Insect. Biochem., Vol. 8, pp. 143 to 148. 0020-1700/78/0601--0!43502.00/0 (~) Pergamon Press Ltd. 1978. Printed in Great Britain

CHARACTERIZATION OF A TRYPSIN-SOLUBILIZED PHENOLOXIDASE FROM LOCUST CUTICLE

SVEND OLAV ANDERSEN

Zoophysiological laboratory C. August Krogh Institute, University of Copenhagen, Denmark

(Received 24 August 1977)

Abstract--A diphenoloxidase has been dissolved from pre-sclerotized locust cuticle by means of trypsin digestion. The solubilized enzyme has been extensively purified and characterized with respect to substrate and inhibitor specificity, pH-optimum and pH-stability and thermostability. The enzyme will oxidize a number of both ortho- and para-diphenols, and in contrast to untreated locust cuticle it is more active in releasing tritium located on the aromatic ring of N-acetyldopamine than in releasing tritium from the aliphatic side-chain of the same compound. The possible relationship between the trypsin-solubilized activity and the insoluble sclerotization enzyme in intact cuticle is discussed.

I N T R O D U C T I O N

SCLEROTIZATION of insect cuticle is assumed to be caused by the introduction of cova len t cross-l inks be tween the protein molecules in the cuticle. The cross-l inks are formed f rom a low-molecular weight diphenol which af ter oxidat ion to a react ive inter- mediate can react with amino- and phenolic groups on the proteins. N-ace ty ldopamine is the diphenol used as sclerot izing agent by many insect s p e c i e s (KARLSON and SEKERIS, 1962), and it can ei ther be oxidized in the ring to give an or tho-quinone , which is a highly react ive compound (reviewed by HACK- MAN, 1971, 1974). or it can be oxidized to another intermediate where the p-posi t ion of the sidechain (the carbon a tom which is c losest to the ring) is act ivated and connec ted to the proteins (ANDERSEN and BARRETT, 1971; ANDERSEN, 1974, 1976).

These oxidat ions are ca ta lyzed by cuticular en- zymes , and while some cuticles apparent ly contain enzymes catalyzing both react ions some cuticles have been found to contain only the / / - ac t iva t ing enzyme in significant amounts (ANDERSEN, 1974). Cuticle f rom the deser t locust, Schistocerca gregaria, belongs to this type, and I have former ly descr ibed some of the propert ies of the p-act ivat ing e n z y m e f rom this insect (ANDERSEN, 1972). The enzyme resisted all a t tempts to be d issolved and the character iza t ion had to be done on pieces of intact cut icle; it was therefore not possible to obtain infor- mation on the molecular propert ies of this enzyme.

I have now, by digesting pre-sclerot ized locust cuticle with trypsin, succeeded in bringing a phenol- oxidase act ivi ty into solution. The propert ies of the solubil ized e n z y m e are descr ibed in this paper, and the relat ionship be tween this soluble act ivi ty and the insoluble act ivi ty present in intact cuticle is discussed.

M A T E R I A L S AND M E T H O D S

Enzyme preparation Locusts (S. gregaria) were reared in the laboratory in

aluminium cages, where the temperature during the day period was about 35°C, whereas during the night it was

about 25°C. The animals were fed on fresh grass, lettuce and wheat bran. When a batch of animals approached the final ecdysis, they were kept under regular observation and taken while in the process of emergence to be frozen and stored at -18°(:-. When about a hundred animals were collected a cuticular preparation was prepare d by homo- genizing them for one minute in 400 ml 1% potassium tetraborate in a Waring blender. The homogenate was poured through a I mm sieve, and the retained material was washed 8-10 times in 2 I. 1% K-borate, twice in distilled water and twice in 96% ethanol. It was air-dried at room temperature and ground to a fine powder in a hammer mill. The powder was stirred with 2 I. of 1% K-borate for at least one hour, filtered and rewashed repeatedly in fresh K-borate until the filtrate appeared completely clear. The residue was washed with 96% ethanol and dried at room temperature. Microscopic ex- amination of the powder showed that it consisted of small

• pieces of cuticle together with a few fragments of muscle fibres. The contamination with non-cuticular material was estimated to be less than 1% by counting random samples under the microscope.

The powder was highly active in releasing tritium from the p-position of N-acetyldopamine when tested in the standard assay (ANDERSEN, 1974). Enzymatic activity was extracted from the powder by a slight modification of YAMAZAKI'S method (1972) which utilizes digestion with trypsin. Ten gram cuticular powder was suspended in 120 ml 1% Na-bicarbonate, 50 rag crystalline trypsin was added, and the suspension was stirred for one hour at 40°C and thereafter filtered. Ten ml 2 mM phenyimethyisui- phonyl fluoride in 5% isopropanol was added to the filtrate to inhibit trypsin activity. The filtrate was cooled to 4*(2, while the residue was resuspended in 120ml 1% Na- bic~bonate and incubated again for one hour at 40°C without the addition of more trypsin. The suspension was filtered and the filtrate combined with the former. A third incubation released only little enzymatic activity from the residue and was not utilized.

Enzyme activity was precipitated from the combined filtrates by the addition of solid ammonium sulphate. The fraction which precipitated between 15 and 30% saturation contained the major part of the enzyme activity and was collected by centrifugation at 2000 rev/min for 20 rain at 4*(2. The precipitate was dissolved in dialysis buffer (0.02 M Na-phosphate buffer, pH 6.8) and dialyzed overnight against 2 1. of the same buffer at 4*(2. Next day the solution was centrifuged at 2000 roy/rain for 20 rain at 4°(2 and the supernatant assayed for enzyme activity.

143

144 S .O . ANDERSEN

Table 1. Summary of the purification of cuticular phenoloxidase

Total Total Total activity Specific activity Recovery of Fraction vol protein (arbitrary units) (units per mg) activity (%)

ml mg MHQ MC MHQ MC MHQ MC Solubilized fraction 200 3045 9973 5480 3.3 1.8 100 100 Am2SO4-fraction 15-30% 26 314 6666 3926 21.2 12.5 66.8 71.6 Ultrogel AcA 34 98 19.9 6679 3744 335.6 188. I 67.0 68.3 DEAE-cellulose 104.5 4.24 4077 2294 961.6 541.0 40.9 41.9

Abbreviations: MHQ: methylhydroquinone: MC: 4-methylcatechol. One unit of enzymatic activity has for both substrates been defined as the amount of enzyme which in the standard

assays gives a change in optical density of 1.000 per l0 mix.

The enzyme was further purified by gel filtration on a column of Ultrogel AcA 34 (2.6 cm × 90 cm), and eluted with dialysis buffer at a rate of 50 ml per hour. Ten ml fractions were collected and assayed for enzyme activity. The fractions containing activity were pooled and further purified on a column of DEAE-cellulose (Whatman DE-52, 1.6 cm × 15 cm). The column wasequilibratedwithdialysis buffer, and elution was performed by a stepwise increase in sodium chloride concentration in the same buffer. The elution rate was 25 ml per hour, and 5 ml fractions were collected. The fractions containing activity were pooled and used directly for characterization of the enzyme.

Enzyme assays Several methods have been used for determining the

enzyme activity. When enzyme activity had to be located in the fractions obtained by column chromatography an assay was used based upon the reaction between glycyl- glycine and the oxidized product of 4-methylcatechol. The coloured reaction product has an absorption maximum at 470 nm. Fifty/~l of the sample to be assayed was added to 3 ml of a solution containing i0 mM methylcatechol and l0 mM glycylglycine in assay buffer (0.2 M acetic acid- sodium acetate buffer, pH 5.5, containing 0.1% Na~-EDTA). The mixture was incubated at 40°C for 30 mix whereafter the absorption at 470 nm was measured.

A modification of the method of KARLSON and LIEBAU (1961) was used to compare the activities of the enzyme towards different substrates. It utilizes the oxidation of ascorbic acid by the oxidation product formed enzymatic- ally from the substrate. In a 3 ml silica cuvette a suitable amount of 10 mM substrate solution in assay buffer was mixed with 0.1 ml 0.05% ascorbic acid solution containing

1% Na~-EDTA and sufficient assay buffer to give a final volume of 3 ml. The sample was placed in the thermo- regulated cell-holder of a recording spectrophotometer, and the reaction was started by adding an appropriate volume of enzyme solution. The reaction was followed automatically by registering the decrease in absorption at 265 nm, where ascorbic acid has its absorption maximum.

Activity towards para-diphenols was determined with methylhydroquinone as substrate. During oxidation of this compound, there is a pronounced increase in absorption at 250 nm, and it is, therefore, not necessary to have a secondary substrate present to follow the reaction spectro- photometrically. In a typical experiment 0.3 ml 10 mM methylhydroquinone was added to 2.65 ml assay buffer, warmed to 40°C, and the reaction started by the addition of 50 #1 enzyme solution. The increase in absorption at 250 nm was recorded automatically for 10-20 min.

Disc gel electrophoresis was performed according to DAVIS (1964) and SDS-gel electrophoresis was performed according to WEBER and OSBORNE (1969). The gels were stained with Coomassie blue and after destaining they were scanned densitometrically by means of a gel scanning unit on a spectrophotometer. Enzyme activity was local- ized in gels by placing them for 30 mix at room temper- ature in 100 ml assay buffer in which either 250mg dopamine or 250 mg 4-methylcatechol plus 250 mg glycyl- glycine were dissolved.

RESULTS

When a cuticular powder , p repared f rom adult locusts which had not quite emerged f rom their

E 20 - c o =E co 08 0 04 40

- / . 8

60 1 = 0.4.8 ° = S o 't,, " #- 8o <

I 0 0 ' " ! . , ~ ~ I I 0 0 200 400 600 800 1000

ml. e f f l u e n t

Fig. 1. Gel filtration of fraction obtained by ammonium sulphate precipitation (15-30% saturation) of trypsin digest of locust cuticle. Column material: Ultrogel AcA 34. Column dimension: 2.6 × 90 cm. Elution: 50 ml 0.02 M- Na-phosphate buffer, pH 6.8, per hour. : transmittance of the eluate at 280

nm. © - - - © : oxidase activity towards 4-methyicatechol.

Phenoloxidase from locust cuticle 145

E 25 c

O

o 5 0

E ~ 7 5

I 00 0

O. I M 0 . 2 M N a C t N o C t

11 I i

o

15 30 45 rnL e f f l u e n t

I O

E c

0 . 8 2

0 . 6

0.4 ~n

0.2

I 6O

Fig. 2. Ion exchange chromatography on DEAE-cellulose of aliquot from pooled fractions (480--600 ml) from Fig. 1. Column dimensions: 1.6cm × 15 cm. Elution scheme: 0.02 M Na-phosphate, pH 6.8, with stepwise increase in NaCI-concentration as indicated. : transmittance of eluate at 280 nm, O-- -O: oxidase activity towards

4-methylcatechol.

exuviae, was treated with trypsin, a diphenoloxi- dase dissolved into solution. The solubilized enzyme has been purified extensively, and the purification scheme is shown in Table I. Figures 1 and 2 show the purifications obtained by gel filtration and by ion exchange chromatography. The purest preparations represent a 300-fold purification of the enzyme when compared with the original digest. It is not possible to compare the specific activities of the soluble preparations with those of the intact cuticle since we

®

(a)

(b)

5 4 3 2 I c m

Fig. 3. Polyacrylamide gel electrophoresis of pooled active fractions from Fig. 2: (a) gel stained with Coomassie blue and scanned densitometrically at 570 nm; (b) gel incubated in dopamine solution for 30 min and scanned at 570 nm. The sharp peaks at the extreme right and left of the figure are due to markers put into the gels to indicate the start and the front. Migration from right towards left.

e

I I I I I I "k~ ' / IL 6 5 4 3 2 I 0

c m

Fig. 4. Polyacrylamide gel electrophoresis in SDS- containing buffers of sample from same preparation as in Fig. 3. The gel wa,: stained with Coomassie blue and

scanned at 570 nm. Migration from right towards left.

cannot estimate how diffusion of substrate into pieces of cuticle will affect the rate of reaction.

By disc electrophoresis the purest preparat2ons gave one major and a few minor bands when the gels were stained for proteins with Coomassie blue. Incubation of unstained gels with dopamine or 4-methylcatechol gave a coloured band localized in the same region as the major protein band (Fig. 3). Some disc-electrophoresis preparations of the soluble enzyme give two closely situated bands both having enzyme activity, and in the preparation shown in Fig. 3 a second component with enzymatic activity can be seen as a small shoulder on the main peak.

Electrophoresis of the same preparation in SDS- containing buffers (Fig. 4) resulted in two majo r bands staining with Coomassie blue and a few minor bands. Comparison of the migration rates with those of standard proteins gave approximate molecular weights of the two major bands at 90,000 and 100,000 daltons.

The partially purified enzyme preparations

, t

b ,'" , 'o

0.75

-~ 0 5 0

0 25 , ' , , ' " b cI

/ / I I 3 6 9 12

m i n

Fig. 5. Disappearance of lag-period during purification of enzyme activity: (a) preparation obtained by ammonium sulphate precipitation between 15 and 30% saturation; (b) preparation obtained by chromatography on DEAE- cellulose. Solid lines: activity towards 4-methylcatechol measured by the decrease in absorbance at 265 nm due to oxidation of ascorbic acid. Broken lines: activity towards methylbydroquinone measured by the increase in absorb-

ance at 2507nm.

146 S .O. ANDERSEN

0.c

a b

c

O.E

E

o

oJ

c

O' 5 IO 15 rain

Fig. 6. Influence of reducing compounds on the develop- ment of colour in the assay for oxidase activity. Assay system: 0.01 M 4-methylcatechol and 0.01 M glycyl- glycine in Na-acetate buffer, pH 5.5: (a) no additions; (b) 2/~1 10 mM mercaptoethanol; (c)5 #110 mM mercapto-

ethanol: (d) 10/~1 10 mM mercaptoethanol.

s h o w e d a signif icant lag per iod in the ox ida t ion of bo th o r tho- and para -d ipheno ls , but the lag per iod was cons ide rab ly r educed or comple te ly abo l i shed w h e n pure r p r epa ra t i ons were used (Fig. 5). The lag per iod is p robab ly not a p roper ty of the e n z y m e but may be caused by some impur i ty affect ing the ra te of r eac t ion . Lag per iods of s ho r t e r or longer dura t ion can be i n t roduced by the addi t ion to the reac t ion mix tu re of small a m o u n t s of r educ ing c o m p o u n d s , such as m e r c a p t o e t h a n o l (Fig. 6).

The e n z y m e will oxidize a wide var ie ty of di- phenols , and the re la t ive veloci t ies ob t a ined wi th a n u m b e r of subs t r a t e s are g iven in Tab le 2. Fo r some

Table 2. Relative velocities and K,.-values for the oxidation of various diphenols by the purified enzyme from locusts

V r c l K m

Catechol 48 1.2 mM 4-methylcatech01 332 0.17 mM 3,4-dihydroxy benzoic acid 13 - - 3,4-dihydroxyphenylacetic acid 23 - -

3,4-dihydroxyphenylpropionic acid 56 - - Dopamine 51 2.7 mM Dopa 5 - - Dope methyl ester 3 - - Noradrenalin 19 - - 3,4-dihydroxyphenylethyl alcohol 137 - - 3.4-dihydroxyphenylethyl acetate 7 i - - N-acetyldopamine 55 1.3 mM N-monochloroacet yldopamine 55 - - N-dichloroacetyldopamine 61 - - N-trichloroacet yldopamine 53 - - Hydroquinone 100 0.93 mM Methylhydroquinone 221 0.19 mM 2,5-dihydroxybenzoic acid 8 - -

2,5-dihydroxyphenylacetic acid 33 4.8 mM Resorcinol 0 - -

The velocities have all been determinedby the ascorbic acid method with a substrate concentration of 3 x 10 -4 M and are caiculatcd relative to the velocity measured for the oxidation of hydroquinone. The K,,-values for the ortho- diphenols were determined by the ascorbic acid method, while the K~,-values for the para-diphenols were deter- mined by the direct method.

Table 3. Influence of various inhibitors on the rate of oxidation of 1 mM methylhydroquinone by the solubilized

locust enzyme

Inhibitor Conc Rel. velocity

o-phenanthroline I 0 -:~ M 100 2,2'-bipyridin 10 -:~ M 94 8-hydroxyquinoli0e 10 -:~ M 10 I Na-diethyldithio- 10 -:~ M 100

carbaminate Na-fluoride 10 -4 M 37 Na-fluoride 10 -:~ M 19 Na-azide 10 -4 M 17 Na-azide 10 -:~ M 5 Na-cyanide 10 -4 M 57 Na-cyanide 10 -:~ M 8

of the subs t r a t e s the K,,,-values have also been de t e rmined .

The effect of inhib i tors has a lso been inves t iga ted . As some of the inhib i tors a b s o r b s t rongly in the ul t ra-viole t it was neces sa ry to use the assay with me thy l ca t echo l and glycylglycine whe re a p roduc t is fo rmed abso rb ing in the visible range. Che la t ing agents , s u c h as o -phenan th ro l ine , E D T A . 8- hyd roxyqu ino l ine , Na -d i e thy ld i t h ioca rbamina t e , and 2 .2 ' -b ipyr id in , have little effect on the e n z y m e : and Na-cyan ide , Na-f luor ide, and Na-az ide inhibi t the ac t iv i ty (Table 3).

W h e n Na-cyan ide was used as an inhibi tor it was o b s e r v e d tha t , apar t f r om the initial inhibi t ion, there was also a gradual r educ t ion in the ra te of r eac t ion , so tha t the inhib i t ion was m u c h more p r o n o u n c e d 15 min a f t e r the s tar t of the reac t ion than it was initially (Fig. 7). The reac t ions were s ta r ted by the addi t ion of e n z y m e to a mix ture of subs t r a t e and Na-cyan ide , but the same resul t was ob ta ined by p remix ing e n z y m e and Na-cyan ide and s ta r t ing the reac t ion by addi t ion of subs t ra te . Wi th Na-f luoride and Na-az ide the p h e n o m e n o n was not obse rved . The reac t ion ra te was d e c r e a s e d but did not change with t ime when these inhib i tors were used.

P h e n y i t h i o u r e a appea r s to inhibi t the reac t ion , but it is appa ren t ly not a t rue inhibi t ion of the enzyme . By add ing the c o m p o u n d , in small a m o u n t s to the reac t ion mix ture one can in t roduce a lag per iod, and a f te r the lag per iod the reac t ion rate inc reases until

0.9

0.6

g .Q

.Q 0 3

0 5 10 t5

rain

Fig. 7. Inhibition by Na-cyanide of the enzyme activity towards methylhydroquinone: (a) no additions; (b) reac- tion mixture contains 10 -4 M NaCN; (c) reaction mixture

contains 10 -2 M NaCN.

Phenoloxidase from locust cuticle 147

0 .4

<~

0.2

o / O - - - - - . - o

o/ \ ° ~ o

\o I I I I

0 4 5 6 7 pH

Fig. 8. pH-dependence of the purified enzyme towards methylhydroquinone (10 -s M). Activity is determined as increase in absorbance at 250 nm per 10 rain. Acetic acid- Na acetate buffers (0.2 M) were used for pH-values at 4-5,5, and Na-phosphate buffers (0.2 M) were used for

pH-values 6-7.

it is nearly the same as the rate in control experi- ments. The explanation is presumably that phenyl- thiourea reacts with or reduces the quinones (or some other oxidized intermediates) which are formed during the reaction. The formation of quinoid reaction products can therefore not be observed before the phenylthiourea h a s been consumed.

The enzyme has a pH-optimum near 5 when as- sayed with methylhydroquinone as substrate (Fig. 8). The enzyme is rather thermostable: after heating a sample of the enzyme to 90°C for 5 min about 50% of the original activity survived (Fig. 9). The ability of the enzyme to release tritium from either the /~-position or from the ring-positions in N-acetyl- dopamine was determined to compare the solubil- ized enzyme with the activity present in intact cuticle. It was found that the solubilized enzyme prefers the ring positions to the #-position, which is in contrast to the preference of the enzyme in the intact cuticle. The ratio between release from the /i-position and release from the ring-positions was about 10 for intact cuticle and about 0.03 for the soluble enzyme.

>.

• 50 g o

0 2 40 610 80 I00 °C

Fig. 9. Thermostability of the purified enzyme. Aliquots of the enzyme were heated for 5 min to the indicated

DISCUSSION

A wide variety of methods for determiningphenol- oxidase activities are described in the literature. Some methods are based upon measurements of the oxygen consumption during the reaction, while in other methods the formation of reaction products is determined. The quinones which are formed by oxidation of the substrate can either be determined directly, or a coloured reaction product can be pro- duced by letting them react with an amino com- pound. Or the quinones can be used to oxidize a secondary substrate, such as ascorbic acid, which is easily determined.

All the methods have their advantages and short- comings and none of them can be recommended generally. The method chosen will depend upon the purpose of the investigation, and in the work reported here it was found to be advantageous to use three different methods to supplement each other.

By these methods it has been established that an oxidase can be liberated from non-hardened locust cuticle by means of trypsin digestion; the oxidase has been extensively purified and it has been par- tially characterized. It is not the first time an in- soluble oxidase has been liberated into solution by the digestion of cuticle with trypsin; YAMAZAKI described in 1972 the solubilization of a diphenoi- oxidase from non-sclerotized pupae of Bombyx mori, and the properties of this enzyme resemble the properties of the enzyme obtained from locusts in many ways, Related enzymes can presumably be obtained from most cuticles.

Digestion with trypsin is necessary to dissolve both the silk moth and the locust enzyme, and it is therefore likely that peptide bonds have to be broken to release the enzyme from some structural element to which it is bound. It cannot, however, be excluded that the insolubility of the enzyme is due to entanglement inside a protein network in the intact cuticle and that it is released when the net- work is opened up by trypsin digestion. About 90% of the protein in unsclerotized locust cuticle is freely extractable with formamide, and this treatment will not release the enzyme into solution; it is therefore not likely that the enzyme is trapped by entangle- ment, but that it is covalenfly bound to some insoluble structure, such as components in the epicuticle or to chitin.

The solubilized enzyme has been purified by a factor of 300, but even the purest preparations give two bands on disc-electrophoresis, and both bands show enzyme activity. The relative amounts of the two components vary from preparation to prepara- tion. It has not been possible to separate them on a preparative scale, and we have therefore not been able to compare them in any detail. Incubation of the gels with various substrates has not shown any differences in substrate specificity between the two bands. YAMAZAKI (1972) describes how the trypsin solubilized phenoloxidase from B. mori pupal cuticle separates into three bands which show dif- ferent substrate specificities. The components pre- sent in the bands could be distinct enzyme entities

temperature and afterwards their activity was determined which occur as such in the cuticle, but it seems more with methylhydroquinone as substrate, likely that they are degradation products of a single

148 S.O. ANDERSEN

cuticular enzyme, which is partly digested during the trypsin treatment, It appears unlikely that the enzyme would not be modified by limited proteo- lysis while being incubated with trypsin.

The stability of the solubilized enzyme is one of its interesting features. It will survive heating to 80°C for short periods and it is difficult to inhibit by chelating agents, although it presumably contains heavy metal ions since it can be inhibited by cyan- ide, fluoride, and azide. The enzyme is not inacti- vated during its action as reported for mushroom tyrosinase (LuDwIG and NELSON, 1939), and the enzyme survives the tanning activity of the quinones which are generated by its own action on diphenols. This may explain why the enzyme activity is un- diminished in fully sclerotized locust cuticle (ANDERSEN, 1972), and is present in locust exuviae.

The enzyme shows a broad substrate specificity. It is active towards both ortho- and para-diphenols, but shows no activity towards monophenols or towards meta-diphenols. The activity is strongly influenced by the nature of the sidechain in the substrate. The best substrates are those having a methyl group as substituent on the r ing--but also rather bulky sidechains can be accommodated. Negatively charged groups in the sidechain appear to diminish the activity and their effect is more pro- nounced the closer the negatively charged group is to the ring. The effect of a positive charge is less pronounced.

An important problem is how this soluble activity is related to the insoluble //-activating enzyme in locust cuticle described earlier (ANDERSEN, 1972, 1976). The two enzymes are similar in pH-optimum and stability, and the only significant difference is that whereas intact cuticle preferably activates the //-position in N-acetyidopamine the solubilized enzyme prefers the ring-positions. This difference in specificity may be due to changes introduced in the enzyme during solubilization; a part of the enzyme molecules may be digested away by the trypsin or the special milieu in the intact cuticle may influence the activity of the enzyme. Another way to explain the difference would be to suggest that the fl-activating enzyme is destroyed during solubil- ization and that at the same time a ring-activating enzyme is generated from an inert precursor in the cuticle, a precursor which is not normally activated

in vivo. There is no evidence to support the latter idea, and the former suggestion, that there is only one enzyme and that it is modified during solubil- ization, appears to be the most attractive hypo- thesis. We are now engaged in a more detailed characterization of the enzyme activity in the intact cuticle in order to compare it with the soluble activity in as many respects as possible.

REFERENCES

ANDERSEN S. O. (1972) An enzyme from locust cuticle involved in the formation of crosslinks from N- acetyldopamine. J. Insect Physiol. 18, 527-540.

ANDERSEN S. O. {1974) Evidence for two mechanisms of sclerotization in insect cuticle. Nature, Lond. 251, 507-508.

ANDERSEN S. O. (1976)Cuticular enzymes and sclero- tization in insects. In The Insect Integument (Ed. by HEPBURN H. R.). Elsevier, Amsterdam.

ANDERSEN S. O. and BARRETT F. M. (1971)The isolation of ketocatechols from insect cuticle and their possible role in sclerotization. J. Insect Physiol. 17, 69--83.

DAvis B. J. (1964) Disc electrophoresis--II. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121, 404-427.

HACKMAN R. H. (1971) The integument of arthropoda. In Chemical Zoology (Ed. by FLORKIN M. and SCHEER B. T.) 6, pp. 1-62. Academic Press, New York.

HACKMAN R. H. (1974) Chemistry of the insect cuticle. In The Physiology of Insecta (Ed. by ROCKSTEIN M.) 6, pp. 215-270. Academic Press, New York.

KARLSON P. and LIEBAU H. (1961) Zum Tyrosinstoff- wechsel der Insekten, V. Reindarstellung, Kristallisa- tion und Substratspezifit~it der o-Diphenoloxydase aus Calliphora erythrocephala. Hoppe-Seyler's Z. physiol. Chem. 326, 135-143.

KARLSON P. and SEKERtS C. E. (1962) N-acetyldopamine as sclerotizing agent of the insect cuticle. Nature, LoAd. 195, 183-184.

LUDWIG B. J. and NELSON J. M. t1939) Inactivation of tyrosinase in the oxidation of catechol. J. Am. chem. Soc. 61, 2601-2606.

WEBER K. and OSBORN M. (1969) The reliability of molecular weight determinations by dodecyl sulphate- polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406--4412.

YAMAZAKI H. I. (1972) Cuticular phenoloxidase from the silkworm, Bombyx mori: properties, solubilization, and purification. Insect Biochem. 2, 431-444.