9
Phenoloxidase in the Periostracum of the Marine Bivalve Modiolus demissus Dillwyn J. HERBERT WAITE AND KARL M. WILBUR Department of Zoology, Duke University, Durham, North Carolina 27706 ABSTRACT Phenoloxidase was extracted from Modiolus demissus mantles and periostraca with the detergent sodium dodecyl sulfate. Partial purification of the periostracal enzyme was achieved by gel filtration and polyacrylamide gel electrophoresis. The enzyme, present in the mantle in latent or proenzyme form, can be chymotryptically activated. Molecular weights of the proenzyme and active enzyme as determined by SDS gel electrophoresis were 80,000 and 70,000 daltons, respectively. A considerable portion of phenoloxidase activity in the periostracum is associated with a large macromolecule that cannot be re- duced into smaller subunits, suggesting enzyme covalently immobilized by quinones. Optimal pH of 8.0-8.5 was found for the active enzyme (MW 70,000). Activity was diminished by adding copper chelators or by reducing disulfide bonds. In the former case, activity was restored by titrating the inhibitor with divalent copper. A hydrophobic active site on the enzyme is suggested by low Km and high reaction velocities for substrates with high partition coefficients in 2actanol:water such as 4-methylcatechol and 4-butylcatechol. The periostracum is a proteinaceous, cuticular layer covering the outer surface of mollusc shells. In addition to its protec- tive contribution to the organism, the perio- stracum is thought to play a significant role as a substratum for the initial deposi- tion of calcium carbonate crystals (Taylor and Kennedy, '69; Meenakshi et al., '74). The mantle, an organ contiguous with the shell, secretes the periostracum as well as all other organic and calcareous compo- nents (general ref., Wilbur, '72). Quinone-tanned proteins or sclerotins have been detected histochemically in the periostracum of a large number of species (Beedham, '58; Brown, '52; Bubel, '73; Hillman, '61; Meenakshi et al., '69) and are presumably related to the durability and chemical resistance of this structure. Besides histochemical data, very little is known about the biogenesis and composi- tion of molluscan sclerotins with the ex- ception of amino acid analyses (Degens et al., '67; Meenakshi et al., '69). In insects, the enzyme phenoloxidase (EC 1.14.18.1 o-diphenol:02 oxidoreduc- tase) has long been accepted as contribut- ing to the sclerotization of the cuticle (Brunet, '67; Pryor, '40). Many insect phenoloxidases are capable of catalyzing two distinct reactions - the ortho hydrox- ylation of phenols and the dehydrogena- tion of catechols to form unstable quinones. Quinones from the second reaction initiate sclerotization by cross-linking proteins in an undetermined manner. In the present investigation, the partial purification, kinetic parameters, and some activation properties of a phenoloxidase from the periostracum of the marine bi- valve Modiolus demissus are reported. MATERIALS AND METHODS Modiolus demissus was collected from intertidal pilings and jetties at Beaufort, North Carolina during April-July, 1974 and maintained in Durham, North Caro- lina for as long as two months in aerated tanks of recirculated seawater at 15°C. Enzyme preparation The valves of 30 specimens were opened by cutting the adductor muscles. After rinsing off tissue fluid with filtered sea water, the mantle margins from both valves were carefully separated from the freshly secreted periostracum and excised. The periostracum was removed by running a sharp scalpel parallel to the shell margin. Approximately one gram (wet weight) of periostraca was collected from 30 animals. The periostraca and mantles were sus- J. ExP. ZOOL., 195; 359-368 359

Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

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Page 1: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

Phenoloxidase in the Periostracum of the Marine Bivalve Modiolus demissus Dillwyn

J. HERBERT WAITE AND KARL M. WILBUR Department of Zoology, Duke University, Durham, North Carolina 27706

ABSTRACT Phenoloxidase was extracted from Modiolus demissus mantles and periostraca with the detergent sodium dodecyl sulfate. Partial purification of the periostracal enzyme was achieved by gel filtration and polyacrylamide gel electrophoresis. The enzyme, present i n the mantle i n latent or proenzyme form, can be chymotryptically activated. Molecular weights of the proenzyme and active enzyme as determined by SDS gel electrophoresis were 80,000 and 70,000 daltons, respectively. A considerable portion of phenoloxidase activity in the periostracum is associated with a large macromolecule that cannot be re- duced into smaller subunits, suggesting enzyme covalently immobilized by quinones. Optimal pH of 8.0-8.5 was found for the active enzyme (MW 70,000). Activity was diminished by adding copper chelators or by reducing disulfide bonds. In the former case, activity was restored by titrating the inhibitor with divalent copper. A hydrophobic active site on the enzyme is suggested by low K m and high reaction velocities for substrates with high partition coefficients in 2actanol:water such as 4-methylcatechol and 4-butylcatechol.

The periostracum is a proteinaceous, cuticular layer covering the outer surface of mollusc shells. In addition to its protec- tive contribution to the organism, the perio- stracum is thought to play a significant role as a substratum for the initial deposi- tion of calcium carbonate crystals (Taylor and Kennedy, '69; Meenakshi et al., '74). The mantle, an organ contiguous with the shell, secretes the periostracum as well as all other organic and calcareous compo- nents (general ref., Wilbur, '72).

Quinone-tanned proteins or sclerotins have been detected histochemically in the periostracum of a large number of species (Beedham, '58; Brown, '52; Bubel, '73; Hillman, '61; Meenakshi et al., '69) and are presumably related to the durability and chemical resistance of this structure. Besides histochemical data, very little is known about the biogenesis and composi- tion of molluscan sclerotins with the ex- ception of amino acid analyses (Degens et al., '67; Meenakshi et al., '69).

In insects, the enzyme phenoloxidase (EC 1.14.18.1 o-diphenol:02 oxidoreduc- tase) has long been accepted as contribut- ing to the sclerotization of the cuticle (Brunet, '67; Pryor, '40). Many insect phenoloxidases are capable of catalyzing two distinct reactions - the ortho hydrox-

ylation of phenols and the dehydrogena- tion of catechols to form unstable quinones. Quinones from the second reaction initiate sclerotization by cross-linking proteins in an undetermined manner.

In the present investigation, the partial purification, kinetic parameters, and some activation properties of a phenoloxidase from the periostracum of the marine bi- valve Modiolus demissus are reported.

MATERIALS AND METHODS

Modiolus demissus was collected from intertidal pilings and jetties at Beaufort, North Carolina during April-July, 1974 and maintained in Durham, North Caro- lina for as long as two months in aerated tanks of recirculated seawater at 15°C.

Enzyme preparation The valves of 30 specimens were opened

by cutting the adductor muscles. After rinsing off tissue fluid with filtered sea water, the mantle margins from both valves were carefully separated from the freshly secreted periostracum and excised. The periostracum was removed by running a sharp scalpel parallel to the shell margin. Approximately one gram (wet weight) of periostraca was collected from 30 animals. The periostraca and mantles were sus-

J. ExP. ZOOL., 1 9 5 ; 359-368 359

Page 2: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

360 J . HERBERT WAITE AND KARL M. WILBUR

pended in 20 ml and 100 ml, respectively, of Tris-HC1 buffer (0.1 M, pH 7.5 at 4°C) and homogenized for five minutes with glass powder in motor-driven ground-glass tissue grinders (A. H. Thomas Co., Phila- delphia, Pa.). The homogenates were cen- trifuged at 1.2 X lo6 g-min. The pellets from periostraca and mantles were resus- pended in 10 ml and 50 ml respectively of Tris-HC1 buffer with 0.5% (w/v) sodium dodecyl sulfate (SDS) (Sigma Chemical Co., St. Louis, Mo.) and centrifuged at 1.2 X lo6 g-min. The resulting supernatants of both the periostracal and mantle ex- tracts were rich in phenoloxidase activity after treatment with chymotrypsin (Sigma).

The supernatant from the periostracal extract was concentrated and equilibrated with Tris-HC1 buffer, 0.5%, in SDS under nitrogen (25 mm ultrafiltration unit (Mil- lipore Corp., Bedford, Mass.) fitted with a membrane having a nominal exclusion limit of 10,000 daltons (Amicon Corp., Lexington, Mass.). The 1 ml concentrate was applied to a column (1.6 X 40 cm) of Sephadex G l O O (Pharmacia, Uppsala, Sweden). Blue Dextran (Pharmacia) was used to measure void volumes. Tris-HC1- 0.5% SDS was the elution buffer. Protein content in the collected fractions was mea- sured turbidimetrically (Davison, '68) or colorimetric ally (Hartree, '72).

SDS gel electrophoresis The procedure for SDS polyacrylamide

gel electrophoresis was essentially identical to that of Weber and Osborn ('69). Highest grade reagents used in the preparation of the gels included acrylamide, N,N' meth- ylenebisacrylamide, N,N,N',N', tetrameth- ylethylene diamine (all Aldrich, Milwaukee, Wis.) and ammonium persulfate (Matheson Coleman and Bell, Raleigh, N.C.). Glass tubes for the gels measured 0.5 X 11.0 cm. Electrophoresis was carried out at 18- 20°C for eight hours at 4-5 mA/gel. Em- pirical molecular weights of unknown pro- teins were determined by comparing their mobilities with the following standards: lysozyme (14,300), myoglobin (17,200), gammaglobulin light and heavy chains (23,500 and 50,000), lactate dehydrogen- ase (36,000), serum albumin (68,000) and phosphorylase A (94,000) (all from Sigma Chemical Co.). Proteins were visualized by fixing and staining gels overnight in

0.5% Coomassie Brilliant Blue (Sigma) dis- solved in methano1:acetic acid:water (40: 15:45). Destaining was achieved with sever- al changes of methano1:acetic acid:water (15: 10:75). Phenoloxidase activity was 10- calized by immersing unfixed gels for two hours in a solution of 10 mM 3,4-D,L-di- hydroxyphenylalanine (Nutritional Bio- chemicals, Cleveland, O.), 0.1 M sodium phosphate (pH 7.5), and 0.01% chymo- trypsin (for gels containing proenzyme). The Beckman Acta CIII double beam spec- trophotometer with gel-scanner adaptor was used to scan Coomassie Blue-stained gels at 580 nm and phenoloxidase gels at 460 nm.

Enzyme assay One unit of enzyme activity is defined

as 1 pmole of 3,4-L-dihydroxyphenylalmine (L-dopa) oxidized per minute in 0.1 M Tris- HC1, 0.01% SDS (pH 7.2, 25OC). Dopa- chrome is an oxidation product formed from two molecules of dopaquinone, and absorbs maximally at 475 nm with an ex- tinction coefficient of 3600 m - lcm - (Mason, '48). Activity was assayed on a double beam spectrophotometer in which the 1 ml sample cuvette contained 0.1 M Tris-HC1 (PH 7.2, 0.01% SDS, 1 mM L- dopa, 25 pg/ml chymotrypsin and phenol- oxidase. The reference cuvette was iden- tically prepared with exception that the enzyme extract was boiled for 30 minutes.

Enzyme kinetics Kinetics of the enzyme-catalyzed oxida-

tion of various catecholic substrates were studied spectrophotometrically by moni- toring the formation of quinones at their

TABLE 1

Extinction coefficients of the oxidation products of catechols

~~ ~

Substrate

3,4-dihydroxyphenylac etic acid

3,4-L-dihydroxyphenyl- . - danine

3,4-dihydroxyphenyl- ethyl amin e

3,4-dihydroxyphenyl- propionic acid

4-tertiary butylcatechol 4-methylcatechol pyroc atechol

Amax E

nm

390

305

305

300 400 400 390

M-1 cm-1

1,200

4,500

4,200

7,200 1,100 1,400 1,400

Page 3: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

PER10 STRACAL PH ENOLOX IDASE 36 1

TABLE 2

Steps in the partial purification of periostracal phenoloxidase

Activity Step units ( U f Specific

Protein Volume X 10-3 activity Yield m g ml Ulmg %

Supernatant of SDS

Supernatant of SDS mantle homogenate 2,880 50 2.30 0.24

periostracum homogenate 108 10 5.00 46.0 100 Dialysis Nz 80 2 4.00 50.0 80

Peak I 31 5 1.53 50.0 12 Peak I1 12 2 2.46 212.0 19

G- 100

wavelengths of maximal absorption. The molar extinction coefficients of the quin- ones listed in table 1 were experimentally derived according to the enzymatic proce- dure of Mason (‘48). Pyrocatechol was ob- tained from Matheson, Coleman and Bell; all other substrates were purchased from Aldrich.

The kinetic parameters, Km and Vmax, calculated for each of the substrates, rep resent the means of five trials, each of which was plotted and evaluated according to the direct linear method of Eisenthal and Cornish-Bowden (‘74). Using the molar extinction coefficients in table 1, Vmax values were converted to pmoles of sub- strate oxidized per minute, normalized with respect to Vmax for L-dopa, and reported as Vrel. Error propagation for Vrel values was handled according to formulations by Ku (‘66).

The enzyme inhibition parameter Ki was determined from Dixon plots of the direct linear data. Compounds investigated for inhibitory effects included, o-phenanthro- line, 3-methoxydopamine, salicylaldoxime, dithiothreitol (all from. Aldrich), sodium azide, sodium diethyldithioc arbamate (Fish- er Scientific, Raleigh, N.C.) and potassium cyanide (Baker, Phillipsburg, N. J.).

RESULTS

Solubilization and activation of enzyme activity

The phenoloxidase activity of the perio- stracum and mantle of M. demissus was extracted in soluble form by SDS. The spe- cific activity of the periostracum, however, was approximately 200 times that of the mantle (table 2). Homogenization of man- tle and periostracum in aqueous buffer

8.5), or buffer in the presence of 1 % Tri- ton X-100, Tween 20 or sodium deoxycho- late did not liberate significant activity into the supernatant. Attempts to remove SDS by dialysis resulted in the precipita- tion of enzyme activity.

A striking feature of the phenoloxidase of M. d a i s s u s is its latency and rapid activation by chymotrypsin. In SDS homo- genates of mantle, no phenoloxidase activ-

0 20 40 6 0 80

time - minutes Fig. 1 Chymotryptic activation of phenoloxi-

dase (0.24 U/mg) in SDS extract of mantle. En- zyme assay was run in 0.1 M Tris (pH 7.5, 25OC), 0.1% SDS, and 1 mM D,L-dopa. Chymotrypsin (25 pglmg mantle protein) was added after eight minutes. Brackets reuresent standard error for

(0.1 M 6is-HCl or phosphate at pH 6.5- four trials.-

Page 4: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

362 J. HERBERT WAITE AND KARL M. WILBUR

0 15 30 4 5 6 0

pg chymotryps in/mg protein

Fig. 2 Effect of chymotrypsin concentrations on the rate of phenoloxidase activation. Substrate used was D,L-dopa; enzyme was present i n 2 mg periostracal protein (46 Ujmg) added to each con- centration of chymotrypsin in 0.1 M Tris (pH 7.5, 25'C), 0.1% SDS. For each trial, latent phenol- oxidase was incubated for five minutes before re- cording the initial reaction velocity. Brackets rep- resent standard error for four trials.

ity can be detected without prior addition of chymotrypsin. Figure 1 illustrates the effect of chymotrypsin on latent mantle phenoloxidase; chymotrypsin was added after eight minutes and maximal activity was achieved after 80 minutes. Even after a 5-hour exposure to chymotrypsin there was no perceptible decrease in the maxi- mal activity of phenoloxidase. In periostra- cum, the enzyme can be found in both active (5040%) and latent forms. When incubated for five minutes with increasing concentrations of chymotrypsin, the latent enzyme achieves one-half maximal activa- tion at 12 pg chymotrypsinlmg periostra- cal protein. Again, chymotryptic deacti- vation of enzyme was not observed at chymotrypsin concentrations as high as 120 pg/mg periostracal protein.

Gel jiltration Periostracal phenoloxidase activity can

be fractionated into two distinct peaks on Sephadex G-100 which elute just after the void volume (fig. 3). As indicated in table 2, however, only Peak I1 exhibited an in- creased specific activity, whereas Peak I possessed a specific activity not signifi- cantly different from the original homo- genate of periostracum. Further fraction- ation of Peak I on Sepharose 4B (range lO5-lO7 daltons), again resulted in the elution of activity at the end of the void

volume collected (mll Fig. 3 Chromatography of periostracal extract on Sephadex G-100. Elution buffer was

0.1 M Tris (pH 7.5), 0.5% SDS. Column had a bed height of 38 cm, pressure head of 10 cm and a flow rate of 0.1 mumin. Void volume was 25 mls. Brackets above Peaks I and 11, re- spectively, represent fractions pooled for Sepharose 4B and kinetics. Protein concentration -. - was measured turbidimetrically; enzyme activity o - - o - - o was assayed with 1 mM D,L-dopa and 25 pg/ml chymotrypsin.

Page 5: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

PERIOSTRACAL PHENOLOXIDASE 36 3

volume. Enzyme from Peak I1 was primar- ily in the latent or proenzyme form but could be activated by chymotrypsin to a maximal activity of 212 Ulmg protein.

SDS gel electrophoresis SDS gel electrophoresis of the periostra-

cal extract with or without added 2-ma- captoethanol (2ME) produced a consistent pattern of five protein-staining bands (fig. 4A,B). Only in the absence of 2ME did Bands 3 and 4 exhibit dopa-oxidase activ- ity (fig. 4C), although Band 5 demonstrated greater stability in the presence of the disulfide reducing agent. Activity in Band 4 was expressed by exposing gels to chymo- trypsin and D,L-dopa after electrophoresis. Note especially the shoulder on Band 4, coinciding in mobility with Band 3 (fig. 4C); this may represent naturally activated, but not yet immobilized enzyme. Activity in Band 3 was enhanced by chymotryptic proteolysis of the periostracal extract or Sephadex Peak I1 before electrophoresis. Band 4, which was probably the proenzyme, and Band 3, the active enzyme, had mo- bilities which corresponded to molecular weights of 80,000 and 70,000 respectively. The accuracy of SDS gel electrophoresis for predicting protein molecular weight is -+ 10% (Weber and Osborn, '69). Sephadex Peak I1 gave rise to two bands after elec- trophoresis corresponding to Band 3 and Band 4 (fig. 4D); protein in Peak I and Band 5 could not be dissociated into small- er subunits by denaturation with 8 M urea, 1 % SDS, 1 % 2ME and 80°C. This sug- gests that enzyme has been immobilized by covalent interactions other than disulfide or hydrogen bonds.

p H optimum The partially purified enzyme (Peak 11)

obtained by Sephadex chromatography was used for determination of the optimal pH. In the range pH 0.8-10.0, maximal activ- ity was observed in the interval pH 8.0- 8.5 (fig. 5). The dotted line in figure 5 in- dicates the formation of anomalous oxida- tion products after pH 9.

Kinetics The Michaelis constant (Km) and max-

imal velocity (relative to L-dopa) (Vrel) for each of the compounds tested are listed in table 3. The lowest Km and highest Vrel

RELATIVE MOBILITY

0 0.2 0.4 0.6 0.8 I . ( I I 1 I I

4 A

I

MOLECULAR WEIGHT x ld4 Fig. 4 Polyacrylamide SDS gel electrophoresis

of the periostracal phenoloxidase. In all gels, elec- trophoresis proceeded toward the positive electrode with Pyronin Y (PY) as the leading tracker. A. Periostracal protein (40 pg) denatured with 8 M urea, 1.0% SDS and 1.0% 2-mercaptoethanol at 80°C before electrophoresis. Gel has been stained with 0.5% Coomassie Blue to visualize proteins. B. Periostracal protein (35 pg) in 0.5% SDS. Stained as in A. C. Profile of enzyme activity i n parallel gels containing periostracal protein (70 pg) in 0.5% SDS. In profile represented by . . . . ., gel was submersed in 0.010% chymotrypsin, 3 mM, D,L-dopa after electrophoresis. Band 3 indicated by the continuous line, was obtained by activating the periostracal protein with 5 pg chymotrypsin for one half-hour previous to electrophoresis. Ac- tivity was visualized by submersing gel in 1 mM D,L-dopa for two hours. D. Sephadex Peak I1 (25 p g protein), denatured and stained as in 4A.

Page 6: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

364 J. HERBERT WAITE AND KARL M. WILBUR

0 2 4 6 B 10

PH Fig. 5 Effect of pH on the activity of perio-

stracal phenoloxidase. Substrate was D,L-dopa; enzyme was previously activated by chymotrypsin (20 pg/mg) for 15 minutes at pH 7.5. Assays at 25OC. Buffers used were 0.1 M KCl (pH 0.8), 0.1 M glycine (pH 3.0; 9.4; 10.0), 1.1 M succinate (pH 4.6), 0.1 M phosphate (pH 6.0; 7.1; 8.0) and 0.1 M Tris-HC1 (pH 8.2; 8.5). All buffers contained 0.01 % SDS.

were observed for 4-tert.-butylcatechol. A significant correlation (r = 0.985) exists between Vrel and the partition coefficient of the catecholic substrates in octanol: water (0.1 M Tris, pH 7.2). It is evident from figure 6 that the lipophilic substrates are more rapidly oxidized than are the polar hydrophilic ones; however, a limiting velocity is approached as the lipophilicity of the catechol becomes very high. Such substrate preference could be the result of a hydrophobic active site, or hydrophobic interactions important for catalysis. No such relationship occurs between the Km and the partition coefficient of the various catechols. For example, 4-methylcatechol and 3,4-dihydroxyphenylethylamine have similar Kms but rather disparate partition coefficients; pyrocatechol has an interme- diate partition coefficient but a very high Km.

Collectively, the kinetic data favor a substrate that is catecholic as opposed to phenolic, with a nonpolar moiety in the variable region R1 of the molecule (table 3). Since, as in 4-butylcatechol, bulkiness in the variable region appears to present no barrier to either the rate of oxidation or affinity of the enzyme-substrate complex, the true substrate could feasibly be bound through its variable region to the hydro- phobic portion of a protein.

TABLE 3

Kinetic parameters of various substrates with periostracal phenoloxidase

Substrate RI ~0 K~ x 1 0 4 S.E. Vr.4 S.E.

Pyrocatec hol -H

4-butylcatec hol - C(CHd3 4-methylcatechol - CH3

3,4-dihydroxyphenyl

3,4-dihydroxyphenyl

3,4-dihydroxyphenyl

3,4-dihydroxyphenyl

3,4-dihydroxybenzoic

alanine - CHzCHNHzCOOH

propionic acid - CHzCHzCOOH

ethylamine - CH~CH~NHZ

acetic acid - CHzCOOH

acid - COOH Nor epinephrine - CHOHCHzNHz Tyrosine - CHzCHNHz COOH Tyramine - CHI CHeNH2

- OH -OH - OH

- OH

-OH

-OH

- OH

- OH - OH -H -H

4.03 0.23 1.70 0.32 1.53 0.06 2.15 0.25 0.92 0.08 2.80 0.10

2.57 0.28 1.00 0.09

3.72 0.14 0.80 0.07

1.32 0.06 1.20 0.12

No reaction

No reaction No reaction No reaction No reaction

Page 7: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

PERIOSTRACAL PHENOLOXIDASE 365

- a i

-CyCYCOOH

L

I.0 100

partition coefficient

Fig. 6 Correlation of VreI with partition coef- ficient of various catechols in 2-octanol: 0.1 M TrisHCl (pH 7.2). Partition coefficients were de- termined by mixing 2 ml 2-octanol with 2 ml of a 1 mM solution of each catechol. Sodium bisulfite was also added (0.1 mM) to prevent oxidation. Absorbance (280 nm) of aqueous phase was mea- sured before and after mixing to determine the relative distribution of the catechol in both phases. Brackets represent standard error of Vre1.

A number of compounds have a profound inhibitory effect on phenoloxidase (table 4). The most potent of these were diethyldi- thiocarbamate (DEDTC), salicylaldoxime (SAD), cyanide and dithiothreitol. All are primarily noncompetitive inhibitors: dithi- othreitol reduces disulfide bonds, cyanide binds copper and interferes with oxygen binding, and DEDTC and SAD both bind copper. Figure 7 demonstrates that inhibi- tion by copper chelators is reversible. Com- plete restoration of enzyme activity can be achieved by titr ating the in hibitor with equimolar amounts of cupric chloride.

Mimosine, 3-methoxydopamine, and o- phenanthroline had no effect on phenol- oxidase at 1 mM concentrations.

DISCUSSION

Phenoloxid ase presumably catalyzes the formation of o-quinones in order to cross- link presclerotin protein subunits in the periostracum. Tyrosinase activity has pre- viously been reported in the mantles of

TABLE 4

Compounds tested for phenoloxidase inhibition

Type of Inhibitof Ki inhibition

CLM Cyanide 10 Noncompetitive Diethyldithio-

carbamate 15 Noncompetitive Salicylaldoxime 5 Mixed Dithiothreitol 10 Noncompetitive

the bivalves Pteria martensii and Cristaria plicata, but this was thought to be related to the formation of melanin pigments (Tsujii, '62). Tyrosinases are versatile phenoloxidases in that they catalyze both the hydroxylation of phenols and the de- hydrogenation of catechols; the former function has not been detected in perio- stracal phenoloxidase in the present study.

In spite of its unusual properties, the enzyme from Modiolus resembles tyrosin- ases and phenoloxidases from other or- ganisms in a number of characteristics. Phenoloxidases from Microbacterium le- prae (Prabhakaran et al., '73) and from the cockroach Periplaneta americana (Pau

0 40 80 120

Inhibitor pmo les I - ' Fig. 7 Restoration of phenoloxidase activity

with cupric chloride after inhibition by SAD and DEDTC. Substrate for each assay was 4-methyl- catechol (400 nm) in 0.1 M Tris (pH 7.8) 0.1 SDS. Phenoloxidase had been maximally activated by chymotrypsin before use in assays. Concentrations in absissa refer to SAD, DEDTC, and copper. Or- dinate corresponds to the inverse of the observed initial velocity.

Page 8: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

366 J. HERBERT WAITE AND KARL M. WILBUR

et al., '71), for example, were solubilized in SDS without relinquishing enzyme ac- tivity. In Periplaneta, the rather limited solubility of phenoloxidase in the absence of SDS was allegedly due to an intimate association with lipids and hydrophobic proteins (Pau et al., '71). Lipophilic phen- oloxidases have also been reported in the fleshfly Sarcophaga barbata (Hughes and Price, '74) and in the mealworm Tenebrio molitor (Aerts and Vercauteren, '64).

The cellular storage of phenoloxidase in zymogen form and proteolytic activa- tion upon secretion are vital biological adaptations because the reaction catalyzed by phenoloxidase is highly deleterious to living cells. As in Modiolus, cuticular phenoloxidases in the blowfly Calliphora mythrocephula (Kalson and Liebau, '61; Munn and Bufton, '73) and the silkworm Bombyx morii (Dohke, '73), and tyrosinase in frog epidermis (Barisas and McGuire, '74) all undergo activation by serine-type proteolytic enzymes. Bovine chymotrypsin appears to be an effective activator of latent Modiolus phenoloxidase, but noth- ing is known about the natural activating mechanism.

Phenoloxidase not only cross-links other proteins by the production of reactive o-quinones but is itself immobilized in the course of sclerotization. The same phe- nomenon was found with the cuticular phenoloxidase in the fruit fly Drosophita virilis (Yamazaki, '69). In Modiolus, up to a third of the total phenoloxidase ac- tivity extracted from the periostracum can be associated with an immense macro- molecule (fig. 3, Peak I) which does not in- crease in specific activity or decrease in size even after extensive fractionation by gel fil- tration and electrophoresis in the presence of SDS, urea, and 2-mercaptoethanol. It would be interesting to erucidate what other proteins have been immobilized in this SDS-soluble sclerotin. Perhaps fur- ther investigation of Bands l and 2 (fig. 4A) will shed some light on this.

The molluscan mantle margin and per- iostracum have been shown to abound in aromatic compounds (Beedham, '58; Brown, '62; Bubel, '73; Hillman, '61; Meenakshi et al., '69), but the identity of only a few of these, including L-dopa (Degens et al., '67), halogenated tyrosine (Hunt and Breuer, '73), and halogenated tryptophan (Andersen, S . 0. personal communication),

has been determined. Although no cate- cholic substrate or quinone product has yet been identified in Modiolus mantle or periostracum, the kinetic data in table 3 and figure 6 do show trends of substrate preference. Clearly, catechols with high partition coefficients in octanol: water (pH 7.2) such as 4-methylcatechol and 4-butyl- catechol exhibit preferential enzyme cata- lysis both in terms of reaction rate and Michaelis constant. Substrates with lower partition coefficients can possess either a high maximal rate or a low Km such as pyrocatechol and 3,4-dihydroxyphenyleth- ylamine respectively, but not both. Al- though tyrosinase activity (towards tyro- sine and tyramine) was not observed, this does not preclude the possibility of phenol- oxidase activity toward other phenols. A tyrosine situated in a hydrophobic pocket on a polypeptide chain, for example, might be a vulnerable target for the enzyme. Phenoloxid ase-c atalyzed oxidation of tyro- syl residues in a polypeptide backbone has been studied in vitro (Dabbous, '66) and in vivo (Kawasaki et al., '74), and consti- tutes an efficient alternative to sclerotiza- tion by means of a discrete tanning agent such as o-quinone.

Quinone-tanned proteins or sclerotins, although poorly understood, are perhaps the most important structural proteins of invertebrate exoskeletons. The advantage of employing quinones to form exoskeletal sclerotins is certainly related to their reac- tivity as crosslinking agents and to the inertness and durability of the crosslinked product. In molluscs, the periostracum is formed before all other portions of the exo- skeleton, and must therefore resist solubili- zation and polymerize rapidly. In Modiolus, solubilization of fi-eshly secreted periostra- cum is retarded by the hydrophobic nature of the phenoloxidase and its substrate until the tanning of the structure is complete.

ACKNOWLEDGMENT

This work was supported by the Office of Naval Research, Oceanic Program, Proj- ect NR 104-194-2.

LITERATURE CITED Aerts, F. E., and R. R. Vercauteren 1964 Spe-

cificity and mode of action of phenoloxidase from larvae of Tenebrio molitor. Enzymologia,

Barisas, B. G., and J. S. McGuire 1974 A prote- 28: 1-20,

Page 9: Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

PERIOSTRACAL PHENOLOXIDASE 367

olytically-activated tyrosinase from frog epider- mis. J. Biol. Chem., 249: 3151-3156.

Beedham, G. E. 1958 Observations on the non- calcareous component of the shell of Lamelli- branchia. Quart. J. Micros. Sci., 99: 341-347.

Brown, C. H. 1952 Some structural proteins of Mytilus edulis. Quart. J. Micros. Sci., 93: 487- 501.

Brunet, P. C. J. 1967 The sclerotins. Endeavor,

Bubel, A. 1973 An EM investigation into the distribution of polyphenols in the periostracum and cells of the inner face of the outer fold of Mytilus edulis. Mar. Biol., 23: 2-14.

Dabbous, M. K. 1966 Inter- and intramolecular crosslinking in tyrosinase-treated tropocollagen. J. Biol. Chem., 244: 53075312.

Davison, P. F. 1968 Proteins in denaturing sol- vents: gel exclusion studies. Science, 161: 906.

Degens, E. T., D. W. Spencer and R. H. Parker 1967 Paleobiochemistry of Molluscan shell pro- teins. Comp. Biochem. Physiol., 20: 553479.

Dohke, K. 1973 Studies on the prephenoloxi- dase-activating enzyme from the cuticle of the silkworm Bombyr mod. Arch. Biochem. Bio-

Eisenthal, R., and A. Cornish-Bowden 1974 A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J., 139: 715.

Hartree, E. F. 1972 Determination of protein. Anal. Biochem., 48: 422427.

Hillman, R. 1961 The formation of periostracum in Mercenada mercenaria. Science, 134: 1754.

Hughes, L., and G. M. Price 1974 The isolation and properties of a lipoprotein fraction possess- ing tyrosinase activity from the hemolymph of the larvae of the fleshfly Sarcophaga barbata. Biochem. Soc. Trans., 2: 336.

Hunt, S. I., and S. W. Breuer 1973 Chlorinated and brominated tyrosine residues in molluscan scleroproteins. Biochem. Soc. Trans., I : 215.

Karlson, P., and H. Webau 1961 Darstellung, Kristallisation und Substratspezifitat der o-Di- phenoloxydase aus Calliphora erythrocephala. 2. Physiol. Chem., 326: 135-146.

Kawasaki, H., H. Sam and M. Suzuki 1974 Structural proteins in the egg envelopes of drag- onflies, Sympetrum infuscatum and s. frequens. Insect Biochem., 4: 99-111.

26: 68-74.

phy~.,157: 203-211.

Ku, H. H. 1966 Notes on the use of propagation of error formulas. J. Res. Nat. Bur. Stand., 70C: 331-341.

Mason, H. S. 1948 Chemistry of melanin: 111. Mechanism of the oxidation of dihydroxyphenyl- alanine by tyrosinase. J. Biol. Chem., 172: 83-99.

Meenakshi, V. R., G. Donnay, P. L. Blackwelder and K. M. Wilbur 1974 Influence of substrata on calcification patterns in molluscan shell. Calc. Tiss. Res., 15: 31-44.

Meenakshi, V. R., P. E. Hare, N. Watabe and K. M. Wilbur 1969 The chemical composi- tion of the periostracum of the molluscan shell. Comp. Biochem. Physiol., 29: 611420.

Munn, E. A., and S. F. Bufton 1973 Purification and properties of a phenoloxidase from the blowfly Calliphora erythrocephala. Eur. J. Bio- chem., 35: 3-10.

Pau, R. N., P. C. J. Brunet and M. J. Williams 1971 The isolation and characteristics of pro- teins from the left colleterial gland of the cock- roach Periplaneta americana. Proc. Roy. Soc. Lond. Ser. B, 177: 565579.

Prabhakaran, K., E. B. Harris and W. F. Kirch- heimer 1973 Particulate nature of o-diphenol- oxidase in Microbacterium leprae and assay of the enzyme by radioisotope technique. Micro- bios, 8: 151-157.

Pryor, M. G. M. 1940 On the hardening of the cuticle in insects. Proc. Roy. Soc. Lond. Ser. B, 128: 378-390.

Taylor, J. D., and W. J. Kennedy 1969 The in- fluence of the periostracum on the shell struc- ture of bivalve mollusks. Calc. Tiss. Res., 3: 274-283.

Tsuji, T. 1962 The mechanism of shell and pearl formation: VIII. Tyrosinase in the mantle. Mie Kenritsu Daigaku Suisan Gakubu Kiyo, 5: 378483.

Weber, K., and M. J. Osborn 1969 The reliabil- ity of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem., 244: 4406-4412.

Wilbur, K. M. 1972 In: Chemical Zoology. VII. Mollusca. M. Florkin and B. T. Scheer, eds. Academic Press, New York, pp. 103-145.

Yamazaki, H. I. 1969 Cuticular phenoloxidase in Drosophila virilis. J. Insect Physiol., 15: 2203-221 1.