Comp. Biochem. Physiol.Vol. 102B,No. 1, pp. 187-191, 1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
THE SEARCH FOR EFFECTORS OF fl-D-GLUCANASES A M O N G MARINE INVERTEBRATES T. N. ZVYAGINTSEVA,L. A. ELYAKOVAand V. B. KRASOHIN Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, the Laboratory of Enzymic Chemistry, Vladivostok 690022, Republic of Russia (Received 23 July 1991) Abstract--1. The search for inhibitors and activators of endo-1--,3- (EC 3.2.1.6) and l--*6-fl-D-glucanases (EC 3.2.1) from marine mollusks in aqueous and ethanol extracts of various types of marine invertebrates, such as sponges, ascidians, coelenterates, echinoderms, dwelling in north seas (the sea of Okhotsk and the sea of Japan) and torrid seas (western coast of Australia) has been made. 2. A comparative analysis has shown that the overwhelming majority of extracts of animals, dwelling near Australia, inhibit fl-D-giucanases. 3. On the contrary, the extracts of animals dwelling in the sea of Okhotsk possess the activating effect, except for sponges of genera Haliclona whose sample extracts display a significant activating effect independently of their place of abode.
INTRODUCTION
Standard testing procedure for the extract's effect on activity of carbohydrases The tests were performed using the solutions of substances in the following concentrations: endo-1--*3- and -1--*6-~-D-glucanases, 1 U/ml, I U = 1 pmol Glc/min; pustulan, 4 mg/ml; laminaran, 5 mg/ml; dried extracts, 4 mg/ml. The mixture, containing 0.05 ml of the extract solution, 0.1 ml of the acetate buffer (0.2 M, pH 5.5) and 5/~1 of LIV or Po was incubated for 15 min, then 0.05 ml of laminaran or pustulan solution, respectively, was added. The mixture was kept for 20 min and the enzymic activities were estimated according to Nelson (1944). The mixture containing no enzymes served as control. The results are represented in Tables 1-3. The enzymic reaction rate in the absence of the effectors was taken for 100%.
The search for the effector sources (inhibitors and activators) of /~-D-glucanases is of great interest because of the practical and scientific importance of both enzymes and effectors. M a n y specific inhibitors of carbohydrases are toxins (Buonocore et al., 1977; Tulsiani et al., 1985). Some of them, in particular amylase inhibitors, are applied in medicine in the treatment of tooth caries, allergy and adiposity (Yoshikuni et al., 1989; Kakegawa et al., 1983). et-Glucosidase inhibitors were established to impede the development of the AIDS virus (Gruters et al., 1987). No information concerning the effectors of 1--,3- and l~6-fl-D-glucanases is available in the literature except for our papers, concerning some inhibitors of endo-1--,3-fl-o-glucanases (Zvyagintseva et al., 1986; Sova et al., 1988).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Enzymes Endo-l--,3-fl-D-giucanase LIV and endo-l-+6-fl-o-giucanase Po were obtained from crystalline styles of mollusks Spisula sachalinensis and Chlamys albidus (Sova et al., 1970; Rudakova et al., 1985), respectively. Substrates Laminaran was obtained from the brown algae Laminaria cichorioides according to the method of Elyakova and Zvyagintseva (1974). Pustulan-l-+6-fl-D-giucan was obtained from Umbillicaria rossica according to the method of Zvyagintseva et al. (1986). Effectors The invertebrates were collected by dredging and SCUBA in the cruises of the research vessel "Aeademik Oparin". Freshly collected animals were completely homogenized in water or ethanol (the weight ratios were 1:1 and 1:5, respectively). Homogenates were subjected to centrifugation, supernatants were dried, and their effect on enzymes was tested.
Ethanol and water, respectively, were employed to extract different substances. We have studied the effect of aqueous and alcohol extracts of animals collected in the sea of Okhotsk and the sea of Japan and in the Indian Ocean near the western coast of Australia on the activity of fl-D-glucanases from marine mollusks: endo-l-~3-//-Dglucanase (LIV from Spisula sachalinensis) and endo1 -,6-fl-D-glucanase (Po from Chlamys albidus). Two hundred and twenty-seven ethanol and 43 aqueous extracts of the animals, residing in the Indian Ocean (Australia) and belonging to various types were tested for the presence of effectors of endo-l-~3-fl-D-glucanase LIV. Ethanol extracts were obtained from different species of sponges (133 samples), ascidians (20 samples), coelenterates: alcyonarians (41), jelly fish (4), gorgonians (6), sea pens (1), actinians (3), hydroid polyps (1), echinoderms: sea lilies (7), holothurians (1), sea stars (1), sea mosses (1) and algae (15). Aqueous extracts were isolated from sponges (32 samples), ascidian (10), coelenterates: alcyonarians (1) (Table 1). Seventy ethanol extracts of animals of the Indian Ocean from sponges (51), ascidian (5), coelenterates:
187
188
T . N . ZVYAGINTSEVAet al.
alcyonarians (3), jelly fish (1), gorgonians (1), echionderms: sea lilies (2), holothurians (1), sea mosses (5) and sea worms (1) were tested for the presence of effectors of endo-1 ~6-fl-D-glucanase Po (Table 1). The effect of animal extracts of the sea of Okhotsk was only tested for LIV activity. Alcohol extracts were isolated from 80 animals, including sponges (36), coelenterates: actinians (5), alcyonarians (3), gorgonians (2), sea pens (1), hydroid polyps (3), echinoderms: sea stars (10), sea mosses (6), sea urchins (2), sea worms (1) and mollusks (11). Among the extracts of animals of the torrid seas, sponges display the most pronounced inhibitory effect on the fl-D-glucanases tested (75 samples out of 133). In this aspect the family Callyspongiidae is the most interesting; all eight samples of the family studied showed an inhibitory effect on fl-D-glucanases. In this case selective inhibition of enzymes is probably due to the action of water soluble low mol. wt compounds as both types of extract (aqueous and alcohol) possess a similar inhibitory effect. Sample No. 25, whose profound inhibitory effect can be found only in aqueous extract, is an exception. More profound inhibitory effect of extracts on 1--*6-fl-t~-
glucanase Po may be noted in comparison with LIV. Four more sponge species, belonging to the families Spirastrellidae and Clathriidae, have the ability for selective inhibition of Po activity. Families Haliclonidae and Spirastrellidae (both are represented by eight species in our work) contain, in contradistinction to Callyspongiidae, practically no inhibitors of endo- 1 --, 3-fl-D-glucanase LIV. A considerable number of alcyonarian species (10 out of 41 tested) inhibited endo-I ~3-/LD-glucanase (see Table 1). These species mainly belong to the genera Sinularia (four out of 14) and Sarcophyton (four out of seven). Almost all Bryozoa samples and several ascidian aqueous extracts tested displayed a low inhibitory effect (25-40%) on fl-D-glucanases. Activity of LIV rises 1.5-2-fold under the action of sponge and alcyonarian alcohol extracts (Table 2). Among 227 samples studied the activating effect was displayed only by the extracts from 25 species of Australian animals while more than 50 extracts showed considerable inhibitory effect (not counting those which showed a low inhibitory effect--less than
50%).
Table 1. The effect of aqueous (1) and ethanol (2) extracts of marine invertebrates on the activity of LIV and Po The activity of enzymes (%) No.
1 2 3 4 5 6 7 8 9 10
11 12 13 14
15 16 17
18 19 20
21
Type, Order, Family Phylum Porifera Order Astrophorida Family Geodiidae Geodinella spherastrosa Family Theneidae Thenea wrighti Order Lithistida Family Theonellidae Theonella swinhoei Order Handromerida Family Spirastrellidae Spirastrella digitata 1 Spirastrella vagabunda 1 Spirastrella vagabunda 1 Family Chondrosiidae Chondrilla australiensis Order Axinellida Family Axinellidae Phakelia cardius Family Desmoxyidae Desmoxya lunata Order Poecilosclerida Family Mycalidae Mycale monanchorata Family Clathriidae Clathria frondifera Clathria lendenfeldi Family Crellidae Crella incrustans 1 Crella incrustans 2 Family Myxillidae Iotrochota baculifera lotrochota coccinea Acarnus thielei Family Raspailidae Echinodictyum mesenterenum Echinodictyum cancellatum Family Phorbasidae Phorbas sp. Order Halichondrida Family Hymehiacidonidae Hymeniacidon conglomerata
LIV (1)
LIV (2)
Po (2)
ND*
60
5
ND
20
ND
ND
15
ND
50 100
23 100
I O0
1O0
10 7
55
25
0
23
30
0
50
38
0
ND
30
30
0
1O0
1O0
15
100
100
17
ND ND
30 15
ND ND
55 ND ND
30 15 20
20 ND ND
ND ND
25 15
ND ND
45
20
0
ND
20
ND
continued
189
]/-D-Glucanases
Table 1---cont. The activity of enzymes (%) No.
Type, Order, Family
LIV (1)
LIV (2)
Po (2)
22
Ulosa sp. Order Haplosclerida Family Callyspongiidae Callyspongia diffusa Callyspongia diffusa Callyspongia sp. Callyspongia subarmigera Callyspongia confoederata Arenosclera sp. 1 Arenosclera sp. 1 Arenosclera sp. 1 Family Niphatidae Amphimedon sp. 1 Arnphimedon sp. 1 Amphimedon sp. 2 Family Oceanapiidae Oceanapia imperfecta Family Petrosiidae Petrosia similis Family Haliclonidae Sigmadocia symbiotica Order Dictyoceratida Family Thorectidae Dysidea herbacea Dysidea fragilis Thorecta cacos Ircinia sp. Cacospongia ridleyi Family Spongiidae Spongia officinalis Hyatella intestinalis Hyatella intestinalis Order Verongida Family lanthellidae Ianthella flabelliformis Phylum Coelenterata Order Alcyonacea Family Alcyonidae Sarcophyton sp. 1 Sarcophyton sp. 2 Sarcophyton sp. 3 Sarcophyton sp. 4 Sinularia sp. I Sinularia sp. 2 Sinularia sp. 3 Sinularia sp. 4 Lemnalia sp. Clavularia sp.
ND
20
ND
20 55 15 ND ND 20 80 50
30 50 100 60 30 20 40 40
0 0 50 60 ND 0 0 20
45 45
20 15
100
100
0 0 10
ND
60
20
ND
10
ND
ND
50
0
23 24 25 26 27 28 29 30 31 32 33 34 35 36
37 38 39 40 41 42 43 44
45
46 47 48 49 50 51 52 53 54 55
53
30
100
100
ND ND ND
25 15 10
0 15 0 15 ND
ND ND ND
5 25 30
ND ND ND
ND
10
ND
ND ND ND ND ND ND ND ND ND ND
30 25 40 40 20 20 15 5 35 30
ND ND ND ND ND ND ND ND ND ND
*Not detected.
A contrary picture was noted for extracts of animals from the sea of Okhotsk. Only two alcohol extracts (Strongylocentrotus sp., Hippasteria leiopelta) out of 80 animals from the sea of Okhotsk showed an inhibitory effect while the other extracts displayed themselves as LIV activators. Thirty-four extracts activated LIV more than 1.5 times including 19 extracts, which activated LIV more than 2.5-3 times (Table 3). It is interesting to note the action of extracts from the family Haliclonidae dwelling in the Indian Ocean and the sea of Okhotsk. No one species among eight torrid sponge species of this family inhibited LIV, but two of them contained some activators (Table 2). Among animal species of the sea of Okhotsk having the best activating effect (Table 3) species of the family Haliclonidae were also noted. Thus, during our study we have found possible sources of activators and inhibitors of fl-glucanases. Some of them have a high specificity for endo-I--*6-
~-D-glucanase. The discovery of these enzymes' sources of activation, which have not been described yet, seems to be most important. The nature of the activating effect of the extracts and the structure of substances which are responsible for them have not yet been elucidated, though some analogies suggest themselves. There are some indications concerning the activating effect exercised by some lipids on enzymes. For example, there is a description of fl-l,2-dioleyldiglyceride, a natural activator of a vegetative origin, containing //-bound residue (or residues of glucose) in the C-3 position (Ross et al., 1988), affecting ~-glucansynthase (membrane enzyme). Fatty acid compositions of lipids of marine invertebrates, living in the north and torrid seas, respectively, are known to considerably differ from each other, which is not surprising as this difference accounts for their thermostability and possibly causes the activating effect of the invertebrate extracts of the north seas.
190
T . N . ZVYAGINTSEVAet al.
Table 2. The activating effect of alcohol extracts of marine invertebrates of the Indian Ocean on the activity of endo-1--*3-/~-D-glucanase LIV No.
Type, Order, Family
Geodia globostellifera Geodinella spherastrosa
150 155
Order Axinellida Family Axinellidae 3 4
Phakellia cactoides Reniochalina stalagmitis
160
Biemna liposigma
6 7
Clathria dura Clathria frondifera
8
Ecinodictyum clathroides
9
Desmacidon arenosa
10
Latrunculia conulosa
1 2 3 4 5 6 7 8
150
11
12 13
14 15 16
17 18 19 20 21 22 23 24 25
Dampia pocillaporaeformis Dendronephthya sp. Sarcophyton sp. 1 Sarcophyton sp. 2 Sinularia sp. 1 Sinularia sp. 2 Sinularia sp. 3 Sinularia sp. 4 Stereonephthya sp. 1 Stereonephthya sp. 2
200
Polymastia kurilensis Polymastia robusta Halichondria panicea Asbestopluma lycopodium Mycale lobata Myxichella fragilis
9
Gellius primitivus Gellius varius
Haliclona gracilis
220 155
12
Haliclona ventilabrum
180
13
150 180 165 180 200 190 190 180 190 150
Acknowledgements--We wish to thank Dr John N. A.
Hooper and Dr Philip Alderslade (Northern Territory Museum Science and Arts, Darwin, Australia) for collecting and identifying some of the marine animals.
220 250 250 260
Order Haplosclerida Family Halielonidae 10 11
215 170
250 260
Family Myxillidae
Family Latrunculidae Order Haplosclerida Family Haliclonidae Haliclona sp. 1 Haliclona sp. 2 Order Dictyoceratida Family Spongiidae Spongia sp. Family Thorectidae Ircinia sp. 1 lrcinia sp. 2 Phylum Coelenterata Order Alcyonacea
300
Family Mycalidae
160
Family Desmacididae
350
Order Poecilosclerida Family Cladorhisidae
Family Clathriidae Family Raspailidae
Tetilla hamatum Tetilla sibirica
LIV activity (%)
Order Halichondrida Family Halichondriidae
165 210 150
Type, Order, Family Phylum Porifera Order Spirophorida Family Tetillidae Order Hadromerida Family Polymastiidae
150
Order Poecilosclerida Family Biemnidae 5
No.
LIV activity (%)
Phylum Porifera Order Astrophorida Family Geodiidae I 2
Table 3. Activating effect of alcohol extracts of marine invertebrates of the sea of Okhotsk on endo-(1--*3)-fl-D-glucanas¢LIV activity
260
220 270 250
Phylum Goelenterata Order Pennatularia Family Virgularidae Pavonaria finmarchica
250
Order Actiniaria Family Metridiidae 14 15 16
17
Metridium dianthus
Phylum Mollusca Order Opistobranchia Opistobranchia gen. sp. Family Tritonidae Tritonia primoriensis
Order Hamiglossa Family Buccinidae Buccinum sp. Phylum Tentaculata Order Ctenostomata Family Vesiculariidae
260 260 250 260
17
Bonerbankia composita
280
18
Phylum Echinodermata Order Forcipulata Family Asteriidae Lepthasterias sp. Phylum Chordata Order Stolidobranchia Family Pyuridae
250
19
Halocynthia aurantium
250
REFERENCES
Buonocore V., Petrucci T. and Silano V. (1977) Wheat protein inhibitors of ~-amylase. Phytochemistry 16, 811-820. Elyakova L. A. and Zvyagintseva T. N. (1974) A study of the laminarins of some far-eastern brown seaweeds. Carbohydr. Res. 34, 241-248. Gruters R. A., Neefjes J. J., Termette M., Goede R. E. V., Tulp A., Guisman H. G., Miedema F. and Ploegh H. L. (1987) Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase. Nature 330, 74-77. Kakegawa H., Mitsuo N., Matsumoto H., Satch T., Akagi M. and Tasaka K. (1983) Hyaluronidase-inhibitory and anti-allergic activities of the photo-irradiated products of tranilast. Chem. Pharm. Bull. 33, 3738-3744. Nelson N. (1944) A photometric adaptation of the Somogyi method of determination of glucose. J. biol. Chem. 153, 375-381. Ross P., Callaghar T., Weinberg-Ohana P., Garden G. and Benziman M. (1988) Glucoside activators o f mung bean UDP-glucose: fl-glucan synthase. 1. Identification of an
endogenous fl-linked glucolipid activator. Plant Physiol. 86, 1099-1103. Rudakova V. Y., Shevchenko N. M. and Elyakova L. A. (1985) Isolation and some properties of endo-/L1,6-glucanase from marine bivalve Chlamys albidus. Comp. Biochem. Physiol. 81B, 677~582. Sova V~ V., Elyakova L. A. and Vaskovsky V. E. (1970) Purification and some properties of fl-l,3-glucanhydrolase from the crystalline style of Bivalvia Sp. sachalinensis. Biochim. biophys. Acta 212, 212-219. Sova V, V., Svetasheva T. G. and Elyakova L. A. (1988) Natural inhibitors of fl-l,3-glucanases. High molecular weight inhibitor from the torrid sponge Myrmekioderma granulata. Khim. prirodn, soedin. No. 4, 566-572 (in Russian). Tulsiani D. R. T., Broquist H. P. and Touster O. (1985) Marked difference in the swaisonine inhibition of rat liver liposomal ct-D-mannosidase and rat liver goldgi mannosidase II. Archs Biochem. Biophys. 236, 427-434. Yoshikuni Y., Ezure Y., Sefo T., Mori K., Watanab¢ M. and Enomoto H. (1989) Synthesis and ~t-glucoside-
B-D-Glucanases inhibiting activity of a new ~,-glucosidase inhibitor, 4-Oc,-v-glucopyranosyl-maraline and its N-substituted derivatives. Chem. Pharm. Bull. 37, 106-109. Zvyagintscva T. N., Makarieva T. N., Stonik V. A. and Elyakova L. A. (1986) Sulphated steroids of sponges of Halichondriidae family--the natural inhibitors of endo-
191
l--,3-~-D-glucanascs. Kh/m. priod, soedin. No. 1, 71-76 (in Russian). Zvyagintscva T. N., Sundukova E. V., Mischenko N. P., Krivoschekova O. E. and Elyakova L. A. (I 986) The method of pustulanisolation from lichens. A.C. 1227189(USSR) Int. CP. A61K35/78. Bull. Otkr. Isobret. V, 16 (in Russian).
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
THE SEARCH FOR EFFECTORS OF fl-D-GLUCANASES A M O N G MARINE INVERTEBRATES T. N. ZVYAGINTSEVA,L. A. ELYAKOVAand V. B. KRASOHIN Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, the Laboratory of Enzymic Chemistry, Vladivostok 690022, Republic of Russia (Received 23 July 1991) Abstract--1. The search for inhibitors and activators of endo-1--,3- (EC 3.2.1.6) and l--*6-fl-D-glucanases (EC 3.2.1) from marine mollusks in aqueous and ethanol extracts of various types of marine invertebrates, such as sponges, ascidians, coelenterates, echinoderms, dwelling in north seas (the sea of Okhotsk and the sea of Japan) and torrid seas (western coast of Australia) has been made. 2. A comparative analysis has shown that the overwhelming majority of extracts of animals, dwelling near Australia, inhibit fl-D-giucanases. 3. On the contrary, the extracts of animals dwelling in the sea of Okhotsk possess the activating effect, except for sponges of genera Haliclona whose sample extracts display a significant activating effect independently of their place of abode.
INTRODUCTION
Standard testing procedure for the extract's effect on activity of carbohydrases The tests were performed using the solutions of substances in the following concentrations: endo-1--*3- and -1--*6-~-D-glucanases, 1 U/ml, I U = 1 pmol Glc/min; pustulan, 4 mg/ml; laminaran, 5 mg/ml; dried extracts, 4 mg/ml. The mixture, containing 0.05 ml of the extract solution, 0.1 ml of the acetate buffer (0.2 M, pH 5.5) and 5/~1 of LIV or Po was incubated for 15 min, then 0.05 ml of laminaran or pustulan solution, respectively, was added. The mixture was kept for 20 min and the enzymic activities were estimated according to Nelson (1944). The mixture containing no enzymes served as control. The results are represented in Tables 1-3. The enzymic reaction rate in the absence of the effectors was taken for 100%.
The search for the effector sources (inhibitors and activators) of /~-D-glucanases is of great interest because of the practical and scientific importance of both enzymes and effectors. M a n y specific inhibitors of carbohydrases are toxins (Buonocore et al., 1977; Tulsiani et al., 1985). Some of them, in particular amylase inhibitors, are applied in medicine in the treatment of tooth caries, allergy and adiposity (Yoshikuni et al., 1989; Kakegawa et al., 1983). et-Glucosidase inhibitors were established to impede the development of the AIDS virus (Gruters et al., 1987). No information concerning the effectors of 1--,3- and l~6-fl-D-glucanases is available in the literature except for our papers, concerning some inhibitors of endo-1--,3-fl-o-glucanases (Zvyagintseva et al., 1986; Sova et al., 1988).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Enzymes Endo-l--,3-fl-D-giucanase LIV and endo-l-+6-fl-o-giucanase Po were obtained from crystalline styles of mollusks Spisula sachalinensis and Chlamys albidus (Sova et al., 1970; Rudakova et al., 1985), respectively. Substrates Laminaran was obtained from the brown algae Laminaria cichorioides according to the method of Elyakova and Zvyagintseva (1974). Pustulan-l-+6-fl-D-giucan was obtained from Umbillicaria rossica according to the method of Zvyagintseva et al. (1986). Effectors The invertebrates were collected by dredging and SCUBA in the cruises of the research vessel "Aeademik Oparin". Freshly collected animals were completely homogenized in water or ethanol (the weight ratios were 1:1 and 1:5, respectively). Homogenates were subjected to centrifugation, supernatants were dried, and their effect on enzymes was tested.
Ethanol and water, respectively, were employed to extract different substances. We have studied the effect of aqueous and alcohol extracts of animals collected in the sea of Okhotsk and the sea of Japan and in the Indian Ocean near the western coast of Australia on the activity of fl-D-glucanases from marine mollusks: endo-l-~3-//-Dglucanase (LIV from Spisula sachalinensis) and endo1 -,6-fl-D-glucanase (Po from Chlamys albidus). Two hundred and twenty-seven ethanol and 43 aqueous extracts of the animals, residing in the Indian Ocean (Australia) and belonging to various types were tested for the presence of effectors of endo-l-~3-fl-D-glucanase LIV. Ethanol extracts were obtained from different species of sponges (133 samples), ascidians (20 samples), coelenterates: alcyonarians (41), jelly fish (4), gorgonians (6), sea pens (1), actinians (3), hydroid polyps (1), echinoderms: sea lilies (7), holothurians (1), sea stars (1), sea mosses (1) and algae (15). Aqueous extracts were isolated from sponges (32 samples), ascidian (10), coelenterates: alcyonarians (1) (Table 1). Seventy ethanol extracts of animals of the Indian Ocean from sponges (51), ascidian (5), coelenterates:
187
188
T . N . ZVYAGINTSEVAet al.
alcyonarians (3), jelly fish (1), gorgonians (1), echionderms: sea lilies (2), holothurians (1), sea mosses (5) and sea worms (1) were tested for the presence of effectors of endo-1 ~6-fl-D-glucanase Po (Table 1). The effect of animal extracts of the sea of Okhotsk was only tested for LIV activity. Alcohol extracts were isolated from 80 animals, including sponges (36), coelenterates: actinians (5), alcyonarians (3), gorgonians (2), sea pens (1), hydroid polyps (3), echinoderms: sea stars (10), sea mosses (6), sea urchins (2), sea worms (1) and mollusks (11). Among the extracts of animals of the torrid seas, sponges display the most pronounced inhibitory effect on the fl-D-glucanases tested (75 samples out of 133). In this aspect the family Callyspongiidae is the most interesting; all eight samples of the family studied showed an inhibitory effect on fl-D-glucanases. In this case selective inhibition of enzymes is probably due to the action of water soluble low mol. wt compounds as both types of extract (aqueous and alcohol) possess a similar inhibitory effect. Sample No. 25, whose profound inhibitory effect can be found only in aqueous extract, is an exception. More profound inhibitory effect of extracts on 1--*6-fl-t~-
glucanase Po may be noted in comparison with LIV. Four more sponge species, belonging to the families Spirastrellidae and Clathriidae, have the ability for selective inhibition of Po activity. Families Haliclonidae and Spirastrellidae (both are represented by eight species in our work) contain, in contradistinction to Callyspongiidae, practically no inhibitors of endo- 1 --, 3-fl-D-glucanase LIV. A considerable number of alcyonarian species (10 out of 41 tested) inhibited endo-I ~3-/LD-glucanase (see Table 1). These species mainly belong to the genera Sinularia (four out of 14) and Sarcophyton (four out of seven). Almost all Bryozoa samples and several ascidian aqueous extracts tested displayed a low inhibitory effect (25-40%) on fl-D-glucanases. Activity of LIV rises 1.5-2-fold under the action of sponge and alcyonarian alcohol extracts (Table 2). Among 227 samples studied the activating effect was displayed only by the extracts from 25 species of Australian animals while more than 50 extracts showed considerable inhibitory effect (not counting those which showed a low inhibitory effect--less than
50%).
Table 1. The effect of aqueous (1) and ethanol (2) extracts of marine invertebrates on the activity of LIV and Po The activity of enzymes (%) No.
1 2 3 4 5 6 7 8 9 10
11 12 13 14
15 16 17
18 19 20
21
Type, Order, Family Phylum Porifera Order Astrophorida Family Geodiidae Geodinella spherastrosa Family Theneidae Thenea wrighti Order Lithistida Family Theonellidae Theonella swinhoei Order Handromerida Family Spirastrellidae Spirastrella digitata 1 Spirastrella vagabunda 1 Spirastrella vagabunda 1 Family Chondrosiidae Chondrilla australiensis Order Axinellida Family Axinellidae Phakelia cardius Family Desmoxyidae Desmoxya lunata Order Poecilosclerida Family Mycalidae Mycale monanchorata Family Clathriidae Clathria frondifera Clathria lendenfeldi Family Crellidae Crella incrustans 1 Crella incrustans 2 Family Myxillidae Iotrochota baculifera lotrochota coccinea Acarnus thielei Family Raspailidae Echinodictyum mesenterenum Echinodictyum cancellatum Family Phorbasidae Phorbas sp. Order Halichondrida Family Hymehiacidonidae Hymeniacidon conglomerata
LIV (1)
LIV (2)
Po (2)
ND*
60
5
ND
20
ND
ND
15
ND
50 100
23 100
I O0
1O0
10 7
55
25
0
23
30
0
50
38
0
ND
30
30
0
1O0
1O0
15
100
100
17
ND ND
30 15
ND ND
55 ND ND
30 15 20
20 ND ND
ND ND
25 15
ND ND
45
20
0
ND
20
ND
continued
189
]/-D-Glucanases
Table 1---cont. The activity of enzymes (%) No.
Type, Order, Family
LIV (1)
LIV (2)
Po (2)
22
Ulosa sp. Order Haplosclerida Family Callyspongiidae Callyspongia diffusa Callyspongia diffusa Callyspongia sp. Callyspongia subarmigera Callyspongia confoederata Arenosclera sp. 1 Arenosclera sp. 1 Arenosclera sp. 1 Family Niphatidae Amphimedon sp. 1 Arnphimedon sp. 1 Amphimedon sp. 2 Family Oceanapiidae Oceanapia imperfecta Family Petrosiidae Petrosia similis Family Haliclonidae Sigmadocia symbiotica Order Dictyoceratida Family Thorectidae Dysidea herbacea Dysidea fragilis Thorecta cacos Ircinia sp. Cacospongia ridleyi Family Spongiidae Spongia officinalis Hyatella intestinalis Hyatella intestinalis Order Verongida Family lanthellidae Ianthella flabelliformis Phylum Coelenterata Order Alcyonacea Family Alcyonidae Sarcophyton sp. 1 Sarcophyton sp. 2 Sarcophyton sp. 3 Sarcophyton sp. 4 Sinularia sp. I Sinularia sp. 2 Sinularia sp. 3 Sinularia sp. 4 Lemnalia sp. Clavularia sp.
ND
20
ND
20 55 15 ND ND 20 80 50
30 50 100 60 30 20 40 40
0 0 50 60 ND 0 0 20
45 45
20 15
100
100
0 0 10
ND
60
20
ND
10
ND
ND
50
0
23 24 25 26 27 28 29 30 31 32 33 34 35 36
37 38 39 40 41 42 43 44
45
46 47 48 49 50 51 52 53 54 55
53
30
100
100
ND ND ND
25 15 10
0 15 0 15 ND
ND ND ND
5 25 30
ND ND ND
ND
10
ND
ND ND ND ND ND ND ND ND ND ND
30 25 40 40 20 20 15 5 35 30
ND ND ND ND ND ND ND ND ND ND
*Not detected.
A contrary picture was noted for extracts of animals from the sea of Okhotsk. Only two alcohol extracts (Strongylocentrotus sp., Hippasteria leiopelta) out of 80 animals from the sea of Okhotsk showed an inhibitory effect while the other extracts displayed themselves as LIV activators. Thirty-four extracts activated LIV more than 1.5 times including 19 extracts, which activated LIV more than 2.5-3 times (Table 3). It is interesting to note the action of extracts from the family Haliclonidae dwelling in the Indian Ocean and the sea of Okhotsk. No one species among eight torrid sponge species of this family inhibited LIV, but two of them contained some activators (Table 2). Among animal species of the sea of Okhotsk having the best activating effect (Table 3) species of the family Haliclonidae were also noted. Thus, during our study we have found possible sources of activators and inhibitors of fl-glucanases. Some of them have a high specificity for endo-I--*6-
~-D-glucanase. The discovery of these enzymes' sources of activation, which have not been described yet, seems to be most important. The nature of the activating effect of the extracts and the structure of substances which are responsible for them have not yet been elucidated, though some analogies suggest themselves. There are some indications concerning the activating effect exercised by some lipids on enzymes. For example, there is a description of fl-l,2-dioleyldiglyceride, a natural activator of a vegetative origin, containing //-bound residue (or residues of glucose) in the C-3 position (Ross et al., 1988), affecting ~-glucansynthase (membrane enzyme). Fatty acid compositions of lipids of marine invertebrates, living in the north and torrid seas, respectively, are known to considerably differ from each other, which is not surprising as this difference accounts for their thermostability and possibly causes the activating effect of the invertebrate extracts of the north seas.
190
T . N . ZVYAGINTSEVAet al.
Table 2. The activating effect of alcohol extracts of marine invertebrates of the Indian Ocean on the activity of endo-1--*3-/~-D-glucanase LIV No.
Type, Order, Family
Geodia globostellifera Geodinella spherastrosa
150 155
Order Axinellida Family Axinellidae 3 4
Phakellia cactoides Reniochalina stalagmitis
160
Biemna liposigma
6 7
Clathria dura Clathria frondifera
8
Ecinodictyum clathroides
9
Desmacidon arenosa
10
Latrunculia conulosa
1 2 3 4 5 6 7 8
150
11
12 13
14 15 16
17 18 19 20 21 22 23 24 25
Dampia pocillaporaeformis Dendronephthya sp. Sarcophyton sp. 1 Sarcophyton sp. 2 Sinularia sp. 1 Sinularia sp. 2 Sinularia sp. 3 Sinularia sp. 4 Stereonephthya sp. 1 Stereonephthya sp. 2
200
Polymastia kurilensis Polymastia robusta Halichondria panicea Asbestopluma lycopodium Mycale lobata Myxichella fragilis
9
Gellius primitivus Gellius varius
Haliclona gracilis
220 155
12
Haliclona ventilabrum
180
13
150 180 165 180 200 190 190 180 190 150
Acknowledgements--We wish to thank Dr John N. A.
Hooper and Dr Philip Alderslade (Northern Territory Museum Science and Arts, Darwin, Australia) for collecting and identifying some of the marine animals.
220 250 250 260
Order Haplosclerida Family Halielonidae 10 11
215 170
250 260
Family Myxillidae
Family Latrunculidae Order Haplosclerida Family Haliclonidae Haliclona sp. 1 Haliclona sp. 2 Order Dictyoceratida Family Spongiidae Spongia sp. Family Thorectidae Ircinia sp. 1 lrcinia sp. 2 Phylum Coelenterata Order Alcyonacea
300
Family Mycalidae
160
Family Desmacididae
350
Order Poecilosclerida Family Cladorhisidae
Family Clathriidae Family Raspailidae
Tetilla hamatum Tetilla sibirica
LIV activity (%)
Order Halichondrida Family Halichondriidae
165 210 150
Type, Order, Family Phylum Porifera Order Spirophorida Family Tetillidae Order Hadromerida Family Polymastiidae
150
Order Poecilosclerida Family Biemnidae 5
No.
LIV activity (%)
Phylum Porifera Order Astrophorida Family Geodiidae I 2
Table 3. Activating effect of alcohol extracts of marine invertebrates of the sea of Okhotsk on endo-(1--*3)-fl-D-glucanas¢LIV activity
260
220 270 250
Phylum Goelenterata Order Pennatularia Family Virgularidae Pavonaria finmarchica
250
Order Actiniaria Family Metridiidae 14 15 16
17
Metridium dianthus
Phylum Mollusca Order Opistobranchia Opistobranchia gen. sp. Family Tritonidae Tritonia primoriensis
Order Hamiglossa Family Buccinidae Buccinum sp. Phylum Tentaculata Order Ctenostomata Family Vesiculariidae
260 260 250 260
17
Bonerbankia composita
280
18
Phylum Echinodermata Order Forcipulata Family Asteriidae Lepthasterias sp. Phylum Chordata Order Stolidobranchia Family Pyuridae
250
19
Halocynthia aurantium
250
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