On the latency and nature of phenoloxidase present in the left colleterial gland of the cockroach Periplaneta americana.код для вставкиСкачать
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. 166 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 INTRODUCTION 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 . 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 .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 167 QUI NO NE TANNING NADA NADA QUINONE cs OH NANE C NADA QUlNONE METHIDE /-- QU I NONE METHIDE - SCLERO 7 I2AT I0N DEHYDRO NADA DEHYDRO NADA QUINONE METHIDE 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. MATERIALS AND METHODS Chemicals 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 . 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. 168 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. RESULTS 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 . 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  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 0.4 169 A 0 2 1 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- 170 Sugumaran and NeElaiappan 0.0 1 2 3 4 5 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 0.01. 1.0 0.1 171 10.0 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 172 w B D 0 1 2 3 4 5 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 173 E c 0 OD P c a w 0 2 4 m a: 0 v) m 4 1.5 0 TIME 3.0 (min) 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.) 174 Sugumaran and Nellaiappan 100 50 0 25 45 55 TEMPERATURE ( * C ) 35 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 175 100 >. + 3: I I- 0 a 50 W 0 K W n 0 1 I 5.0 I I 6.0 7.0 8 .O P" 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. DISCUSSION In accordance with the earlier studies by Brunet and his associates , 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 176 Sugumaran and Nellaiappan 0 10 20 11s 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 . 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 . Two types of activation processes have been identified in the literature. Bodine  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  reported the activation of prophenoloxidase by SDS in Musca, while Heyneman and Vercauteren  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 . Hackmann and Goldberg  reported the activation of prophenoloxidase from the hemolymph of Lucilia ctrprina by SDS, as well as oleate. Cockroach Colleterial Gland Phenoloxidase 0.0 2.0 4.0 6.0 177 8.0 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 . In Drosophila, he observed that lipids did not activate the proenzyme, but a protein factor did. Schweiger and Karlson  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 . 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 178 Sugumaran and Nellaiappan TABLE 1. Effect of Various Inhibitors on Phenoloxidase Activity Inhibitors added None Phenylthiourea Diethyldithiocarbamate Potassium cyanide Mimosine Ethylenediamine tetraacetate Sodium oxalate Sodium azide Sodium fluoride o-phenanthroline a,a'-dipyridyl Tiron Neocuproine 8-hydroxy quinoline Concentration 1 PM 5 PM 20 pM 20 pM 40 pM 50 pM 0.1 mM 0.5 rnM 1 mM 1mM 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 0 82 94 100 23 66 100 16 65 93 18 45 50 83 61 85 91 17 49 50 38 18 35 0 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. 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 179 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.  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.  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. 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 . Oxalate has been identified as the endogenous inhibitor of phenoloxidase in the spinach chloroplasts . In addition it has also been demonstrated that oxalate is a potent inhibitor of mushroom tyrosinase . 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 , 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 180 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. NOTE ADDED IN PROOF At 50 kg/rnl concentration, all phospholipids tested activated the prophenoloxidase. 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