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On the latency and nature of phenoloxidase present in the left colleterial gland of the cockroach Periplaneta americana.

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Archives of Insect Biochemistry and Physiology 15:165-181 (1990)
On the Latency and Nature of Phenoloxidase
Present in the Left Colleterial Gland of the
Cockroach Periplanefa americana
Manickam Sugumaran and Kaliappan Nellaiappan
Department of Biology, University of Massachusetts, Boston, Massachusetts
The phenoloxidase system responsible for the sclerotization of cockroach
ootheca is found to be present as an inactive form in the left colleterial gland
of Periplaneta americana. The supernatant fraction obtained by centrifugation of the milky white secretions contained the inactive phenoloxidase which
required both sodium dodecyl sulfate (SDS) and the insoluble sediment for
exhibitingenzymeactivity. Bovineserum albumin could replace the sediment
in the activation process. Proteins separated from the supernatant fraction by
molecular sieve chromatography on Sephadex C-25 did not require either
albumin or the sediment, but required SDS for exhibiting the phenoloxidase
activity. Among the detergents tested, SDS (anionic) and cetylpyridinium
chloride (cationic) activated the phenoloxidase, but CHAPS (mitterionic) or
nonionic detergents failed to activate the enzyme. The activation caused by
SDS occurred well below the critical micellar concentration of SDS indicating that SDS i s causing the activation by binding to the protein and altering
its conformation. Chloroform-methanol extracts of vestibulum or right gland
could replace SDS confirming the presence of endogenous activator@) of
phenoloxidase system. A variety of exogenously added lipids could activate
the latent enzyme, among which linoleate, oleate, laurate, linolenate,
phosphatidylethanolamine, and phosphatidylglycerol proved to be the effective activators of the latent phenoloxidase.
Partially purified phenoloxidase was found to be extremely labile and lost
its activity on a) freezing and thawing, b) dialysis, and c) heating for 10 min at
55°C. It exhibited a pH optimum of 7 and was inhibited drastically by phenylthiourea and diethyldithiocarbamate.It readily oxidized a number of o-diphenols
such as 3,4-dihydroxybenzylalcohol, 3,4-dihydroxyphenethyl alcohol, catechol,
N-acetyldopamine, N-acetylnorepinephrine, dopa, dopamine, etc., but failed
to oxidize both 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde.
It neither converted the typical laccase substrate syringaldazineto its quinone
methide product, nor oxidized the p-diphenols, hydroquinone and methylhydroquinorie. Therefore, the enzyme participating in the quinone tanning
Acknowledgments: This research was supported by grants from National Institutes of Health
(R01-AI-14753)and University of Massachusettsat Boston (BRSG, Ed. Needs, and Fac. Dev.).
Received June1,1990; accepted August 6,1990.
Address reprint requests to Dr. Manickam Sugumaran, Department of Biology, University of
Massachusettsat Boston, Harbor Campus, Boston, MA 02125.
0 1990 Wiley-Liss, Inc.
Sugumaran and Nellaiappan
of cockroach ootheca appears to be a typical o-diphenol oxidase and not a
laccase as previously thought.
Key words: ootheca sclerotization, cockroach egg case, o-diphenoloxidase, quinone tanning
Nearly five decades ago, Pryor examined the mode of tanning of cockroach
ootheca and proposed the quinone tanning hypothesis for sclerotization of
cuticle [l-51. Subsequent workby Andersen’s group [6-121 and our group [13-201
paved the way to a better understanding of the tanning mechanisms in insect
cuticle. While Pryor discovered the participation of phenoloxidase and
3,4-dihydroxybenzoicacid in tanning reactions and proposed the quinone tanning hypothesis, Andersen’s group identified an alternate mechanism in which
the side chain of catecholamine derivatives (such as N-acetyldopamine) participates in addition reaction with cuticular components [6-121. Our group identified quinone methides as a new type of sclerotizing agents and discovered
two new enzymes, viz., 4-alkyl-o-quinone:2-hydroxy-p-quinone
methide isomerase and N-acetyldopaminequinone methide/l,2-dehydro-N-acetyldopamine
tautomerase, as key components in the sclerotization machinery of insect cuticle [13-201. Moreover, our studies also led to the unification of all sclerotization mechanisms as shown in Figure 1. In spite of the rapid strides made on
the mechanism of tanning of insect cuticle in recent years, the studies on the
tanning of ootheca received little attention, although cockroach ootheca is considered to be an ideal model system for the study of tanning reaction [21].
Cockroach ootheca, which seems to be devoid of chitin [l],is synthesized
by intermingling of secretions from the left and right colleterial glands at the
genital vestibulum [1,3,5,21]. The milky white secretion of the left colleterial
gland contains the 4-0-p-D-glucosides of DHM+and DHBAlc [21-251. In addition, this gland also secretes structural proteins and the enzyme phenoloxidase
[26].The smaller right gland is known to secrete a P-glucosidase [21-231. When
the secretions are mixed at the genital vestibulum, the P-glucosidase liberates
the catechols which are then oxidized by phenoloxidase. The quinonoid products thus formed react with the structural proteins to render them hard and
rigid in the vestibular pouch [1,5,21-261.
Brunet’s group and later Pau’s group investigated the structural proteins of
the ootheca [26,27], and the former has studied the phenoloxidase system from
the left colleterial gland of Periplanefa arnericana [28-301. The enzyme has been
characterized to be a laccase type (p-diphenoloxidase)oxidase based on inhibition and substrate specificity studies [28-301. In this context, it is interesting
to note that in insect cuticle also, increasing evidence indicates that laccases
are responsible for tanning reactions [see ref. 31 for review]. During our continued studies on the sclerotization mechanisms, we began to reinvestigate
*Abbreviations used: DHBA = 3,4-dihydroxybenzoic acid; DHBAlc = 3,4-dihydroxybenzylalcohol; DHBAld = 3,4-dihydroxybenzaldehyde; K, = activation constant; MBTH = 3methyl-2-benzothiazolinonehydrazone; NADA = N-acetyldopamine; NANE = N-acetylnorepinephrine; SDS = sodium dodecyl sulfate.
Cockroach Colleterial Gland Phenoloxidase
7 I2AT I0N
Fig. 1. Unified mechanism for the sclerotization of insect cuticle. Catecholic sclerotizing precursor ( R = CH,, NADA) i s oxidized by cuticular phenoloxidases (A = either o-diphenol oxidase or laccase or both) to o-benzoquinone derivative which participates in quinone tanning.
Quinone isomerase (6)converts quinone to quinone methide which undergoes Michael
1,6-addition reaction with either water to form NANE or cuticular nucleophiles to account for
quinone methide sclerotization ( = p-sclerotization).NADAquinone methide-I ,Zdehydro NADA
tautomerase (C)converts NADA-quinone methide to 1,2-dehydro NADA which upon oxidation by phenoloxidases generates quinone methide imineamide which reacts with both side
chain carbon atoms (quinone methide sclerotization or a, p-sclerotization). These reactions
are also possible for other N-acyldopamiederivatives, but not for C6-C1compounds.
the mechanism of tanning of cockroach ootheca and in this paper present our
results which show the latency and a-diphenoloxidase nature of the oxidase
present in the colleterial gland.
DHBAlc was prepared by sodium borohydride reduction of DHBAld. Sodium
borohydride (0.4g) was slowly added to a stirred solution of DHBAld (1.38 g)
in 10 ml of water at room temperature for 2 h. At the end of this period, the
contents were acidified with acetic acid and lyophilized. The residue was subjected to molecular sieve chromatography on Sephadex G-25 with 0.2 M acetic acid as the eluant. The fractions containing DHBAlc were pooled, lyophiliied,
and used. White solid from alcohol; mp 133-134°C; literature mp 134°C [25].
DHBA, DHBAld, catechol, and MBTH were obtained from Sigma Chemical
Co., St. Louis, MO. SDS and reagents for polyacrylamide gel electrophoresis
were obtained from Bio-Rad Laboratories, Richmond, VA.
Sugumaran and Nellaiappan
Enzyme Preparation
The left colleterial glands of Periplanetu arnericana were dissected in 0.9% (w/v)
NaCl containing 0.015% (w/v) CaC12and placed in distilled water containing
1mM phenylmethylsulfonylfluoride (PMSF) (100 kl/gland) for 15min at room
temperature. The lumen of the tubules of the left gland was removed from the
milky suspension after gentle homogenization in an Eppendorf tube. The milky
suspension (8.2 mg/ml) was centrifuged at 48,0009 for 10 min and the clear
supernatant (2.75 rng/ml) obtained was desalted on a Sephadex G-25 column
(2 x 6 cm) by using 10 mM sodium phosphate buffer, pH 7.0. The desalted
protein fraction (200 pg/ml), which is fivefold purified, was used as the
enzyme source.
Enzyme Assay
The phenoloxidase activity was routinely assayed spectrophotometrically
by using a reaction mixture (1ml) containing 0.2 M sodium phosphate buffer,
pH 7.0, 2 mM catechol, 0.002% MBTH, and aliquots of the enzyme sample.
The reaction was initiated by the addition of 20 pl of 10% (w/v) SDS and monitored continuously by following the increase in absorbance at 480 nm of the
MBTH-quinone adduct formed in the reaction mixture by using a Gilford model
2600 spectrophotometer.
Oxygen Uptake Studies
For some experiments, phenoloxidase activity was assayed by using a YSI
model 53 Clark type oxygen electrode. The reaction mixture contained 0.2 M
sodium phosphate buffer, pH 7.0, aliquots of the enzyme, 2 mM substrate
(except syringaldazine which was used at 15 pM), and 0.2% (w/v) SDS. The
reaction was started by the addition of SDS and the oxygen consumed in the
reaction mixture was continuously monitored at 30°C.
The Latency and Activation of Phenoloxidase System
The milky white secretions of the left gland did not exhibit any phenoloxidase
activity as such when tested either by the spectrophotometric method or by
the oxygen uptake techniques indicating that the phenoloxidase is probably
present as an inactive proenzyme form. Phenoloxidase is present in several
organisms as an inactive proenzyme and is known to be activated either
proteolytically or by detergents [32]. Attempts to activate the phenoloxidase
with proteases ended in marginal activation of phenoloxidase only. However,
detergents caused rapid activation of phenoloxidase activity in the milky white
secretion. Among the reagents tested, SDS caused tremendous activation of
phenoloxidase activity (Fig. 2). Therefore, routinely SDS was used to activate
the inactive phenoloxidase from the left colleterial gland of P. urnericunu.
Large portions of the left gland secretions seem to be comprised of protein
globules [26] which can be separated by simple centrifugation. After separation of insoluble protein globules, when the phenoloxidase was checked in
the presence of SDS, neither the supernatant fraction nor the sediment exhibited any phenoloxidase activity (Fig. 2). However, when these two fractions
Cockroach Colleterial Gland Phenoloxidase
TIME ( m i d
Fig. 2. Distribution of phenoloxidase in the secretion of left gland. A reaction mixture (1ml)
containing 2 mM catechol, 0.002% MBTH, different fractions, 0.2% S D S in 0.2 M sodium phosphate buffer, pH 7.0, was incubated at room temperature and the increase in absorbance at
480 nrn was continuously monitored. A: Whole left gland secretion. B: Sediment plus supernatant fraction. C: Supernatant plus bovine serum albumin (1 rng). D: Supernatant alone. E:
Sediment alone. F: Sediment with bovine serum albumin (1 mg).
were mixed together and then activated, phenoloxidase activity could be readily observed. In order to determine whether any other protein could be substituted in the activation process, phenoloxidase activity was checked after
adding bovine serum albumin to both the sediment and supernatant fractions.
As shown in Figure 2, phenoloxidase activity was detected in the soluble fraction, while the sediment did not possess any detectable activity. Although
crude supernatant fraction required both SDS and either the sediment fraction or an inert protein like bovine serum albumin to exhibit phenoloxidase
activity, after desalting on Sephadex G-25, the exogenous addition of protein
was found to be obsolete for phenoloxidase activity. Therefore, the rest of the
studies were carried out with the desalted supernatant fraction.
Figure 3 shows the activation of phenoloxidase obtained from Sephadex G-25
column, by different detergents. While SDS (anionic) caused maximum activation, followed by cetylpyridinium chloride (cationic), other detergents such
as caprylic acid (anionic), deoxycholate (anionic), sodium laurylsarcosidate
(anionic),CHAPS (zwitterionic),Triton X-100 (nonionic),Tween-20 (nonionic),
Nonidet P-40 (nonionic), octyl-p-D-glucopyranoside (nonionic), or Brij 58
(nonionic)caused either marginal activation or no activation at all. The activation of phenoloxidase activity by detergents could occur either as a consequence
of binding to the protein in low concentrations and causing correct conforma-
Sugumaran and NeElaiappan
Fig. 3. Activation of phenoloxidase by detergents. A reaction mixture (1 ml) containing2 m M
catechol, 0.002% MBTH, enzyme protein (250 bg), 0.2% detergent in 0.2 M sodium phosphate
buffer, pH 7.0 was incubated at room temperature and the increase in absorbance at 480 nm
was continuously monitored. Detergents used are: (A) SDS;(B)cetylpyridinium chloride; (C)
deoxycholate; (D) sodium N-laurylsarcosidate; and (El caprylic acid, digitonin, Triton X-100,
CHAPS, Tween 20, Nonidet P-40, Brij 58, or octyl-P-D-glucopyranoside.
tional changes for exhibiting the enzyme activity or as a consequence of binding into the detergent micelles. These two mechanisms can easily be
distinguished by examining the activation process at different concentrations
of detergent. If the former hypothesis is correct, then one should observe the
activation well below the critical micellar concentration of the detergent; if the
latter proposal is correct one should observe activation only after the detergent micelles have formed. The results of such an experiment are shown in
Figure 4. The activation of phenoloxidase by both SDS and cetylpyridinium
chloride exhibited a sigmoidal kinetics. The K, value for SDS was found to be
approximately 0.8 mM, which is well below the critical micellar concentration
of SDS (8 mM). In the case of cetylpyridinium chloride, the K, value was about
0.6 mM, which is of the same order of its critical micellar concentration
(0.8 mM). Therefore, it appears that the detergent molecules are binding to a
site on the enzyme and causing conformational changes that culminate in the
activation of the latent phenoloxidase.
If this is true, it is also likely that in vivo, such an activation is occurring. To
test whether endogenous chemicals can cause the activation of latent
phenoloxidase, chloroform-methanol extracts of the right gland, left gland,
and the vestibulum were added to the standard assay in the place of detergents. As shown in Figure 5, it is evident that both right gland and vestibu-
Cockroach ColleterialGland Phenoloxidase
conc (mM)
Fig. 4. Effect of different concentration of detergents on the activation of phenoloxidase.Assay
conditions as outlined for Figure 3, with the exception of changing detergent concentration.
Detergents used are: (A) SDS and (B) cetylpyridiniurn chloride.
lum possess endogenous activator(s),while the left gland extracts did not have
any activating effect, understandably. Since lipids were the primary candidates
to serve the endogenous activator function, both fatty acids and phospholipids
were tested for their ability to activate the latent phenoloxidase. Among the
fatty acids tested (Fig. 6A), linoleate (CI8:2)proved to be the most effective
activator followed by oleate (CI8:l),laurate (el,),and linolenate (CI8:3). Although stearate (C18)exhibited the same initial rate of activation, the total phenoloxidase activated in 3 min by this fatty acid was less than half activated by
SDS. Other fatty acids such as myristate (Cl4), palmitate (C16),nonadecanoate
(CI~),behenate (C22), and lignocerate (C24)
caused marginal activation only,
Of the phospholipids tested (1 mg/ml) (Fig. 6B), both phosphatidylethanolamine and phosphatidylglycerol activated the la tent enzyme effectively, while
phosphatidylcholine and sphingomyelin failed to activate the phenoloxidase.
Phosphatidylinositol and phosphatidylserine caused intermediate activation.
Sugumaran and Nellaiappan
TIME ( m i d
Fig. 5. Activation of phenoloxidase by endogenous activators. Assay conditions as outlined
for Figure 3, with the exception of replacing detergent with endogenous activators. For preparation of extracts, the tissues (four right gland, one left gland, or two vestibulum) were gently
homogenized in 100 pI of chloroform: methanol (2:l) and centrifuged. An aliquot of the supernatant (25 pl) was added to 100 pI alcohol and used for activatingphenoloxidasewith appropriate
controls. A: Control with 0.2% SDS. B: Extract from vestibulum. C: Extract from right gland.
D: Extract from left gland.
Properties of Phenoloxidase
The phenoloxidase activity of the left colleterial gland was found to be labile
and lost its activity completely upon freezing and thawing or dialysis. Hence,
most of the studies were carried out with freshly prepared enzyme only. Whitehead et al. I291 reported that the left gland phenoloxidase is remarkably stable towards heat (98°C for 10 min). Heat inactivation studies were conducted
to determine whether or not the left gland phenoloxidase was exhibiting such
an unusual stability. As shown in Figure 7, heat treatment of the partially purified enzyme at 55°C for 10 min resulted in irreversible loss of phenoloxidase
activity. The enzyme activated by SDS exhibited a pH optimum of 7 in sodium
phosphate buffer (Fig. 8). The effect of varying concentrations of DHBAlc on
the velocity of the enzyme catalyzed oxidation is shown in Figure 9 (inset). As
is evident, the enzyme follows typical Michaelis-Menten kinetics. From the
Lineweaver-Burk plot (Fig. 9)) the V,,, for the reaction was calculated to be
0.76 pmol of oxygen consurned/min/mg protein and a K, of 0.8 mM.
Earlier work by Brunet's group [28-301 indicated that the phenoloxidase is
a laccase type. In order to assess this claim, we conducted both inhibition studies and substrate specificity studies. As shown in Figure 10, the phenoloxidase
exhibited a wide substrate specificity and oxidized a variety of o-diphenolic
compounds such as DHBAlc, NADA, 3,4-dihydroxyphenethyl alcohol, 4methylcatechol, dopamine, catechol, 3,4-dihydroxyphenylglycol, NANE, etc.
Cockroach Colleterial Gland Phenoloxidase
Fig. 6 . Activation of phenoloxidase by lipids. Assay conditions as outlined for Figure 3, with
the exception of replacing detergents with different lipids (1 mg/ml final concentration). The
reaction mixture also contained 20% ethanol to facilitate solubilization of lipids. A: Fatty acids
used are: (2) linoleic acid (C18:*);(3) lauric acid (C12);(4) oleic acid (C,8:,); (5) linolenic acid
(C18:d; ( 6 )stearic acid (C18);(7) palmitic acid (Clb) and lignoceric acid (CZ4);(8) behenic acid
(C2d; (9) myristic acid (C14);and (10) nonadecanoic acid (CI9).(Curve 1 is control experiment
with SDS.)B: Phospholipids used are: (2) phosphatidylethanolamine; (3)phosphatidylglycerol;
(4) phosphatidylserine; (5) phosphatidylinositol; ( 6 ) phosphatidylcholine; and (7) sphingomyelin. (Curve 1 is control experiment with SDS.)
Sugumaran and Nellaiappan
Fig. 7. Temperature stability of phenoloxidase. The phenoloxidase was heated at indicated
temperatures for 5 min, cooled, and assayed for enzyme activity as outlined in Figure 3.
Interestingly, the enzyme failed to oxidize syringaldazine-a typical laccase
substrate-when tested either by oxygen uptake studies or by spectrophotometric techniques. Moreover, both hydroquinone and methylhydroquinone
served as poor substrates for the enzyme indicating that it is not a laccasetype enzyme. The enzyme also oxidized tyramine marginally and failed to
attack tyrosine. Therefore, it appears that the phenoloxidase found in the
colleterial gland is a typical o-diphenoloxidase. Surprisingly, the enzyme
activated by SDS did not attack DHBA, one of the putative sclerotizing precursors of cockroach ootheca. To see whether phenoloxidase activated by
different reagents could attack DHBA, the substrate specificity of enzyme
activated by a) SDS; b) oleate; and c ) vestibular extract was checked for its
ability to oxidize DHBA. All three preparations failed to oxidize DHBA, but
attacked DHBAlc readily.
Among the inhibitors tested (Table l),phenylthiourea-a typical o-diphenoloxidase inhibitor-drastically inhibited enzyme activity. Chelators such as
diethyldithiocarbamate, potassium cyanide, sodium oxalate, and ethylenediamine tetraacetate also inhibited the enzyme activity drastically, while iron-
Cockroach Colleterial Gland Phenoloxidase
0 1
8 .O
Fig. 8. Effect of pH on the activity of phenoloxidase. A reaction mixture (1 ml) containing
2 mM DHBAlc, 0.2 M sodium phosphate buffer at specified pH, enzyme protein (200 hg), and
0.2% SDS was incubated at 30°C and the oxygen consumed during the reaction was continously
monitored by using any oxygen electrode. The reaction was initiated by the addition of SDS.
Control experiments without SDS addition did not show any oxygen consumption at these pH
ranges tested.
specific chelators such as o-phenanthroline, a-a'-dipyridyl, and tiron showed
marginal inhibition only.
In accordance with the earlier studies by Brunet and his associates [26], the
milky white secretions from the left colleterial gland of P. nmericuna contained
the phenoloxidase. When the protein globules of the secretions were separated
from the supernatant proteins by centrifugation, and then checked for enzyme
activity, only the supernatant possessed the enzyme activity. The globules were
devoid of any phenoloxidase activity. However, the phenoloxidase required
both the protein globules and a detergent such as SDS to exhibit its activity in
the crude preparations. Although an inert protein like bovine serum albumin
could substitute for the protein globules for phenoloxidase activation, such
constraints were not observed with the partially purified preparation. The partially purified phenoloxidase required only a detergent to exhibit its activity.
SDS could either directly activate prophenoloxidase or indirectly activate other
components which in turn activate the prophenoloxidase. However, lack of
Sugumaran and Nellaiappan
Fig. 9. Lineweaver-Burk plot for DHBAlc oxidation by phenoloxidase.Assay conditions were
the same as described for Figure 8 with the exception of varying the concentration of DHBAlc.
Amount of enzyme protein used is 132 kg. Inset: Effect of varying concentration of DHBAk on
the velocity of phenoloxidase reaction.
any lag period during the activation process indicates that the latter possibility is less likely to operate. Moreover, inactive homogenous prophenoloxidase
is known to be activated by a detergent such as SDS or oleate [32,33]. Elegant
studies on direct activation of a plant phenoloxidase by SDS have been reported
recently [33]. The authors concluded from their studies that SDS, at levels
below the critical micellar concentrations, bound to the enzyme allosterically
and caused the conformational changes in the protein which resulted in the
activation of the latent form. In the present study also, it could be demonstrated that SDS caused the activation of latent phenoloxidase at below the
critical micellar concentrations, probably by binding to the protein allosteric d y and causing conformationalchanges to expose the active site of the enzyme.
Activation of insect prophenoloxidase has been extensively reviewed by
Brunet [32]. Two types of activation processes have been identified in the
literature. Bodine [34] described the activation of latent phenoloxidase from
the diapausing eggs of the grasshopper, Melanoplus differentialis, by surface
active reagents or heat or heavy metals. Later, Funatsu and Inaba [35] reported
the activation of prophenoloxidase by SDS in Musca, while Heyneman and
Vercauteren [36] demonstrated the activation of latent phenoloxidase by
sodium oleate. The latter authors also examined the activation caused by
different fatty acids and concluded the oleate is the most effect activator among
the fatty acids tested [37]. Hackmann and Goldberg [38] reported the activation of prophenoloxidase from the hemolymph of Lucilia ctrprina by SDS, as
well as oleate.
Cockroach Colleterial Gland Phenoloxidase
TIME ( m i d
Fig. 10. Substrate specificity of phenoloxidase. A reaction mixture (1 rnl) containing 2 m M
substrate, 0.2 M sodium phosphate buffer, pH 7.0, enzyme protein (200 pg), and 0.2% SDS was
incubated at 30°C and the oxygen consumed in the reaction was continously monitored by
using Clark-type oxygen electrode. Substrates used are: (1) DHBAlc; (2) 3,4-dihydroxyphenethylalcohol; (3) NADA; (4) 4-methylcatechol; (5) dopamine; ( 6 ) catechol; (7) norepinephrine; (8) 3,4-dihydroxyphenylglycol; (9) NANE; (10) dopa; (1 1) 3,4-dihydroxyphenylacetic acid,
or tyramine; (12) hydroquinone, or rnethylhydroquinone; (13) syringaldazine, DHBA, DHBAld,
or tyrosine.
A different type of activation was characterized by Ohnishi [39]. In Drosophila, he observed that lipids did not activate the proenzyme, but a protein factor did. Schweiger and Karlson [40] not only confirmed this observation in
Calliphora, but provided evidence that activation of prophenoloxidase is achieved
by limited proteolysis. Similar observations were made on Drosophilu [41]. However, extensive studies have been carried out only on the activation of the proenzyme from the silkworm Bombyx mori [42, and the references cited therein].
Ashida and coworkers demonstrated that proteases isolated from both cuticle
and hemolymph removed a M, 5,000 peptide from the prophenoloxidase,
thereby activating the enzyme. In the present studies, we could not observe
activation of this kind. However, ample evidence could be collected for the
activation of the latent enzyme by surfactants.
Among the various detergents tested, both SDS and cetylpyridinium chloride activated the latent phenoloxidase while both nonionic and zwitterionic
detergents failed to activate the enzyme. SDS caused activation of insect
Sugumaran and Nellaiappan
TABLE 1. Effect of Various Inhibitors on Phenoloxidase Activity
Inhibitors added
Potassium cyanide
Ethylenediamine tetraacetate
Sodium oxalate
Sodium azide
Sodium fluoride
8-hydroxy quinoline
1 PM
5 PM
20 pM
20 pM
40 pM
50 pM
0.1 mM
0.5 rnM
1 mM
5 mM
5 mM
9 mM
1 mM
5 mM
8 mM
9.1 mM
5 mM
5 mM
9.1 mM
9.1 mM
9.1 mM
9.1 mM
% inhibition
phenoloxidase has already been reported [35,38]; but to the best of our knowledge, insect phenoloxidase activation caused by a cationic detergent, viz., cetylpyridinium chloride, has not been reported. Whitehead et al. [30]reported
the activation of phenoloxidase from the left colleterial gland of l? arnericana
by both deoxycholate and Tween 85. In the present study, we could not find
any significant activation by these two nonionic detergents. Chloroform-methanol
extracts of vestibulum or right gland could replace the SDS in the activation
process indicating the presence of endogenous activators. Since lipids were
suspected to be the prime candidates, attempts were also made to activate the
phenoloxidase system by exogenous addition of different lipids. Among the
fatty acids tested, linoleate, laurate, oleate, and linolenate activated the latent
phenoloxidase. Although stearate activates the enzyme at nearly the same initial rate as that of SDS or linoleate, the activated enzyme seemed to be unstable, and showed less than half of the activity exhibited by SDS at the end of
the reaction (Fig. 6A). Of the phospholipids tested, phosphatidylethanolamine
and phosphatidylglycerolwere the best activators followed by phosphatidylserine
and phosphatidylinositol, Phosphatidylcholine and sphingomyelin failed to
activate the enzyme. Further studies on the role of lipids in the activation process and the characterization of endogenous activator(s) are in progress.
In insect cuticle, both a-diphenoloxidase and laccase seem to be present. In
recent years, it has been suggested that laccase is the enzyme which participates in sclerotizationreactions and that phenoloxidase may simply be involved
in defense mechanism ((31) and references cited therein]. In regard to cockroach egg case, previous studies indicated that the enzyme involved in scle-
Cockroach Colleterial Gland Phenoloxidase
rotization of ootheca is a laccase [28-301. However, present studies demonstrate
that the enzyme is not a laccase but a typical o-diphenoloxidase. Thus, the
enzyme from left colleterial gland failed to oxidize syringaldazine-a typir%l
substrate for laccase. In addition, it did not attack o-diphenols such as hydroquinone or methylhydroquinone. Finally, inhibition studies indicated that the
enzyme is a typical o-diphenoloxidase. Among the various inhibitors tested,
phenylthiourea-a potent inhibitor of phenoloxidase-drastically inhibited the
reaction even at the 5 pM level. At the 20 pM level, complete inhibition was
observed, whereas Whitehead et al. [30] failed to observe any inhibition by
phenylthiourea at this concentration. In our hands, diethyldithiocarbamate-a
copper-specific chelator-also inhibited the enzyme strongly, Thus, at a concentration of 50 bM, it showed total inhibition of phenoloxidase activity, while
Whitehead et al. [30] did not observe any inhibition even at the 200 pM level.
Mimosine, a structural analog of dopa, showed 45% inhibition at the 5 mM
level. While the debate regarding the nature of the enzyme participating in the
sclerotization of insect cuticle is still going on, the above considerations clearly
indicate that at least in cockroach ootheca, o-diphenoloxidase and not the laccase is the enzyme involved in the tanning process. The difference between our
results and that of Whitehead et al. [30]could be due to the fact that the earlier
workers used the enzyme preparation without activation for extended assay
times (up to 8 h). With SDS-activated enzyme, we could monitor the reaction
easily within minutes. This coupled with the highly sensitive nature of our
assay procedure seems to have generated more accurate results.
The potent inhibition of phenoloxidase activity by sodium oxalate is quite
intriguing. Oxalate crystals have been identified as important components of
egg cases and their presence in the secretions of the left gland has been demonstrated as early as 1960 [43]. Oxalate has been identified as the endogenous
inhibitor of phenoloxidase in the spinach chloroplasts [44]. In addition it has
also been demonstrated that oxalate is a potent inhibitor of mushroom tyrosinase [45]. In the present studies, we could demonstrate the inhibition of
phenoloxidase by oxalate. Therefore, the probable function of oxalate in the
left gland is to inhibit any undesired phenoloxidase action.
Previous workers claimed that DHBA is the best substrate for left gland
phenoloxidase and concluded that DHBA is the sclerotizing precursor for the
tanning of ootheca [3,5,21-23,28-301. Their results were based on oxygen consumption studies using Warburg apparatus on the left gland phenoloxidase
for extended periods of time. They reported the oxidation of DHBA, DHBAld,
and hydroquinone, but did not test DHBAlc, as they were not aware of its
presence at that time. However, we could not find any evidence for the facile
oxidation of DHBA, DHBAld, or hydroquinone. On the contrary, DHMlc, which
is also found in the left gland (as a glucoside), proved to be the best substrate
tested for this enzyme. DHBA inhibited the oxidation of DHBAlc at 10 times
the concentration of substrate to about 25%, while DHBAld neither served as
substrate nor inhibited the enzyme. Since there could be differences in the
substrate specificity of the enzyme activated differently [38], phenoloxidase
activated by a) SDS, b) oleate, and c) extracts of vestibulum was checked for
its specificity towards DHBAlc and DHBA. All three modes of activated
phenoloxidase readily oxidized DHBAlc but failed to attack DHBA. Thus DHWc
Sugumaran and Nellaiappan
seems to be the only endogenous substrate for this enzyme. DHl3A may be
formed as an end product of DHBAlc oxidation as its concentrations steadily
increased with the progress of tanning without any significant decrease
(Sugumaran and Nellaiappan, unpublished results). Hence it may serve other
functions such as a bacteriocide rather than participating in the tanning of
exoskeleton. Currently, we are investigating this possibility.
At 50 kg/rnl concentration, all phospholipids tested activated the prophenoloxidase. At higher concentration, some lipids precipitated the enzyme resulting in apparent lack of activation (Fig. 6'8).
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cockroaches, nature, periplaneta, phenoloxidase, gland, present, colleterial, american, left, latency
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