
50
22
14
28
4
14
16
0
8
20
14
8
2
299
35
28
10
20
17
42
23
4
28
2
6
22
0
6
13
14
7
0
277
Ponyfish'
38
20
19
28
12
46
33
-
20
5
13
21
2
11
22
13
6
3
312
Fruit
flf
30
15
15
18
10
44
20
8
26
2
14
12
-
10
18
14
6
2
264
House-
flye
40
16
25
27
17
38
27
18
10
5
9
19
0
11
21
10
9
4
306
aWeisiger and Fridovich [31].
bAsada et a1 [32].
'Martin and Fridovich [27].
dLee et a1 141.
eDetermination of residues made on a solution containing three housefly CuZnSOD isozymes.
ence of four tyrosine residues per dimer. The 18 cysteine and five methionine
residues exceed the numbers typically observed in CuZnSOD, whereas the
10 histidines and 10 valines were fewer than typically observed. A PI of 4.85.1 was measured from the matrix of the isoelectric focusing gel and reflects
a protein containing a high percentage of aspartic acid and glutamic acid
residues.
Housefly CuZnSOD, incubated in the dark for 30 min and illuminated for
60 min, was not affected by RB generated ' 0 2 (Table 3). This lack of effect by
*02
was not surprising considering that the substrate for SOD is the high
energy oxygen radical, 02-.
LDH lost no activity in the presence or absence of RB when not illuminated
(data not shown) but was rapidly deactivated when illuminated (Table 4).At
3 pM housefly CuZnSOD provided no protection for LDH, though this level
of SOD is sufficient to almost completely inhibit the conversion of cytochrome cox to cytochrome c,,d in the bioassay. To ensure that the concentration of housefly CuZnSOD was not too low to provide protection, 15 pM
additional bovine heart CuZnSOD was added to the reaction vessel with no
observable effect.
DISCUSSION
The purification procedure used in this study was more complex than the
original isolation of CuZnSOD from bovine erythrocytes by McCord and
Housefly Superoxide Dismutase
39
TABLE 3. Effects of Rose Bengal on Purified
Housefly CuZnSOD From Newly Emerged
Adults In Vitro
Timea
(min)
Activityb
(unitsiml f SD)
0
15
30
45
60
25.8
26.2
26.8
26.2
25.8
+ 1.6
f 0.7
f 1.8
f 0.9
i 0.9
aTest solution containing rose bengal (1 mM) and
housefly CuZnSOD (all 3 isozymes present) was
preincubated in the dark for 30 min and then
exposed to a 650-W light source for 60 min.
bActivity measured using method of Asada et a1
19,101. See text for details. Each value represents
triplicate determinations.
TABLE 4. Effect of Rose Bengal on Lactic Dehydrogenase in
the Presence and Absence of Purified Housefly CuZnSOD*
Time
(min)
0
2
4
6
8
10
12
% Inhibition
Controla
0
7.1 f 2.9
32.1 8.3
46.6 6.3
60.8 f 7.1
75.0 k 6.7
84.1 + 8.1
*
*
+ SD
Plus CuZnSODb
0
7.3 f 2.1
29.6 + 2.2
49.8 3.3
61.3 f 1.0
73.8 f 2.1
80.5 + 4.9
*Purified housefly CuZnSOD from newly emerged adults was
actually a solution of three electromorphs (see text).
"Test solution containing RB (Abs = 0.8 at 556 nm) and LDH
(550 units) in 15 ml phosphate buffer (50 mM, pH 7.5)
illuminated with a 650-W light. Assay after Callaham et a1 1201.
milliliter of solution from "a" plus 3 pM (96 p g ) housefly
CuZnSOD. See text for details.
Fridovich [5]. Contaminants were observed at the final gel filtration step
when the unmodified method was used, and as a result, cation exchange
chromatography on CM-Sepharose and preparative electrophoresis were
incorporated. M . domestim CuZnSOD proved to be highly stable to salting
out with dibasic potassium phosphate in the presence of chloroform-ethanol,
whereas D. melanogaster enzyme quickly lost activity at this step [4]. The level
of purification and specific activity using DEAE-Sephadex was lower in this
study than that in studies using erythrocyte sources. Typically, 60- to 300fold purification is observed at this step [5,22-241; however, only a 41-fold
purification was measured here. Sephadex gel filtration gave a 132-fold
purification with a specific activity of approximately 880. Gel filtration is the
last step in many CuZnSOD purifications, and specific activities approaching
2,600 were frequently observed; however, inclusion of preparative electrophoresis was required to attain a comparable level of purity [2,5,24]. Lee et
40
Bird et al
a1 141 reported a much higher specific activity for D. melanoguster of approximately 4,800. In parallel studies with the bovine enzyme, D. lnelunogasfer
CuZnSOD was found to be 1.6 times more active. This high activity in the
fruit fly led them to conclude that the high level of resistance to ionizing
radiation observed in Drosophilu is due to this unusually active CuZnSOD.
SOD activity in the Lee et a1 [4] study was measured by the method of
McCord and Fridovich [5], whereas in this study it was expressed in the form
of Asada units [9]. Therefore, caution should be taken when comparing the
results of these two studies because of difference in the expression of assay
results.
The aggregation phenomenon reported by Salin and Wilson [3] for the
porcine enzyme was also observed in the housefly protein. Enzyme purified
by gel filtration and homogeneous with respect to molecular weight at approximately 34,800 was stored at 4°C. Subsequent chromatography of the
fraction on Sephadex G-200 revealed the presence of a high-molecularweight, 254-nm absorbing peak that possessed no SOD activity and consistently eluted in the void volume. It was assumed that this high-molecularweight fraction was aggregate CuZnSOD, which could be seen as a bluegreen precipitate on standing in purified sample. Protein measurement of a
filtered sample of this purified CuZnSOD sample indicated that protein loss
had occurred while the specific activity was unchanged. Salin and Wilson 131
suggested that the self-association was due to interchain sulfhydryl interactions and could be prevented by addition of a thiol reagent. We also observed
that 2-mercaptoethanol or dithiothreitol prevented formation of the precipitate, though it was not possible to reverse the process.
MnSOD and CuZnSOD were both found in housefly tissues. It is now
known that MnSOD is a mitochondria1enzyme and that CuZnSOD is located
in the cytosol, though exceptions have been noted 113. Three CuZnSOD
isozymes were identified at all ages and in each of the body tagma along
with a single MnSOD. These results indicated that all isozymes are present
in each of the body divisions. No attempt was made to determine the
presence of the isozymes in individual organs either quantitatively or
qualitatively.
The relationship of the CuZnSOD isozymes to one another has been
investigated previously [14,24-281. Multiple isozymes have been reported but
not characterized in other insect species. Lorimer [29] observed three isozymes in the forest tent caterpillar, Mulucossomu disstriu, with as many as four
in some individuals, and Bartlett 1301 reported two isozymes in the pink
bollworm, Pectinorphoru gossypielh. Neither study specified the forms as
CuZnSOD or MnSOD, but it is likely that the most common isozymes
observed were analogous to the CuZnSOD electromorphs and MnSOD of
this study. Lee et a1 [4] reported only one CuZnSOD and one MnSOD in D.
melanoguster.
The amino acid profile of housefly CuZnSOD is presented in Table 2 along
with those of several other species. It is obvious that the housefly enzyme
contained a greater number of aspartic acid, serine, proline, and cysteine
residues but fewer valine residues. Four tyrosines were present per dimer of
housefly CuZnSOD, whereas only two per dimer are generally reported.
Housefly Superoxide Dismutase
41
Drosophilu and chicken liver are representative of those enzymes. Martin and
Fridovich [27l examined seven marine fish species and typically found 3-5
tyrosine residues in each of these. Tryptophan was absent from Muscu and
all other species in Table 2 except ponyfish. The absence of the tryptophan
in these species including housefly is reflected by the UV-absorbance
maximum centered at 258 nm while ponyfish has a higher maximum at 265
nm (Table 5). The extinction coefficient of housefly CuZnSOD is only onehalf the value of fruit fly enzyme but is in the same range as that in spinach
and chicken liver. The 1.6 mol of copper and 1.7 mol of zinc per mol of
enzyme dimer are in close agreement with the values reported for the other
species. Determination of subunit size by SDS-PAGE led to the conclusion
that each molecule of active enzyme is composed of two associated subunits
of approximately 16,000. Based on this, the molecular weight of the dimer
would be in the 32,000 range rather than 34,800 measured by gel filtration.
M . domesticu CuZnSOD is not significantly different from CuZnSODs
isolated from other organisms. Indeed the similarities are striking with respect to amino acid composition, molecular weight, metal prosthetic groups,
PI, and ultraviolet absorbance spectrum as shown in Tables 2 and 3. The PI
is the most dissimilar value, reflecting the variation in acidic amino acid
residues. The housefly CuZnSOD data presented here actually represent a
heterogeneous mixture of at least three resolvable isozymes.
Rose bengal is one of a large group of dye compounds known to generate
*02
in illuminated aqueous solutions. lo2generated in this manner is responsible for many biological effects including reactions with amino acids,
nucleic acids, lipids, tocopherols, polysaccharides, and various enzymes [33].
Housefly CuZnSOD, however, was found to be unaffected when exposed to
'02for a period of 1h. Forman et a1 [34] investigated bovine CuZnSOD and
found it was also unaffected by lo2when its full metal complement was
present. Removal of the metals led to a rapid, irreversible deactivation of the
apoenzyme by the destruction of 3.6 histidine residues per molecule. Presumably, photooxidizable amino acid residues are not accessible to ' 0 2 as a
TABLE 5. Comuarison of CuZnSOD From Musca domesticu and Several Other Sources
Chicken
livera
Spinach'
Ponyfish'
Molecular weight
Metal content per dimer
Copper
Zinc
UV-absorbance
peaks (nm)
Extinction coefficient
M-cm-*
30,400
32,200
32,000
8,750
9,920
-
VI
5.35-6.75
-
7.8-8.2
1.8
1.6
258
2.2
2.2
258
-
265
'Weisiger and Fridovich 1311.
'Asada et a1 [32].
'Martin and Fridovich [27].
dLee et a1 [4].
eValues measured from a solution of three CuZnSOD isozymes
Fruit flyd
32,000
2.1
2.2
258
13,200
5.3
Houseflye
32,000
1.6
1.7
258.9
7,890
4.8-5.1
42
Bird et at
result of evolutionary pressures to eliminate residues that react with highenergy oxygen.
Studies on the relationship of lo2and 02-have led at least two groups
[6,nto suggest that SOD is capable of interacting with lo2and inhibiting its
reaction. This was not the case in this study, since LDH exposed to lo2was
rapidly inactivated when 3 pM housefly CuZnSOD was present or absent.
The possibility of too low a CuZnSOD level was also precluded by addition
of 15 pM bovine CuZnSOD. The results were identical, indicating that either
02-was not involved in the deactivation of LDH, though its generation by
RB could not be ruled out, or housefly CuZnSOD was unable to detoxify the
lo2molecule.
Use of the xanthene dyes as pesticides presents another mechanism for
toxicological study. In two in vitro tests we saw no indication that housefly
CuZnSOD affords protection to the insect against an oxygen species (lo2)
related to its primary substrate (02-);
however, the possibility exists that the
in vivo situation is different. Preliminary results have indicated that the total
SOD levels do, in fact, increase in the presence of RB and light in houseflies
allowed to feed on sugar-water solutions containing the dye. Studies are
continuing in this area.
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