Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

j? Insect Physiol., 1975, Vol. 21, pp. 1373 to 1384. Pergamon Press. Printed in Great Britain.

TYROSINASE

ACTIVITY

IN THE LARVA

SARCOPHAGA

OF THE FLESHFLY,

BARBARTA

LYNDEN HUGHESand GARJZTH M. PRICE Agricultural

Research Council,

Unit of Invertebrate Brighton BNl 9QJ,

Chemistry and Physiology, Sussex, England

University

of Sussex,

(Received 2 January 1975) Abstract-When

plasma from third instar larvae of the fleshfly, Sarcophaga barbavta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 6.5% to ammonium sulphate, and it was found that the 50% fraction contained 950,0 of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration. The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.0.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme. The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 5096 fraction, 620,& now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in wivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.

MATERIALS

INTRODUCTION TYROSINASEactivity in insects has been a muchinvestigated subject (reviews by BRUNET, 1963, 1965; COTTRELL, 1964; HACKMAN, 1964). In dipterous larvae (OHNISHI, 1953, 1954, 1958, 1959; LEWIS and LEWIS, 1963 ; KARLSON et al., 1964) and in lepidopterous larvae (ASHIDAand OHNISHI, 1967 ; ASHIDA, 1971) the enzyme has been shown to be present in the blood as an inactive precursor or proenzyme while an activator of the proenzyme has been found in the cuticle (KARLSON et al., 1964; LAI-FOOK, 1966; ASHIDA et al., 1974). However, it is still not clear whether in vivo the enzyme found in the blood plays a r81e in the hardening and darkening of the larval cuticle at the time of puparium formation and whether the proenzyme in the blood is activated by the cuticle activator. In the present work the level of proenzyme in the blood during the third larval instar and early puparial stages of the fleshfly has been measured, and its behaviour during isolation and activation has been examined.

AND

METHODS

Chemicals The following chemicals were purchased. Acryland N,N’-methylene-bis-acrylamide of amide electrophoresis-purity grade from Bio Rad Laboratories, 32nd and Griffin, Richmond, Calif., U.S.A., and 4-methyl catechol from Koch-Light Laboratories Ltd., Colnbrook, Bucks. This was recrystallized from light petroleum (B.P. 60 to 80°C) before use. Other chemicals were of Analar grade and glass-distilled water was used throughout. Breeding of Sarcophaga Larvae of the fleshfly were bred essentially as previously described for the blowfly, Calliphora erythrocephala (PRICE, 1969). Fractionation

of larval haemolymph

The anterior end of a larva was pricked with a fine pin and the haemolymph released (cu. SO/d)

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LYNDENHUGH= ANDGARETHM. PRICE

was collected in an ice-cold tube. By this method about 4 ml were collected and centrifuged at 2700 g for 15 min to remove the haemocytes. The cell-free supernatant (plasma) was diluted tenfold with glass-distilled water (PRICE, 1967) and left to stand on ice for 2 to 3 hr during which tune a yellow precipitate formed. Occasionally it was

activator was dissolved in 1 ml of Ringer. This fraction is subsequently referred to as the activator. Determination

of tyrosinase actiwity

Tyrosinase activity was measured essentially as previously described (HUGHES and PRICE, 1974). The assay mixture consisted of appropriate volumes

Plasma (4 ml) diluted tenfold with cold distilled water, left to stand overnight at 4°C. Centrifuged at 2700 g for 15 min

-_:;%$;;I

(NH&SO, in the

Dissolvid

in 4 ml of

cold. Left for 5 hr on ice and centri, O fuged as above

:tI:

Supernatant: Rendered 50% to (NH&SO, and left overnight on ice. Centrifuged as above

Residue : Dissolved in 4 ml of Ringer to give the 307; (NH,),SOI fraction

Super&ant: Rendered 65 % to (NH&SO, and left overnight on ice. Centrifuged as above

Residue: Dissolved in 4 ml of Ringer to give the SO?b (NH,),SOI fraction

Ringer to give the lipoprotein ,

I

Supernatant: Discarded

Residue: Dissolved in 4 ml of Ringer to give the 65% (NH,),SOI fraction

Scheme 1. Flow diagram showing fractionation

found necessary to adjust the pH of the diluted plasma to 6.0 before precipitation occurred. Further treatment was carried out as shown in Scheme 1. Isolation of cuticle activator Cuticles, isolated from larvae as previously described (PRICE, 1972), were each cut longitudinally to give a sheet of tissue. Twenty of these were incubated overnight at 4’C in 10 ml of Ringer after which they were removed and the medium was centrifuged at 2700 g for 15 min. The supernatant was rendered 30% to ammonium sulphate in the cold, left for 5 hr or overnight at 4”C, and the protein which precipitated out was centrifuged down as before. The protein pellet which contained the

of Surcophaga plasma.

of plasma fractions (see Scheme 1) and activator in 3 ml of 0.1 M phosphate buffer, pH 6.5, containing methyl catechol to 0.01 M. Oxygen uptake was measured with a Rank oxygen electrode and tyrosinase activity is expressed as pmole of 0, absorbed per min at 25°C. Determination

of protein

Protein was determined by the method of LOWRY et al. (1951). y-Globulin was used as standard. Disk electrophoresis on polyauylamide

gel

Disk electrophoresis was carried out in a conventional manner as previously described (PRICE, 1974).

Tyrosinase

activity in Surcophagu larvae

RESULTS Distribution

of tyrosinase activity

We have previously reported (HUGHES and PRICE, 1974) that tenfold dilution of plasma from third instar larvae of the fleshfly with cold distilled water results in precipitation of lipoproteins and most of the active tyrosinase. However, it was possible that the water supernatant and the lipoprotein fraction also contained tyrosinase in its inactive form or proenzyme. Proenzyme has previously been isolated from the blood of third instar larvae of the blowfly, Calliphora erythrocephala, by ammonium sulphate fractionation (KARLSONet al., 1964; MUNN and BUFTON, 1973). In the present work the water supernatant was examined for proenzyme after fractionation with ammonium sulphate as shown

0

2

4

6

6

Time, days Fig. 1. Development of tyrosinase activity in 30% ammonium sulphate fraction (C-0, units O-20) and in the lipoprotein fraction (A-A, units O-1.0). These fractions were obtained as shown in Scheme 1. In this figure and in subsequent figures tyrosinase activity was assayed as described in Materials and Methods, and the activities shown relate to l,O ml of plasma.

in Scheme 1. Fig. 1 shows the development of tyrosinase activity at 4°C in the 30% fraction and in the lipoprotein fraction. The activity in the 30% fraction rose to a maximum after 1 day, then fell Table 1. Tyrosinase

Fraction

Distribution

1.02

316

2.21 3.23

68.4

Lipoprotein Total

slightly over the next 2 days and subsequently remained constant for up to 1 week, while that in the lipoprotein fraction rose to a maximum after 2 days and remained constant for up to 1 week. The activity in both the 50 and 65% fractions was initially very low and remained so for up to 1 week. In subsequent experiments, fractions were stored for 48 hr at 4°C before they were assayed. Table 1 (results of one experiment) shows that the combined activity of the water supernatant and lipoprotein fraction was 3.2 @mole of oxygen absorbed/min per ml of plasma). However, when the supernatant was fractionated with ammonium sulphate (Scheme 1) the level of activity rose to 12.8 and the 30% fraction now accounted for 80% of the activity. This increase indicated that the water supernatant contained proenzyme, some of which had been activated by treatment with ammonium sulphate. To determine the full potential of the proenzyme in the fractions they were incubated overnight (18 hr) at 4°C either with activator (see Materials and Methods) or with the same volume of Ringer as control. Table 2 shows that in the presence of activator there was a large increase in activity (12.8-255.7) with the 50% fraction now accounting for 95 y0 of all the activity. Over the same period the activity of the controls did not change, This result showed that the water supernatant contained proenzyme, the vast amount of which was precipitated out in the 50% fraction. This fraction will subsequently be referred to as the proenzyme. The level of activity in the 30% fraction was virtually the same before and after incubation with activator, indicating that this fraction contained mostly active enzyme and little, if any, proenzyme. Table 2 also shows that on a protein basis the 50% fraction was the most active. All activities shown in Table 2 are corrected for the small amount of enzyme activity in the activator itself.

Effect of activator

concentration

Fig. 2(a) shows that when a fixed amount of proenzyme was incubated at 4°C with various amounts

activity in various fractions of Surcophugu larval haemolymph

Tyrosinase activity (pmole of 0, absorbed/min)

Water supernatant

1375

(%)

Fraction

Tyrosinase activity @mole of 0, absorbed/min)

Distribution

30% Ammonium sulphate 10.30 fraction of water 0.14 50% supernatant 65% 1 { 0.15 2.21 Lipoprotein Total 12.80

Haemolymph (blood) was centrifuged to remove the blood cells and the plasma was fractionated by water dilution and with ammonium sulphate as shown in Scheme 1. The levels of activity shown relate to 1.0 ml of plasma.

(%) 80.4 1.1 1.2 17.3

LYNDEN HUG=

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AND GARFZTHM. PRICE

Table 2. Effect of activator on tyrosinase activity in various fractions of Sarcophaga larval haemolymph Tyrosinase

activity of fraction With activator

Without activator

Fraction

(%)

11.0

4.3

6.19

0.02

242.9

95.0

37.61

< 0.01 0.41

0.4 1.4 255.7

0.2 0.5

0.01 0.26

pmole of 0s absorbed/ min

Distribution

Protein content of fraction

(%)

(mg)

10.30

80.4

1.77

5.81

0.14

1.1

6.46

0.15 2.21 12.80

1-2 17.3

63.60 544

Lipoprotein Total

-

pmole of 0, absorbed/ min per mg of protein

pmole of 0, absorbed/ min per mg of protein

pmole of Or absorbed/ min

Total

Distribution

Haemolymph was fractionated as shown in Scheme 1. Activator was prepared as described in Materials and Methods. The levels of activity shown relate to 1-O ml of plasma.

(b)

0

2

4

6

.24

Time; hr

0

1

2

3

4

Time, hr

Fig. 2. (a) Activation of proenzyme in the presence of various amounts of activator. Proenzyme and activator were mixed in the proportion 1 : 1 (O-O), 1 : 0.75 (A-A), 1 : 0.5 (O--O), and 1 : 0.25 (o-0) by volume, then incubated at 4°C for various periods, and the tyrosinase activity of the mixture assayed. (b) Time taken to reach rapid phase of activation plotted against relative concentration of activator.

of activator there followed a lag period before activation took place, the duration of which was dependent upon the activator concentration (Fig. 2b). Depending upon the activator concentration activation was complete in 0.5 to 5 hr. Furthermore, in the presence of a fixed amount of proenzyme the maximum activity attained was the same for all concentrations of activator tested, but at 24 hr the activity had fallen below the maximum.

Effect of proenzyme

concentration

When a fixed amount of activator was incubated at 4°C with various amounts of proenzyme there followed a lag period before activation took place,

the duration of which was dependent upon the proenzyme concentration (Fig. 3). However, in this case the activity attained at 24 hr was dependent upon the concentration of proenzyme in the incubation mixture.

E#ect of temperature of incubation When a 1 : 1 by volume mixture of proenzyme and activator was incubated at 4°C and at 25”C, activation commenced sooner in the mixture at 25°C than in the one at 4°C (Fig. 4). However, the maximum activity attained at 4°C was greater than that attained at 25°C.

Tyrosinase

activity in Sarcophugu larvae

1377

m

a

z

I

a

0

2

0

0

2

4 Time,

6

24

hr

Fig. 3. Activation of various amounts of proenzyme in the presence of a fixed amount of activator. Activator and proenzyme were mixed in the proportion 1 :1 (O-O), 1 : 0.75 (A-A), 1 : 0.5 (O----O), and 1 : 0.25 (0-0) by volume then incubated at 4°C for various periods, and the tyrosinase activity of the mixture assayed.

4

I

I

6

8

hr

Time,

Fig. 5. Effect of further addition of activator on tyrosinase activity. Proenzyme and activator were incubated for various periods at 4°C and tyrosinase activity assayed. Maximum activity was attained at 2) hr and a further addition of activator was made at 4) hr (arrow). Tyrosinase activity was subsequently assayed.

Effect of further addition of proenzyme The above experiment was repeated except that at 44 hr more proenzyme and not activator was added to the mixture. Fig. 6 shows that addition of proenzyme resulted in a further increase in tyrosinase activity indicating that activator was still present in the mixture. The rapidity with which activation took place suggests that the original activation process, between zero time and 4+ hr (Fig. 6), did not result in a measurable depletion of any of the activator present. ;

100

o-o-0-0

-

0 r x 7c

4

75

-

50

-

25

-

E

0

15

30 Time,

45

60

min

Fig. 4. Effect of incubation temperature on the rate of activation of proenzyme by cuticle activator. Activator and proenzyme were mixed in the proportion 1 : 1 by volume and incubated at 4°C (0-O) and at 25°C (0-O) for various periods after which the tyrosinase activity of the mixture was assayed.

P d z

I 0

0 I 0”

f-“-“-!

; s x

/ 6

?,/o

0

2

I

1

I

4

6

8

Time,

Effect of further

addition of activator

It was shown above (Fig. 3) that the maximum activity attained is proportional to the proenzyme concentration and it is assumed that the attainment of maximum activity indicates that all the proenzyme has been converted into active enzyme. To check this assumption a 1 : 1 by volume mixture of proenzyme and activator was incubated at 4°C. Maximum activity was attained after 24 hr (Fig. 5). Further addition of activator at 44 hr did not result in any increase in tyrosinase activity so indicating that there was no proenzyme left to be activated.

hr

Fig. 6. Effect of further addition of proenzyme on tyrosinase activity. Proenzyme and activator were incubated at 4°C for various periods and tyrosinase activity assayed. Maximum activity was attained at 24 hr and a further addition of proenzyme was made at 4$ hr (arrow). Tyrosinase activity was subsequently assayed.

Distribution of activator fractions of cuticle

between ammonium sulphate

Cuticles from 7 day larvae were incubated in Ringer at 4°C overnight after which they were removed and the medium rendered 30, 50, and

LYNDFZN HUGHFS AND GARETH M. PRICE

1378

65% to ammonium sulphate, the protein precipitating out at each concentration being dissohed in Ringer. Samples of each fraction were incubated with an equal volume of proenzyme for various periods and their tyrosinase activity assayed. Fig. 7 shows that a high percentage of the activator was in the 30% fraction, there being only a small amount in the 50 and 65% fractions.

results show that the outer and inner layers of the cuticle contained more activator than did the middle However, during separation of the cuticle layer. into layers, activator was released into the medium (A-A, Fig. 8) and, since it is not known whether it was released more rapidly from one layer than from another, it is not possible to say with certainty that the distribution shown truly reflects the in vivo distribution.

Level

of activator

in cuticles of different

ages

When activator, isolated from cuticles of 3 to 8 day larvae and rounded-off white puparial stage (r.o.), was incubated with proenzyme from 7 day larvae the rate of activation was similar for all these ages, indicating that the level of activator in the cuticle was fairly constant over most of the third instar.

5 =t

0

15

45

30 Time,

60

Tyrosinase

min

Fig. 7. Activation of proenzyme in the presence of different ammonium sulphate fractions of cuticle exudate. Ammonium sulphate fractions, 30% (C-C), 50% (A-A), and 65% (O-Cl), were prepared as described in Materials and Methods, incubated with proenzyme at 4°C for various periods, and their tyrosinase activity assayed. Distribution

of activator

in cuticle

Cuticles from 7 day larvae were split into three layers, outer, middle, and inner. Activator was isolated from each layer by incubation in Ringer at 4°C overnight and from the medium in which the layers were separated. When a 1 : 1 by volume mixture of activator and proenzyme was incubated at 4”C, activity developed as shown in Fig. 8. The

in plasma

Haemolymph, isolated from 3 to 8 day larvae golden-brown (g-b.), and darkand from r.o., brown (d.b.) puparial stages, was fractionated as shown in Scheme 1. The tyrosinase activity of each fraction, before and after addition of activator from 7 day larvae, was assayed and the results are shown in Fig. 9 and Table 3. The level of proenzyme (tyrosinase activity in 50% ammonium sulphate fraction after addition of activator) and of total tyrosinase (total activity of all fractions after addition of activator) increased steadily throughout the third instar reaching a maximum in 7 day larvae and remained at this level in 8 day larvae and in the r-0. white puparial stage. The level of enzyme

7 0

;

activity

40

x 7 .E E B p

30

20

8 -z 0”

10

I z =.

5 7

0

15

45

30 1 Time,

60

min

FIG. 8. Activation of proenzyme by various cuticle layers. Cuticle was separated into three layers, outer (O-O), middle (a---O), and inner (O-0). These were incubated separately in Ringer overnight at 4°C and the activator released (30% ammonium sulphate fraction), and that previously released into the medium during dissection of the cuticles (A-A), was incubated with proenzyme at 4°C for various periods and the tyrosinase activity assayed.

3

4

5 Larval

age,

7

6 days

and

puparial

6

gb

db

stage

Fig. 9. Level of proenzyme (A-A) and of active ~.. . _ enzyme (m-0) in plasma fractions (see Scheme 1 and Table 3) during the third larval instar and early puparial stages of S. barbarta. The curve (0-C) is the sum of the activities present in all the fractions after activation (see Table 3). r.o., Rounded-off white puparial stage; g.b., golden-brown puparial stage; d.b., dark-brown puparial stage. Tyrosinase activity was assayed as described in Materials and Methods.

3.88

6.70

3.77

0.51

55.62

9.03

0.55

_*

1.40

0.32

0.26

50.38

0.91

0.40

0.28

0.28 7.99

lZ4 3.70

I;0 3.19

0.40 2.76

0.28 2.70

-

4

-

3

:45 7.60

129.83

-*

117.78

5

activity, prior to and after activation,

26.10

0.54

0.24

0.27 25.05

6

225.44

6.42

190.56

+ 5.26 23.20

22.09

0.62

0.44

0.23 20.80

7

287.57

-*

261.90

;07 IS.60

26.57

0.62

168

0.32 23.95

-

8

277.49

-*

253.00

+ 0.66 23.83

25.13

0.56

0.52

0.18 23.87 of67 21.60

284.27

-*

262.00

r.0. -

0.00 _-__ 14.68

1.80

0.77 12.11

--

97.02

:44 10.62

291.08

182.00

g.b. -

357.41

0.67

70.73

189.98 96.03

d.b.

in plasma fractions from third instar larvae and early puparial stages of the fleshfly, S. barbarta

Larval age in days, and early puparial stages. Tyrosinase activities in pmoles of oxygen absorbed/min. - , Prior to activation; + , after activation. Plasma was fractionated as shown in Scheme 1. Tyrosinase activity was assayed as described in Materials and blethods and relates to 1.0 ml of plasma. r.o., g.b., and d.b.; rounded-off white, golden-brown, and dark-brown puparial stages, respectively. * Activity beneath the limits of detectability.

Total

Lipoprotein 30% Ammonium sulphate fraction 500/, of water supernatant 1 65% I

Fraction

Table 3. Tyrosinase

375.36

-*

83.26

20:70 87.40

3 r a b

$

: 9

?:

$, <

+ z 0 8. 2 $

LYNDEN HUCHFZSAND

1380

isolated in the form of active enzyme remained low over this period. At each age up to and including the r.o. stage, approximately 90% of the total tyrosinase was isolated in the SOO;, ammonium sulphate fraction as proenzyme (Table 3). However, at the g.b. puparial stage there was a redistribution of activity between the ammonium sulphate fractions, the 500,; fraction now accounting for only 33% of the activity and the 65% fraction accounting for 62.5tb of the total activity, the activity in both these fractions being ascribed to proenzyme in that it appeared only after the addition of activator. At the d.b. stage 12 hr later there was yet a further redistribution of activity, the lipoprotein fraction accounting for 545%, and the 30 and 50% fractions accounting for 23.3 and 22.2%, respectively. There was no measurable activity in the 65%fraction. However, not only was there a redistribution of activity but the activity was no longer attributable to proenzyme but to active enzyme, in that the activity was not increased by the addition of activator (Table 3). Thus, the level of total enzyme activity in the plasma at the dark-brown puparial stage is higher than at the 8 day larval stage and is apparently all present as active enzyme (Fig. 9).

Distribution cytes

of tyrosinase

between plasma

activity

Disk electrophoresis

on polyacrylamide

gel

Samples (approximately 150 pg of protein) of the 30, 50, and 65% ammonium sulphate fractions and of the lipoprotein fraction from plasma of 7 day larvae were subjected to eIectrophoresis (PRICE, 1974) after which the gels were washed in phosphate buffer, pH 6.5 (three changes, 30 min each) and

immersed overnight in 0.01 M dihydroxyphenylalanine (dopa). With the liprotein fraction (Fig. 10a) a band of enzyme activity was discernible only at the surface of the stacking gel. With the 30”/& fraction (b), activity was discernible at the surface

and haemo-

Haemolymph, isolated from 7 day larvae, was centrifuged at 2700 g for 15 min to precipitate the haemocytes. The plasma was decanted and the haemocytes were washed by resuspending and centrifuging them in the same volume of Ringer. The washed haemocytes were resuspended in Ringer, sonicated (PRICE, 1974), and centrifuged at 28,000 g for 30 min. The supernatant was decanted and the pellet resuspended in an equal volume of Ringer. When samples of the plasma, washings, cell-sonicate supernatant, and pellet were incubated with activator and assayed for enzyme activity, it was found that the first three fractions accounted for 98.6, 1.0, and 0.4% of the total activity, respectively, none being detectable in the cell-sonicate pellet.

Tyrosinase

GARETH M. PRICE

in cuticle

Ammonium sulphate fractions of cuticle exudates from 7 day larvae, prepared as described above, were assayed for tyrosinase activity. The 30 and SO”/6 fractions contained 91.8 and 8,2% of the activity, respectively, while activity could not be detected in the 65% fraction under the conditions of assay. During the period from the 4 day to the 8 day larva the level of enzyme activity in the cuticle remained fairly constant at 0.06 @mole of 0, absorbed/min per cuticle) but fell at the r.o. white puparial stage to O-03.

a

b

c

d

*

Fig. 10. Localization of tyrosinase activity on polyacrylamide gels after electrophoresis of plasma fractions (a-e) and of cuticle fraction (f) obtained from Sarcophaga larvae as described in the text. After electrophoresis all the gels were washed in buffer (see text), and then incubated in 0.01 M dopa solution. (a) Lipoprotein fraction; (b) and (c) 30 and 50% ammonium sulphate fractions, respectively; (d) 50% ammonium sulphate fraction incubated with activator prior to electrophoresis; (e) gel of 50% ammonium sulphate fraction cut in half longitudinally, left half incubated with dopa solution and right half incubated with 0.002 M tyrosine solution; (f) 30% ammonium sulphate fraction of cuticle. 0, Origin of stacking gel; I, stacking-separating gel interface. Arrow shows direction of migration. Band staining intensity is illustrated by density of shading.

of the stacking gel, at the stacking-separating gel interface, and a short distance along the separating gel. It was possible that the enzymes on the surface of the stacking gel and at the stacking-separating gel interface had not penetrated further into the gel because they were in a highly aggregated state. This possibility was substantiated when it was found that after centrifuging the 30% and lipoprotein fractions at 100,000 g for 30 min, the activity remaining in the supernatant was only 18 and

16%,

respectively,

of

that

present

before

Tyrosinase

activity in Sarcophaga larvae

centrifugation. Sedimentation under such conditions indicates the presence of a large aggregate of at least microsomal dimensions. With the 50% fraction (c), three major bands of activity were observed in the separating gel after 18 hr incubation, but none in the stacking gel, indicating that the 50% fraction contained some active enzyme which was not aggregated to as great an extent as was the enzyme in the 30% (b) and lipoprotein fractions (a). However, when the SOY/, fraction was incubated with activator prior to electrophoresis, bands of activity were now discernible at the surface of the stacking gel and at the stacking-separating gel interface (d). The result showed that activation is accompanied by aggregation of the enzyme. When gels of the 50% fraction were incubated with tyrosine (0.002 M) only one band of activity was discernible and this corresponded to the slowest-running catecholase band (e). No bands of activity were discernible on gels of the lipoprotein fraction when they were incubated with tyrosine, or on gels of the 65% ammonium sulphate fraction when they were incubated with dopa or tyrosine. In other experiments gels of the lipoprotein fraction and of the 30, 50, and 65% ammonium sulphate fractions were washed in buffer and then incubated for 2 hr in cuticle activator before being incubated in dopa solution. This procedure gave results identical to those shown in Fig. 10 for gels a, b, and c whereas for the 65y0 ammonium sulphate fraction (not shown) a single band of activity was now discernible in the separating gel at a position approximating the slowestrunning catecholase band of the 50% fraction. These results indicate that incubation with activator subsequent to electrophoresis had a detectable effect only with the 65% ammonium sulphate fraction. The results also indicate that, with the 500,$ fraction, the process of electrophoresis may itself result in activation of some of the proenzyme. When gels of the 30% ammonium sulphate fraction of cuticle were incubated with dopa, one band of activity was discernible (f) but with tyrosine no bands were discernible. The result indicates that the soluble enzyme in the cuticle behaves as a diphenoloxidase and not as a monophenoloxidase. DISCUSSION

It was previously reported (HUGHES and PRICE, 1974) that tenfold dilution of plasma from SUYCOphugu larvae with cold distilled water resulted in precipitation of a lipoprotein fraction possessing tyrosinase activity. Since efforts aimed at separating enzyme activity from the lipoprotein were unsuccessful it was suggested that the tyrosinase might be a lipoprotein. However, it was not known whether the tyrosinase associated with the lipoprotein accounted for all the potential tyrosinase activity in the plasma, or whether the water supernatant also

I381

contained some enzyme, but in an inactive form or proenzyme. To investigate these possibilities the supernatant was fractionated with ammonium sulphate and each fraction, as well as the lipoprotein fraction, was incubated with a cuticle activator preparation. The results showed that the lipoprotein fraction accounted for less than 1 y0 of the total potential enzyme activity while the 50% ammonium sulphate fraction (proenzyme) accounted for 95 9 h of the total activity. Since the water treatment resulted in precipitation of all the lipoprotein, and since 95 yb of the potential enzyme activity remained in the supernatant, then the enzyme could not be a lipoprotein. It is possible that water dilution of the plasma resulted in the activation of some of the proenzyme present and that the active enzyme was adsorbed onto the flocculent precipitate of lipoprotein, co-sedimenting with it during centrifugation. Co-sedimentation of phenoloxidase with particulate material and with mitochondria has previously been observed by SEKJZRISand MERGENHAGEN (1964). The development of enzyme activity that occurred on standing at 4°C (Fig. 1) indicates that proenzyme may also adsorb onto the lipoprotein precipitate, and be subsequently converted to the active enzyme in an undetermined manner. A similar development of activity was observed with the 30% ammonium sulphate fraction (Fig. 1). Thus, these two fractions became active without the addition of cuticle activator in contrast to the 50 and 65% fractions which attained activity only after the addition of activator. The activation of tyrosinase by means other than treatment with a cuticle activator has also been observed in eggs of the grasshopper, Melanoplus diferentialis (BODINE et al., 1937), in larvae of the mealworm Tenebrio molitor (HEYNEMAN and VERCAUTEREN,1968), in acetone-powder preparations of plasma from the Chinese oak silkmoth, Antherea pernyi (EVAKS, 1967), in haemolymph of the European cornborer, Ostriniu nubiZuZis (BRENNAN and BECK, 1972), and in extracts of whole parasitic wasps, Movmoniella vitripennis (FIRTEL and SAUL, 1967). EVANS (1967) also observed that tenfold dilution of fresh blood and plasma with enzyme activity,

water resulted in a stimulation of and PRESTON and TAYLOR (1970)

working with the cockroach, Leucophaea maderae, and ISHAAYA (1972) working with the Egyptian cotton worm, Spodoptera littoralis, have also used blood diluted with distilled water as phenoloxidase Our results suggest that ammonium preparations. sulphate treatment also results in some activation of the 30% fraction attaining activity proenzyme, without the addition of activator (Table 1). hRTS and VERCAUTEFGN(1964) reported that the proen-

zyme from T. molitor larvae required the presence of 0.1 M ammonium sulphate for solubility and for the development of activity. In the present work the effect of the ammonium sulphate appears to be quite

different.

1382

LYNDENHUGH= AND GARETHM. PRICE

When the activator and proenzyme were mixed there followed a lag period before the rapid phase of activation, the duration of the lag period being dependent upon the activator concentration (Fig. 2a, b) and the proenzyme concentration (Fig. 3). The final activity attained was independent of the activator concentration but was dependent upon the concentration of proenzyme. This result is in contrast to that of KARLSON et al. (1964) who, working with the blowfly, C. erythrocephula, found that the final activity attained was dependent upon the concentration of activator added. On the other hand, similar results to ours have been obtained with preparations also from Culliphora (MUNN and BUFTON, 1973) and from the silkworm, Bombyx mori (OHNISHI et al., 1970; DOHKE, 1973). The time taken for activation to commence was also dependent upon the temperature, activation commencing more rapidly at 25°C than at 4°C although the maximum activity attained at 25 “C was less than that at 4°C. Similar temperature effects have also been observed by OHNISHI (1959), EVANS (1967), FIRTEL and SAUL (1967), and BRENNANand BECK (1972). When activation was complete as indicated by the attainment of maximal enzyme activity, the addition of more activator did not increase the level of activity (Fig. 5) indicating that there was no proenzyme left to be activated. On the other hand, addition of more proenzyme resulted in an increase in activity (Fig. 6) indicating that activator was still present. Similar results were obtained by DOHKE (1973) working with the silkworm, B. mori. To obtain activator, cuticles were incubated in Ringer overnight at 4°C and the protein released into the medium was fractionated with ammonium sulphate, most of the activator being found in the 30% fraction (Fig. 7). Many authors (KARLSON et al., 1964; ASHIDAand OHNISHI, 1967 ; OHNISHI et al., 1970; THOMSON and SIN, 1970; DOHKE, 1973) have homogenized cuticles prior to fractionation with ammonium sulphate. However, using the incubation techniques described above, dficulties experienced in homogenizing Sarcophaga larval cuticles were avoided and ample amounts of activator were obtained. When the cuticle was split into three layers, the outer and inner layers were found to contain most activator, the middle layer the least. However, for reasons already given, it is not possible to say with certainty that this is a true reflection of the in vivo distribution. The level of proenzyme in the haemolymph rose steadily during the third instar (Fig. 9) reaching a maximum in 7 to 8 day larvae, pupariation commencing about 24 hr later (rounded-off white puparial stage). The level of proenzyme was still maximal 1 hr later (golden-brown puparial stage), but its distribution was different from that observed in earlier stages in that now the 50% ammonium sulphate fraction accounted for only 33% of the

activity and the 65% fraction accounted for 62% of the total activity (Table 3). The activity in both these fractions is ascribed to proenzyme in that it appeared only after addition of activator. At the dark-brown puparial stage 12 hr later none of the enzyme isolated in any of the fractions behaved as proenzyme but as active enzyme, in that the activity before addition of the activator was similar to that after its addition. A fall in the level of proenzyme immediately following pupariation has previously been observed in extracts of whole Calliphora larvae (KARLSONand SCH~EIGER, 1961). Other workers (OHNISHI, 1953; MITCHELL, 1966; MITCHELL et al., 1967) found that the phenoloxidase activity in extracts of whole Drosophila larvae and early pupae rose to a maximum at the time of pupariation and then fell rapidly over the next 3 hr reaching a minimum level 12 hr after pupariation. Our results with Sarcophaga indicate that the total level of tyrosinase activity in the blood at 12 hr after pupariation is slightly higher than it is in the late larval stage (Table 3). However, a high percentage of this activity was co-precipitated with the lipoprotein fraction, probably in an aggregated state as indicated by its behaviour on gel electrophoresis and centrifugation. It may be that in the work on Drosophila, active enzyme could have been precipitated down during the preparation of the extracts so resulting in the low level of activity in the extract used. The fact that most of the proenzyme was ‘salted out’ by a different ammonium sulphate concentration (65%) at the golden-brown stage from that at the rounded-off white puparial stage (50%) could reflect changes in the composition of the haemolymph, perhaps the appearance of activator, which could further result in a rapid change in the physicochemical properties of the proenzyme (change in molecular weight) during this 1 hr period. When active enzyme is produced from proenzyme in the silkworm, B. mori, a change in molecular weight of several thousand has been observed (ASHIDAet al., 1974). Further, several authors (HOROWITZ and FLING, 1955; OHNISHI, 1958; AERTS and VERCAUTEREN,1964; ASHIDAand OHNISHI, 1967) have noted the tendency of active tyrosinase to aggregate to particulate dimensions while maintaining catalytic activity. In the present work, evidence that aggregation of the enzyme takes place during activation was provided by the difference in electrophoretic behaviour of the proenzyme fraction before and after its incubation with activator. Similar observations have been made with preparations from D. melanogaster (MITCHELL and WEBER, 1965 ; MITCHELL et al., 1967) and C. erythrocephala (MUNN and BUFTON, 1973). The soluble enzyme in the cuticle behaved as a diphenoloxidase in that it oxidized dopa but not tyrosine and is thus similar to the enzyme extracted from whole Cdiphora larvae (KARLSONand LIEBAU, 1961) and from cuticles of Luciliu larvae (HACKMAN

Tyrosinase

activity iIn Sarcnphaga

and GOLDBERG, 1967), and Periplaneta larvae (MILLS et al., 1968). An insoluble lactase-type enzyme has been found in the pupal cuticle of B. mori (YAMAZAKI, 1969, 1972) but whether such an enzyme is present in Sarcophaga larval cuticle is not known. The level of activity in the cuticle during the third instar remained fairly constant but fell at the rounded-off white puparial stage. A fall at that stage in Lucilia larvae was also observed by HACKMAN and GOLDBERG (1967). When cuticles were separated into outer, middle, and inner layers most of the enzyme activity was found in the outer layer. In a previous study on Sarcophaga cuticle, DENNELL (1947) located most of the enzyme activity in the inner epicuticle and outer endo-cuticle, both of which would be included in the outer layer in the present work. It is to be emphasized that in the present work extracts have been made of plasma and of cuticle, but not of whole insects. At the dark-brown puparial stage all the enzyme in the various plasma fractions behaved as active enzyme. At earlier stages it is probable that in viva all the enzyme in the plasma is present as proenzyme and that the small amount of active enzyme isolated resulted from the activation of some proenzyme during the fractionation procedure. If the fractionation procedure activates only a small amount of proenzyme, then, since at the dark-brown puparial stage virtually all the enzyme was isolated as active enzyme, much of this may well have been present as such in vivo. If the enzyme is active in the blood then it may be possible to detect there oxidation products of tyrosine. The presence of an active enzyme in the blood also raises the question as to how it became active. Is the cuticle activator implicated in this and/or is there a factor released by the blood cells at the time of pupariation ? These aspects are currently under investigation. Acknowledgements-We thank Mr. S. E. LEWIS for critically reading the manuscript and we are indebted to Mrs. ELIZABETHHUGHESfor skilled technical assistance.

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BODINE J. H., ALLEN T. H., and BOELL E. J. (1937) Enzymes in ontogenesis (Orthoptera)-III. Activation of naturally occurring enzymes (tyrosinase). Proc. Sot. exp. Biol. Med. 37, 450-453. BRENNANJ. J. and BECK S. D. (1972) Activation of tyrosine hydroxylase in haemolymph from diapausing larvae of the European corn borer, Ostrinia nubilalis. Insect Biochem. 2, 451459. BRUNET P. C. J. (1963) Tyrosine metabolism in insects. Ann. N.Y. Acad. Sci. 100, 1020-1034. BRUNET P. C. J. (1965) The metabolism of aromatic compounds. In Aspects of Insect Biochemistry: Biothem. Sot. Symp. (Ed. by GOODWIN T. W.), 25, 4977. Academic Press, London. COTTRELL C. B. (1964) Insect ecdysis with particular emphasis on cuticular hardening and darkening. Adv. Insect Physiol. 2, 175-218. DENNELL R. (1947) A study of an insect cuticle: the formation of the puparium of Sarcoplzaga falculatn Pand. (Diptera). PYOC. R. SOL. (B) 134, 79-110. DOHKE K. (1973) Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori-I. Activation reaction by the enzyme. Archs Biochem. Biophys. 157, 203-209. EVANS J. T. (1967) The activation of prophenoloxidase during melanization of the pupal blood of the Chinese oak silkmoth, Antheraea _ perti._ .?. _ Insect Phvsiol. 13. 1699-1711. FIRTEL R. A. and SAUL G. B. (1967) Characteristics of ohenol oxidases in Mormoniella vitribennis (Walker). J. Insect Physiol. 13, 1197-1206. HACKMANR. H. (1964) Chemistry of the insect cuticle. In The Physiology of Insecta (Ed. by ROCKSTEINhf.), 3, 471-506. Academic Press, New York. HACKMAN R. H. and GOLDBERG M. (1967) The -Odiphenoloxidases of fly larvae. r. Insect Ph,ysioZ. 13, 531-544. HEYNEMAN R. A. and VERCAUTERENR. E. (1969) Evidence for a lipid activator of prophenoloxidase in Tenebrio molitor. 7. Insect Phvsiol. 14. 409-415. HOROWITZ N. H. and FLING i% (195Sj The autocatalytic production of tyrosinase in extracts of Drosophila melanogaster. In Metabolism of Amino Acids (Ed. by MCELROY W. D. and GLASS B.). Johns Hopkins University Press, Baltimore. HUGH= L. and PRICE G. M. (1974) The isolation and properties of a lipoprotein fraction possessing tyrosinase activity from the haemolymph of the Aeshfly, Sarcophaga barbarta. Biochem. Sot. Trans. 2,336-338. ISHAAYA I. (1972) Studies on the haemolymph and cuticular phenoloxidase in Spodoptera littoralis larvae. Insect Biochem. 2, 409-419. KARLSON P. and LIEBAU H. (1961) Zum Tyrosinstoffwechsel der Insekten-V. Reindarstellung, Kristallisation und SubstratspezifitHt der O-Diphenoloxydase aus Calliphora. Hoppe-Seyler’s Z. physiol. Chem. 326, 135-144. KARLSON P., MERCENHAGEND., and SEKERIS C. E. (1964) Zum Tyrosinstoffwechsel der Insekten-XV. Weitere Untersuchungen iiberdas 0-Diphenoloxydase-System Hoppe-Seyler’s 2. von Calliphora erythrocephala. physiol. Chem. 338, 42-50. KARLSON P. and SCHWEICER A. (1961) Zum Tyrosinstoffwechsel der Insekten-IV. Das PhenoloxydaseSystem von Calliphora und seine Beeinflussung durch das Hormon Ecdyson. Hoppe-Seyler’s Z. physiol. Chem. 323, 199-210.

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OHNISHI E., DOHKE K., and A~HIDA M. (1970) Activation of prephenoloxidase-II. Activation by oichymotrypsin. Archs Biochem. Biophys. 139, 143-148. PRFSTON J. W. and TAYLOR R. L. (1970) Observations on the phenoloxidase system in the haemolymph of the cockroach, Leucophaea maderae. J. Insect Physiol. 16, 1729-1744. PRICE G. M. (1967) Studies on tyrosinase. In Pest Infestation Research. Agricultural Research Council, H.M.S.O., London. PRICE G. M. (1969) Protein synthesis and nucleic acid metabolism in the fat body of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 15, 931-944. PRICE G. M. (1972) Tyrosine metabolism in the larva of the blowfly, Calliphora erythrocephala. Insect Biothem. 2, 175-185. PRICE G. M. (1974) Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 20, 329347. SEKERIS C. E. and MERGENHAGEND. (1964) Phenoloxidase system of the blowfly, Calliphora erythrocephala. Science, Wash. 145, 68-69. THOMSON J. A. and SIN Y. T. (1970) The control of prophenoloxidase activation in larval haemolymph of Calliphora. J. Insect Physiol. 16, 2063-2074. YAMAZAKIH. I. (1969) The cuticular phenoloxidase in Drosophila virilis. J. Insect Physiol. 15, 2203-2211. YAMAZAKI H. I. (1972) Cuticular phenoloxidase from the silkworm, Bombyx mori: properties solubilization and purification. Insect Biochem. 2, 431444.