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Evidence for a lipid activator of prophenolase in Tenebrio molitor
J. InsectPhysbl., 1968,Vol. 14,pp. 409 to 415. Pergamon Press. Printed in Great Britain
EVIDENCE FOR A LIPID ACTIVATOR OF PROPHENOLASE IN ZJ?.UV~B~IO MOLITOR* R. A. HEYNEMAN and R. E. VERCAUTEREN Department of Physiological Chemistry, Veterinary College, University of Ghent, Belgium (Receked 17 Jury 1967) Abstract-It has been shown that neither the soluble proteins nor the separated soluble low molecular material of Tenebrio tissue homogenates could activate samples of latent phenolase. Only the precipitate after centrifugation of a larval homogenate contained a thermostable factor able to convert the prophenolase into an active enzyme. This factor, the presence of which could be demonstrated in larval integuments, was a lipid. Further analysis showed that especially C-18 fatty acids exercise a high activating power on latent phenolase. As lipase activity was detected in the subcuticular fat and tracheal oenocytes, a hypothetical mechanism for the activation process was drawn up considering published data. INTRODUCTION
SINCE the existence of phenolases as latent proenzymes in intact tissues has been shown in Melanoplus eggs (BODINE and ALLEN, 1938), most activation processes of the proenzyme in witro are carried out with some slightly denaturating agents such as thymol, chloroform, ether, ethylurethan, and some metal ions, or with some surface active compounds such as detergents. Although the proenzyme has been isolated in a very purified form (KARLSON et al., 1964; HEYNEMAN, 1965), the activation in &o still remains doubtful. KARLSONet al. (1964) have isolated an activator from Calliphora larvae which was proteinaceous and was localized in the integument. Recently GEIGER and MITCHELL (1966) have demonstrated that one of the five protein components involved in phenol oxidase production in extracts of Drosophila melanogaster is rate limiting in the activation process. This natural activator is initially localized in the larval salivary gland and released afterwards in the haemolymph. On the other hand, there is some mention of a lipoidal activator in Mekznoplus, Tenebrio, and Drosophila (BODINE and ALLEN, 1941; LEWIS and LEWIS, 1963). Thus, while it is well established that the presence of a proenzyme of phenolase is probably general in insects, the nature and the mode of action of a natural activator, and the general occurrence of a common type of activator are still uncertain. The approach to this problem was to find out whether or not there exists in Tenebro an activator comparable with the one found by several authors in Diptera, and to look for a possible activation mechanism in eriwo. * Experimental data presented here are part of Programme No. 76 ‘Enzymology supported by a grant of the Foundation for Basic Research of the Belgian Government.
410
R. A. HEYNEMANANDR. E. VERCAUTEREN MATERIALS
All experiments were performed maintained in laboratory culture. Analytical
AND METHODS upon
last instar
larvae
of Tenebrio molitor
methods
Enzyme preparations of the inactive prophenolase were made following a previously developed method (HEYNEMAN, 1965). All activity determinations were carried out with the Warburg respirometer according to AERTS and VERCAUTEREN(1964). The specific activities of our enzyme samples showed, after activation with 1 x 10es M Na-oleate in 0.1 M phosphate buffer and with 4-methylcatechol as substrate, an average value of 690 pmole O,/min per mg N. The protein content of all samples was estimated by the micromethod described by MATTENHEIMER (1960). Lipid extractions of isolated integuments were made after the method of JENSEN et al. (1951). The phosphatide fractions of these extracts were precipitated with acetone and MgCl, (MACLACHLAN, 1944). After centrifugation, the clear supernatant was concentrated under reduced pressure in an atmosphere of nitrogen. For characterization of the lipid components in the extracts thin-layer chromatography with the following solvent systems was used: Solvent 1: light petroleum (b.p. 60-80”C)-ether-acetic acid (70 : 30 : 2). Solvent 2 : acetic acid-acetonitrile (1 : 1). Silica Gel G (Merck) was the adsorbent. Lipid substances were detected by spraying with either 10% phosphomolybdic acid in ethanol or a O*OSo/osolution of Rhodamine B. Gas chromatographic analyses were carried out with the Argon Chromatograph. Methylesters of the isolated fatty acids were made following the micromethod of DE FRANCESCO (1962). The histochemical localization of lipase in dissected animals was demonstrated with the classical or-naphthyl acetate method (PEARSE, 1960) and the more specific Tween method (GOMORI, 1952). Preparation
of larval homogenate and particulate
fractions
The larvae were chilled and ground in a mortar with a known volume of cold 0.15 M NaCl solution. The brei was filtered through four layers of cheese cloth and centrifuged during 20 min at 15,000g. Three layers were obtained: an upper Samples layer, a precipitate, and a clear supernatant containing the latent enzyme. of these fractions were tested for their activating capacity on purified phenolase. Acetone powders for the preparation of an eventual proteinaceous activator were made according to the method of KARLSON and SCHMID (1955). The dried powder was extracted with O-067 M phosphate buffer, pH 7.0, containing O-20/, ascorbic acid. The homogenate was centrifuged and the supernatant chromatographed on a Sephadex G-25 column. Finally, traces of the active phenolase were removed by ultracentrifugation at 120,OOOg for 90 min.
LIPIDACTIVATOR OFPROPHENOLASE IN TENEBRIO
MOLZTOR
411
RESULTS
As already reported by ARRTS and VERCAUTEREN (1964), the proenzymecontaining supernatant of a centrifuged homogenate can be stabilized by gel filtration on Sephadex G-25. The whole bulk of soluble larval proteins coming off the Sephadex G-25 column can be stored for several weeks at 4°C without conversion of the proenzyme into an active phenolase. No activating action on the latent enzyme was detected after incubation with the separated low molecular material ; nor with the lipid upper layer obtained after centrifugation of the homogenate. Only with the resuspended precipitate was a considerable activation of the proenzyme obtained. In order to solubilize an eventual activator protein, the precipitate was treated with the butanol-NaHCO, solution of MORTON(1953), and the extract tested as an activator. Values for phenolase activity after incubation of the latent enzyme with these different fractions are summarized in Table 1. TABLE~-RELEASED
PHENOLASE ACTIVITY AFTER INCUBATION OF PROPHENOLASE SEVEFWLEXTRACTS TESTED AS POSSIBLE ACTIVATORS
Activator 0.5 ml suspended precipitate O-5 ml precipitate after 30 min at 100°C 0.5 ml butanol extract 1 /Imole Na-oleate
WITH
Activity in &mole O.Jmin per ml) 2.06 1.72 0 4.25
These results clearly show that the precipitate obtained after centrifugation of a larval homogenate contains a factor able to activate the proenzyme. This factor, which could not be solubilized, was found to be thermostable. Other attempts to demonstrate an eventual proteinaceous activator of prophenolase in Tenebrio molitor were made by incubating a purified phenolase sample with various volumes of an acetone powder extract (KARLSONand SCHMID, 1955). No activation of the latent enzyme could be detected. The minimal uptake of oxygen after a few hours of incubation was easily shown to be due to small traces of active phenolase present in the acetone powder extract. Only after 24 hr of incubation was an increase of the phenolase activity observed. As these mixtures turned slowly brownish, probably the tyrosyl residues of the latent enzyme were oxidized by the noted traces of active enzyme (YASUNOBU et al., 1959). As already noticed, after storage of a partly inactive phenolase, this effect seems to benefit the conversion of the latent enzyme into an active enzyme. After microdissection of Tenebrio larvae and careful isolation of the integuments with the adherent subepidermal fat, it could be demonstrated that the previously mentioned thermostable activator was present in a homogenate of larval integuments. Lipid extraction procedure was carried out as already described, and the
412
K. A. HEYNEMAN AND R. E. VERCAUTEREN
concentrated extract was subjected to thin-layer chromatography. When the plate was developed, using solvent 1, several spots could be recognized after spraying with phosphomolybdic acid reagent (Fig. 1). The spots were scraped off, extracted in ether, air dried, resuspended in 0.1 M phosphate buffer, pH 7, and tested as possible activators for the latent enzyme. Two spots, 2 and 5, gave a reaction in the presence of proenzyme and substrate. The previously isolated phosphatide fraction did not have any activating action on the proenzyme sample. In order to obtain a larger recovery of separated material, the lipid extract was chromatographed on a Silica Gel H column and eluted with light petroleum etherether-acetic acid 90 : 10 : 1. Fig. 2 represents the elution curve measured at 254 mp. When peaks III and VI were transferred to a buffer system and assayed,
70
1
0
60
50
0
0
0
40 30 20 I 0 0
6
c
i
k
FIG. 1. Thin-layer chromatogram of a lipid extract of isolated integuments from Terrebrio larvae developed with 10% phosphomolybdic acid in ethanol. Solvent: light petroleum-ether-acetic acid (70 : 30 : 2). a: Lipid extract; b : oleic acid; c: phosphatide fraction ; d : cholesterol; e : linoleic acid.
Effluent,
mC
FE. 2. Elution curve after column chromatography on Silica Gel H of a lipid The cross-hatched peaks represent extract of isolated larval integuments. fractions with activating capacity on latent phenolase.
LIPID ACTIVATOROF PROPHRNOLASEIN TENEBRIO
413
MOLITOR
they showed a high activating capacity. Whereas, after thin-layer chromatography (solvent 2), peak III could be recognized as consisting of free fatty acids; the chemical nature of peak VI is still unknown. After methyl esterification the material of peak III was injected in a gas chromatographic column and the fractions were automatically recorded. As shown in Table 2, most of the fatty acids could be identified. TABLE Z-COMPOSITION
OF THE FATTY
EXTRACTBASED ON GAS CHROMATOGRAPHIC ANALYSIS
ACID
Palmitic acid Branched fatty acid Stearic acid Oleic acid Linoleic acid Linolenic acid Branched fatty acid Unsaturated fatty acid Unsaturated fatty acid Behenic acid
C-16 C-18 C-18:0 C-181 C-182 C-l 83 c-20 c-20 c-20 c-22
After incubation of the proenzyme with equimolecular concentrations of some natural occurring fatty acids, the resulting enzymic activity showed a maximum in the presence of C-18 fatty acids (except linolenic acid) (Fig. 3). > s IOr
Fo tty FIG. 3.
Activating
acids
influence of higher fatty acids at equimolar (1 x 10-a M) on latent phenolase.
concentrations
In order to find out a possible mechanism for the release of fatty acids in situ, we have submitted the isolated integuments and tracheae to histochemical tests for lipase activity. The ar-naphthyl acetate staining gave a strong positive reaction for
414
R. A. HIXYNEMAN ANDR. E. VERCAUTERFN
the tracheal oenocytes and the subcuticular fat. Using the more specific Tween method after GOMORI (1952), lipase activity in both tissues could be affirmed. The small oenocytes of the subepidermis (WIGGLESWORTH, 1948) did not contain any lipase activity. Staining for free fatty acids (without Tween 60) gave a positive reaction for the large oenocytes in the neighbourhood of the spiracles, whereas the subcuticular fat was only weakly coloured. DISCUSSION The presence of phenolase in a special type of blood cell, called an oenocytoid, has been proved by several authors (DENNELL, 1947; DECLEIR et al., 1960). It is undoubtedly this same type of cell which JONES (1949) describes in the larva of Sarcophaga bullata under the name of ‘spherule cell’. Early in the moulting process the oenocytoids increase greatly in number and size. During the period of cuticle formation, the oenocytoids are broken down and ingested by amoebocytes (WIGGLESWORTH, 1965). Exactly the same process was described by POYARKOFF (1910) with the oenocytoids of Galerucella. It is quite probable that the liberated phenolase diffuses into the cuticle (DENNELL, 1958). Activation of the proenzyme may take place by contact with the fatty acids present and set free by lipase activity or by the unknown lipoid activator mentioned above, probably occurring in the cuticle itself. After activation, the enzyme becomes insoluble after a few hours so that diffusion of a fatty-acid-activated phenolase through the new cuticle towards the epicuticle is still possible. As no protein activator for the latent phenolase could be detected in larval Tenebrio homogenates, it would be interesting to follow the effect of the proteinaceous activator of other species on our purified proenzyme. Further investigation on the activation mechanism and a comparative physical and chemical study of the prophenolase and active phenolase would also be profitable. REFERENCES F. and VERCAUTEREN R. (1964) Specificity and mode of action of phenoloxidase from larvae of Tenebrio molitor. Enzymologia 28, l-20. BODINEJ. H. and ALLEN T. H. (1938) Further studies on the activation of the enzyme
AERTS
tyrosinase. J. cell. [email protected]. 11, 409-415. BODINEJ. H. and ALLEN T. H. (1941) Some properties of protyrosinase. J. cell. camp. Physiol. 18, 151-156. DECLEIRW., AERTS F., and VERCAUTJXREN R. (1960) The localization of polyphenoloxidase in hemocytes. Proc. 11th int. Congo. Ent. (B) 3, 176-179. DE FRANCESCOF. (1962) Sulla transesterificazione stechiometrica dei gliceridi mediante alcool met&o. Riv. ital. Sod. GY. 5, 245-252. DENNELL R. (1947) The occurrence and significance of phenolic hardening in the newly formed cuticle of crustacea decapoda. Proc. R. Sot. B, 134,485503. DENNELL R. (1958) The hardening of insect cuticles. Biol. Rev. 33, 178-186. GEIGERH. and MITCHELL H. (1966) Salivary gland function in phenoloxidase production in Drosophila. J. Insect Physiol. 12, 747-754. GOMORIG. (1952) Microscopic Histochemistry, pp. 203-206. University Press, Chicago. HEYNEMANR. A. (1965) Final purification of a latent phenolase with mono- and diphenolase activity from Tenebrio molitor. Biochem. biophys. Res. Cmnm. 21, 162-169.
LIPID ACTIVATOR OF PROPHENOLASE IN
TENEBRIO
MOLITOR
415
JENSEND., CHAIKOFFI., and TARVERH. (1951) The ethionine-induced fatty liver: dosage, prevention and structural specificity. J. biol. Chem. 192, 395-2. JONESJ. C. (1949) Hemocyte types of Tenebrio. Iowa St. Co&J. Sci. 24, 355361. KARLSONP., MERGENHAGEND., and SEKEF~S C. (1964) Weitere Untersuchungen fiber das o-Diphenoloxydase system von Calliphora erythrocephala. Hoppe-Seyler’s Z. physiol. Chem. 338,42-50. KARLSONP. and SCHMIDH. (1955) Uber die Tyrosinase der Calliphora Larven. HoppeSeyler’s Z. physiol. Chem. 300, 3541. LEWIS H. W. and LEWIS H. S. (1963) Genetic regulations of dopa oxidase activity in Drosophila. Ann. N.Y. Acad. Sci. 100, 827-839. MACLACHLANP. L. (1944) Determination of the iodine number of whole phospholipid. J. biol. Chem. 92, 395402. MATTENHEIMERH. (1960) Mihromethoden ft?r das Klinisch-chemische und Biochemische Laboratorium. Walter de Gruyter, Berlin. MORTON R. K. (1953) Microsomal particles of normal cow’s milk. Nature, Lond. 171, 734-735. PEARSEE. (1960) Histochemistry, p. 886. Little, Brown, Boston. POYARKOFFE. (1910) Recherches histologiques sur la metamorphose d’un Coleopdre. Arch. Anat. Microsc. 12, 333474. WIGGLESWORTH V. B. (1948) The structure and deposition of the cuticle in Tenebrio molitor. Quart. J. micr. Sn’. 89, 197-216. WIGGLESWORTH V. B. (1965) The Principles of Insect Physiology, p. 394. Methuen, London. YASUNOBUK. T., PETERSONE. and MASONH. (1959) Th e oxidation of tyrosine-containing peptides by tyrosinase. J. biol. Chem. 234, 3291-3295.
EVIDENCE FOR A LIPID ACTIVATOR OF PROPHENOLASE IN ZJ?.UV~B~IO MOLITOR* R. A. HEYNEMAN and R. E. VERCAUTEREN Department of Physiological Chemistry, Veterinary College, University of Ghent, Belgium (Receked 17 Jury 1967) Abstract-It has been shown that neither the soluble proteins nor the separated soluble low molecular material of Tenebrio tissue homogenates could activate samples of latent phenolase. Only the precipitate after centrifugation of a larval homogenate contained a thermostable factor able to convert the prophenolase into an active enzyme. This factor, the presence of which could be demonstrated in larval integuments, was a lipid. Further analysis showed that especially C-18 fatty acids exercise a high activating power on latent phenolase. As lipase activity was detected in the subcuticular fat and tracheal oenocytes, a hypothetical mechanism for the activation process was drawn up considering published data. INTRODUCTION
SINCE the existence of phenolases as latent proenzymes in intact tissues has been shown in Melanoplus eggs (BODINE and ALLEN, 1938), most activation processes of the proenzyme in witro are carried out with some slightly denaturating agents such as thymol, chloroform, ether, ethylurethan, and some metal ions, or with some surface active compounds such as detergents. Although the proenzyme has been isolated in a very purified form (KARLSON et al., 1964; HEYNEMAN, 1965), the activation in &o still remains doubtful. KARLSONet al. (1964) have isolated an activator from Calliphora larvae which was proteinaceous and was localized in the integument. Recently GEIGER and MITCHELL (1966) have demonstrated that one of the five protein components involved in phenol oxidase production in extracts of Drosophila melanogaster is rate limiting in the activation process. This natural activator is initially localized in the larval salivary gland and released afterwards in the haemolymph. On the other hand, there is some mention of a lipoidal activator in Mekznoplus, Tenebrio, and Drosophila (BODINE and ALLEN, 1941; LEWIS and LEWIS, 1963). Thus, while it is well established that the presence of a proenzyme of phenolase is probably general in insects, the nature and the mode of action of a natural activator, and the general occurrence of a common type of activator are still uncertain. The approach to this problem was to find out whether or not there exists in Tenebro an activator comparable with the one found by several authors in Diptera, and to look for a possible activation mechanism in eriwo. * Experimental data presented here are part of Programme No. 76 ‘Enzymology supported by a grant of the Foundation for Basic Research of the Belgian Government.
410
R. A. HEYNEMANANDR. E. VERCAUTEREN MATERIALS
All experiments were performed maintained in laboratory culture. Analytical
AND METHODS upon
last instar
larvae
of Tenebrio molitor
methods
Enzyme preparations of the inactive prophenolase were made following a previously developed method (HEYNEMAN, 1965). All activity determinations were carried out with the Warburg respirometer according to AERTS and VERCAUTEREN(1964). The specific activities of our enzyme samples showed, after activation with 1 x 10es M Na-oleate in 0.1 M phosphate buffer and with 4-methylcatechol as substrate, an average value of 690 pmole O,/min per mg N. The protein content of all samples was estimated by the micromethod described by MATTENHEIMER (1960). Lipid extractions of isolated integuments were made after the method of JENSEN et al. (1951). The phosphatide fractions of these extracts were precipitated with acetone and MgCl, (MACLACHLAN, 1944). After centrifugation, the clear supernatant was concentrated under reduced pressure in an atmosphere of nitrogen. For characterization of the lipid components in the extracts thin-layer chromatography with the following solvent systems was used: Solvent 1: light petroleum (b.p. 60-80”C)-ether-acetic acid (70 : 30 : 2). Solvent 2 : acetic acid-acetonitrile (1 : 1). Silica Gel G (Merck) was the adsorbent. Lipid substances were detected by spraying with either 10% phosphomolybdic acid in ethanol or a O*OSo/osolution of Rhodamine B. Gas chromatographic analyses were carried out with the Argon Chromatograph. Methylesters of the isolated fatty acids were made following the micromethod of DE FRANCESCO (1962). The histochemical localization of lipase in dissected animals was demonstrated with the classical or-naphthyl acetate method (PEARSE, 1960) and the more specific Tween method (GOMORI, 1952). Preparation
of larval homogenate and particulate
fractions
The larvae were chilled and ground in a mortar with a known volume of cold 0.15 M NaCl solution. The brei was filtered through four layers of cheese cloth and centrifuged during 20 min at 15,000g. Three layers were obtained: an upper Samples layer, a precipitate, and a clear supernatant containing the latent enzyme. of these fractions were tested for their activating capacity on purified phenolase. Acetone powders for the preparation of an eventual proteinaceous activator were made according to the method of KARLSON and SCHMID (1955). The dried powder was extracted with O-067 M phosphate buffer, pH 7.0, containing O-20/, ascorbic acid. The homogenate was centrifuged and the supernatant chromatographed on a Sephadex G-25 column. Finally, traces of the active phenolase were removed by ultracentrifugation at 120,OOOg for 90 min.
LIPIDACTIVATOR OFPROPHENOLASE IN TENEBRIO
MOLZTOR
411
RESULTS
As already reported by ARRTS and VERCAUTEREN (1964), the proenzymecontaining supernatant of a centrifuged homogenate can be stabilized by gel filtration on Sephadex G-25. The whole bulk of soluble larval proteins coming off the Sephadex G-25 column can be stored for several weeks at 4°C without conversion of the proenzyme into an active phenolase. No activating action on the latent enzyme was detected after incubation with the separated low molecular material ; nor with the lipid upper layer obtained after centrifugation of the homogenate. Only with the resuspended precipitate was a considerable activation of the proenzyme obtained. In order to solubilize an eventual activator protein, the precipitate was treated with the butanol-NaHCO, solution of MORTON(1953), and the extract tested as an activator. Values for phenolase activity after incubation of the latent enzyme with these different fractions are summarized in Table 1. TABLE~-RELEASED
PHENOLASE ACTIVITY AFTER INCUBATION OF PROPHENOLASE SEVEFWLEXTRACTS TESTED AS POSSIBLE ACTIVATORS
Activator 0.5 ml suspended precipitate O-5 ml precipitate after 30 min at 100°C 0.5 ml butanol extract 1 /Imole Na-oleate
WITH
Activity in &mole O.Jmin per ml) 2.06 1.72 0 4.25
These results clearly show that the precipitate obtained after centrifugation of a larval homogenate contains a factor able to activate the proenzyme. This factor, which could not be solubilized, was found to be thermostable. Other attempts to demonstrate an eventual proteinaceous activator of prophenolase in Tenebrio molitor were made by incubating a purified phenolase sample with various volumes of an acetone powder extract (KARLSONand SCHMID, 1955). No activation of the latent enzyme could be detected. The minimal uptake of oxygen after a few hours of incubation was easily shown to be due to small traces of active phenolase present in the acetone powder extract. Only after 24 hr of incubation was an increase of the phenolase activity observed. As these mixtures turned slowly brownish, probably the tyrosyl residues of the latent enzyme were oxidized by the noted traces of active enzyme (YASUNOBU et al., 1959). As already noticed, after storage of a partly inactive phenolase, this effect seems to benefit the conversion of the latent enzyme into an active enzyme. After microdissection of Tenebrio larvae and careful isolation of the integuments with the adherent subepidermal fat, it could be demonstrated that the previously mentioned thermostable activator was present in a homogenate of larval integuments. Lipid extraction procedure was carried out as already described, and the
412
K. A. HEYNEMAN AND R. E. VERCAUTEREN
concentrated extract was subjected to thin-layer chromatography. When the plate was developed, using solvent 1, several spots could be recognized after spraying with phosphomolybdic acid reagent (Fig. 1). The spots were scraped off, extracted in ether, air dried, resuspended in 0.1 M phosphate buffer, pH 7, and tested as possible activators for the latent enzyme. Two spots, 2 and 5, gave a reaction in the presence of proenzyme and substrate. The previously isolated phosphatide fraction did not have any activating action on the proenzyme sample. In order to obtain a larger recovery of separated material, the lipid extract was chromatographed on a Silica Gel H column and eluted with light petroleum etherether-acetic acid 90 : 10 : 1. Fig. 2 represents the elution curve measured at 254 mp. When peaks III and VI were transferred to a buffer system and assayed,
70
1
0
60
50
0
0
0
40 30 20 I 0 0
6
c
i
k
FIG. 1. Thin-layer chromatogram of a lipid extract of isolated integuments from Terrebrio larvae developed with 10% phosphomolybdic acid in ethanol. Solvent: light petroleum-ether-acetic acid (70 : 30 : 2). a: Lipid extract; b : oleic acid; c: phosphatide fraction ; d : cholesterol; e : linoleic acid.
Effluent,
mC
FE. 2. Elution curve after column chromatography on Silica Gel H of a lipid The cross-hatched peaks represent extract of isolated larval integuments. fractions with activating capacity on latent phenolase.
LIPID ACTIVATOROF PROPHRNOLASEIN TENEBRIO
413
MOLITOR
they showed a high activating capacity. Whereas, after thin-layer chromatography (solvent 2), peak III could be recognized as consisting of free fatty acids; the chemical nature of peak VI is still unknown. After methyl esterification the material of peak III was injected in a gas chromatographic column and the fractions were automatically recorded. As shown in Table 2, most of the fatty acids could be identified. TABLE Z-COMPOSITION
OF THE FATTY
EXTRACTBASED ON GAS CHROMATOGRAPHIC ANALYSIS
ACID
Palmitic acid Branched fatty acid Stearic acid Oleic acid Linoleic acid Linolenic acid Branched fatty acid Unsaturated fatty acid Unsaturated fatty acid Behenic acid
C-16 C-18 C-18:0 C-181 C-182 C-l 83 c-20 c-20 c-20 c-22
After incubation of the proenzyme with equimolecular concentrations of some natural occurring fatty acids, the resulting enzymic activity showed a maximum in the presence of C-18 fatty acids (except linolenic acid) (Fig. 3). > s IOr
Fo tty FIG. 3.
Activating
acids
influence of higher fatty acids at equimolar (1 x 10-a M) on latent phenolase.
concentrations
In order to find out a possible mechanism for the release of fatty acids in situ, we have submitted the isolated integuments and tracheae to histochemical tests for lipase activity. The ar-naphthyl acetate staining gave a strong positive reaction for
414
R. A. HIXYNEMAN ANDR. E. VERCAUTERFN
the tracheal oenocytes and the subcuticular fat. Using the more specific Tween method after GOMORI (1952), lipase activity in both tissues could be affirmed. The small oenocytes of the subepidermis (WIGGLESWORTH, 1948) did not contain any lipase activity. Staining for free fatty acids (without Tween 60) gave a positive reaction for the large oenocytes in the neighbourhood of the spiracles, whereas the subcuticular fat was only weakly coloured. DISCUSSION The presence of phenolase in a special type of blood cell, called an oenocytoid, has been proved by several authors (DENNELL, 1947; DECLEIR et al., 1960). It is undoubtedly this same type of cell which JONES (1949) describes in the larva of Sarcophaga bullata under the name of ‘spherule cell’. Early in the moulting process the oenocytoids increase greatly in number and size. During the period of cuticle formation, the oenocytoids are broken down and ingested by amoebocytes (WIGGLESWORTH, 1965). Exactly the same process was described by POYARKOFF (1910) with the oenocytoids of Galerucella. It is quite probable that the liberated phenolase diffuses into the cuticle (DENNELL, 1958). Activation of the proenzyme may take place by contact with the fatty acids present and set free by lipase activity or by the unknown lipoid activator mentioned above, probably occurring in the cuticle itself. After activation, the enzyme becomes insoluble after a few hours so that diffusion of a fatty-acid-activated phenolase through the new cuticle towards the epicuticle is still possible. As no protein activator for the latent phenolase could be detected in larval Tenebrio homogenates, it would be interesting to follow the effect of the proteinaceous activator of other species on our purified proenzyme. Further investigation on the activation mechanism and a comparative physical and chemical study of the prophenolase and active phenolase would also be profitable. REFERENCES F. and VERCAUTEREN R. (1964) Specificity and mode of action of phenoloxidase from larvae of Tenebrio molitor. Enzymologia 28, l-20. BODINEJ. H. and ALLEN T. H. (1938) Further studies on the activation of the enzyme
AERTS
tyrosinase. J. cell. [email protected]. 11, 409-415. BODINEJ. H. and ALLEN T. H. (1941) Some properties of protyrosinase. J. cell. camp. Physiol. 18, 151-156. DECLEIRW., AERTS F., and VERCAUTJXREN R. (1960) The localization of polyphenoloxidase in hemocytes. Proc. 11th int. Congo. Ent. (B) 3, 176-179. DE FRANCESCOF. (1962) Sulla transesterificazione stechiometrica dei gliceridi mediante alcool met&o. Riv. ital. Sod. GY. 5, 245-252. DENNELL R. (1947) The occurrence and significance of phenolic hardening in the newly formed cuticle of crustacea decapoda. Proc. R. Sot. B, 134,485503. DENNELL R. (1958) The hardening of insect cuticles. Biol. Rev. 33, 178-186. GEIGERH. and MITCHELL H. (1966) Salivary gland function in phenoloxidase production in Drosophila. J. Insect Physiol. 12, 747-754. GOMORIG. (1952) Microscopic Histochemistry, pp. 203-206. University Press, Chicago. HEYNEMANR. A. (1965) Final purification of a latent phenolase with mono- and diphenolase activity from Tenebrio molitor. Biochem. biophys. Res. Cmnm. 21, 162-169.
LIPID ACTIVATOR OF PROPHENOLASE IN
TENEBRIO
MOLITOR
415
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