Comp. Biochem. PhysioL, 1975, Vol. 5013,pp. 209 to 210. PergamonPress. Printed in Great Britain
SHORT COMMUNICATION THE DISTRIBUTION OF THE D-ASPARTATE OXIDASE ACTIVITY IN CEPHALOPODA A. D'ANIELLO, R. PALESCANDOLOAND V. SCARDI Department of Physiology and Biochemistry, Stazione Zoologica, 80121 Naples, Italy (Received 26 November 1973) Abstract--1. A survey of the o-aspartate oxidase activity in the digestive gland of four species of octopods and six species of decapods has been made. 2. Enzyme activity has been found only in the octopods. 3. The results indicate that the presence of the D-aspartate oxidase activity in a species is probably related to its feeding habits.
INTRODUCTION ALTHOUGH o-aspartate oxidase (E.C. 1.4.3.1),
a flavoprotein catalysing specifically the oxidative deamination of o-aspartate and o-glutamate, was obtained in a high state of purity from the hepatopancreas of the octopus (Octopus vulgaris Lam.) by D'Aniello & Rocca (1972), no work was done on its distribution in other cephalopods. A survey of the o-aspartate oxidase activity in the digestive gland of ten species of cephalopods (four octopods and six decapods) was therefore undertaken with the aim of finding a more convenient enzyme source and of understanding the physiological significance of this unusual oxidase. MATERIALS AND METHODS The following species of cephalopods, collected from Naples Bay, were examined: Octopus vulgaris Lain., Eledone moschata Lam., Eledone aldrovandii Rafinesque, Argonauta argo L., Loligo vulgaris Lam., Allotheutis subulata Lam., lllex coindeti Verany, Sepia officinalis L., Sepia elegans D'Orbigny and Sepiola rondeleti Steenstrap. Except for A. argo, L coindeti and S. elegans, the animals were maintained in the laboratory in circulating sea water until dissection. The digestive gland or "liver" of the decapods and the hepatopancreas (freed of the "pancreatic" part) of the octopods were used as an enzyme source. The tissues from several animals of each species were pooled and homogenized with 4 vol. of 0-05 M potassium phosphate buffer, pH 7.0, and centrifuged for 30 min in a Sorvall centrifuge (SS-1) at ca. 12,000 rev/min in the cold room (+ 5°C); the supernatants were dialysed overnight against distilled water, clarified if necessary and used as the enzyme solution. In the case of .4. argo only two specimens were available, whereas in the case of S. rondeleti more than fifty specimens were necessary to obtain a reasonable amount of material. The enzyme activity was measured by adaptation of the micromethod of Soda (1968) and expressed in milliunits 209
(mU) per g of moist tissue, a unit being defined as the amount of enzyme catalysing the formation of a/~mole of keto acid per min at 30°C. Specificactivity was calculated as mU/mg of protein; protein being measured by the biuret method (Gornall et al., 1949) after precipitation with 5% trichloroacetic acid and dissolution in 0.1 N NaOH. The apparent Km values for D-aspartate and Dglutamate were calculated according to the procedure of Niebergall et al. (1966) by using mesotartrate as a fully competitive inhibitor of the D-aspartate oxidase activity (Palescandolo, Rocca, Scardi & Tosi, unpublished data). RESULTS AND DISCUSSION Activity measurements indicated that the Daspartate oxidase is present in all the octopods examined (Table 1) and in none of the decapods examined. The absence of enzyme activity in the six species of decapods examined cannot be attributed to a possible dissociation of the enzyme into apoenzyme and coenzyme, as no activity was recovered by incubation with F A D prior to enzyme assay. From Table 1 it can be seen that the octopus (O. vulgaris) represents by far the best source for Daspartate oxidase, even though it was previously chosen only because it is a common, easily available, large-sized animal whose hepatopancreas was reported to contain an enzyme oxidizing o-glutamate and, at a slower rate, o-aspartate (Blascb_ko & Himms, 1955). In this connection it should be noted that o-aspartate oxidase from O. vulgaris acts on Dglutamate at a higher rate than on o-aspartate (see Table 1). For this reason the enzyme was initially referred to as D-glutamate oxidase (Blaschko & Himms, 1955; Rocca & Ghiretti, 1958). The average ratio of the Km values for D-glutamate and Daspartate, respectively, is 1.39, independently of the enzyme source; this is in good agreement with the ratio calculated from the data obtained by D'Aniello
210
A. D'ANIELLO,R. PALESCANDOLOAND V. SCARDI Table 1. D-Aspartate oxidase distribution in the digestive gland of octopods Activity (mU/mg of tissue) Species
Octopus vulgaris Eledone moschata E. aldrovandii Argonauta argo
Specific activity (mU/mg of protein)
A
G
A
G
729 440 187 16
915 326 193 --
21 "5 12'5 6.0 1"3
24.7 9"3 6'1 .
.
KmA (mM)
gm G (raM)
2"5 1-2 3"6 .
3.5 1'7 4"9
KmG/KmA 1.40 1"42 1.36
.
--, Not determined. Column A: data obtained with D-aspartate as substrate. Column G: data obtained with D-glutamate as substrate. KmA and KING:apparent Michaelis constant for D-aspartate and D-glutamate, respectively. & Rocca (1972) with the purified enzyme and by an assay method different from the present one. The slight enzyme activity found in A. argo seems to indicate this free swimming octopod, which feeds only on small fishes (a food common to all cephalopods), as being a link between octopods and decapods. The presence of D-aspartate oxidase in a species is probably related to its feeding habits. REFERENCES
BLASCHKO H. & HIMMS J. M. (1955) o-Glutamic acid oxidase in cephalopod liver. J. PhysioL, Lond. 128, 7. D'ANrELLO A. & ROCCA E. (1972) D-Aspartate oxidase from the hepatopancreas of Octopus vulgaris Lam. Comp. Biochem. PhysioL 41B, 625-633.
GORNALLA. G., BARDAWILLC. J. & DAVIDM. M. (1949) Determination of serum protein by means of the biuret reaction. J. biol. Chem. 177, 751-766. NIEBERGALLP. J., SIGITAE. T. & DOLUISIOJ. T. (1966) A modified method for determing Ki/Kra. Analyt. Biochem. 16, 189-192. ROCCA E. & GHIRETTI F. (1958) Purification and properties of D-glutamic acid oxidase from Octopus vulgaris Lam. Archs Biochem. Biophys. 77, 336-349. SODAK. (1968) Microdeterminationof D-amino acids and D-amino acid oxidase activity with 3-methyl-2benzothiazolone hydrazone hydrochloride. Analyt. Biochem. 25, 228-235. Key Word Index--D-Aspartate oxidase; Cephalopoda; Octopus; Eledone; Sepia; Allotheutis; lllex.
SHORT COMMUNICATION THE DISTRIBUTION OF THE D-ASPARTATE OXIDASE ACTIVITY IN CEPHALOPODA A. D'ANIELLO, R. PALESCANDOLOAND V. SCARDI Department of Physiology and Biochemistry, Stazione Zoologica, 80121 Naples, Italy (Received 26 November 1973) Abstract--1. A survey of the o-aspartate oxidase activity in the digestive gland of four species of octopods and six species of decapods has been made. 2. Enzyme activity has been found only in the octopods. 3. The results indicate that the presence of the D-aspartate oxidase activity in a species is probably related to its feeding habits.
INTRODUCTION ALTHOUGH o-aspartate oxidase (E.C. 1.4.3.1),
a flavoprotein catalysing specifically the oxidative deamination of o-aspartate and o-glutamate, was obtained in a high state of purity from the hepatopancreas of the octopus (Octopus vulgaris Lam.) by D'Aniello & Rocca (1972), no work was done on its distribution in other cephalopods. A survey of the o-aspartate oxidase activity in the digestive gland of ten species of cephalopods (four octopods and six decapods) was therefore undertaken with the aim of finding a more convenient enzyme source and of understanding the physiological significance of this unusual oxidase. MATERIALS AND METHODS The following species of cephalopods, collected from Naples Bay, were examined: Octopus vulgaris Lain., Eledone moschata Lam., Eledone aldrovandii Rafinesque, Argonauta argo L., Loligo vulgaris Lam., Allotheutis subulata Lam., lllex coindeti Verany, Sepia officinalis L., Sepia elegans D'Orbigny and Sepiola rondeleti Steenstrap. Except for A. argo, L coindeti and S. elegans, the animals were maintained in the laboratory in circulating sea water until dissection. The digestive gland or "liver" of the decapods and the hepatopancreas (freed of the "pancreatic" part) of the octopods were used as an enzyme source. The tissues from several animals of each species were pooled and homogenized with 4 vol. of 0-05 M potassium phosphate buffer, pH 7.0, and centrifuged for 30 min in a Sorvall centrifuge (SS-1) at ca. 12,000 rev/min in the cold room (+ 5°C); the supernatants were dialysed overnight against distilled water, clarified if necessary and used as the enzyme solution. In the case of .4. argo only two specimens were available, whereas in the case of S. rondeleti more than fifty specimens were necessary to obtain a reasonable amount of material. The enzyme activity was measured by adaptation of the micromethod of Soda (1968) and expressed in milliunits 209
(mU) per g of moist tissue, a unit being defined as the amount of enzyme catalysing the formation of a/~mole of keto acid per min at 30°C. Specificactivity was calculated as mU/mg of protein; protein being measured by the biuret method (Gornall et al., 1949) after precipitation with 5% trichloroacetic acid and dissolution in 0.1 N NaOH. The apparent Km values for D-aspartate and Dglutamate were calculated according to the procedure of Niebergall et al. (1966) by using mesotartrate as a fully competitive inhibitor of the D-aspartate oxidase activity (Palescandolo, Rocca, Scardi & Tosi, unpublished data). RESULTS AND DISCUSSION Activity measurements indicated that the Daspartate oxidase is present in all the octopods examined (Table 1) and in none of the decapods examined. The absence of enzyme activity in the six species of decapods examined cannot be attributed to a possible dissociation of the enzyme into apoenzyme and coenzyme, as no activity was recovered by incubation with F A D prior to enzyme assay. From Table 1 it can be seen that the octopus (O. vulgaris) represents by far the best source for Daspartate oxidase, even though it was previously chosen only because it is a common, easily available, large-sized animal whose hepatopancreas was reported to contain an enzyme oxidizing o-glutamate and, at a slower rate, o-aspartate (Blascb_ko & Himms, 1955). In this connection it should be noted that o-aspartate oxidase from O. vulgaris acts on Dglutamate at a higher rate than on o-aspartate (see Table 1). For this reason the enzyme was initially referred to as D-glutamate oxidase (Blaschko & Himms, 1955; Rocca & Ghiretti, 1958). The average ratio of the Km values for D-glutamate and Daspartate, respectively, is 1.39, independently of the enzyme source; this is in good agreement with the ratio calculated from the data obtained by D'Aniello
210
A. D'ANIELLO,R. PALESCANDOLOAND V. SCARDI Table 1. D-Aspartate oxidase distribution in the digestive gland of octopods Activity (mU/mg of tissue) Species
Octopus vulgaris Eledone moschata E. aldrovandii Argonauta argo
Specific activity (mU/mg of protein)
A
G
A
G
729 440 187 16
915 326 193 --
21 "5 12'5 6.0 1"3
24.7 9"3 6'1 .
.
KmA (mM)
gm G (raM)
2"5 1-2 3"6 .
3.5 1'7 4"9
KmG/KmA 1.40 1"42 1.36
.
--, Not determined. Column A: data obtained with D-aspartate as substrate. Column G: data obtained with D-glutamate as substrate. KmA and KING:apparent Michaelis constant for D-aspartate and D-glutamate, respectively. & Rocca (1972) with the purified enzyme and by an assay method different from the present one. The slight enzyme activity found in A. argo seems to indicate this free swimming octopod, which feeds only on small fishes (a food common to all cephalopods), as being a link between octopods and decapods. The presence of D-aspartate oxidase in a species is probably related to its feeding habits. REFERENCES
BLASCHKO H. & HIMMS J. M. (1955) o-Glutamic acid oxidase in cephalopod liver. J. PhysioL, Lond. 128, 7. D'ANrELLO A. & ROCCA E. (1972) D-Aspartate oxidase from the hepatopancreas of Octopus vulgaris Lam. Comp. Biochem. PhysioL 41B, 625-633.
GORNALLA. G., BARDAWILLC. J. & DAVIDM. M. (1949) Determination of serum protein by means of the biuret reaction. J. biol. Chem. 177, 751-766. NIEBERGALLP. J., SIGITAE. T. & DOLUISIOJ. T. (1966) A modified method for determing Ki/Kra. Analyt. Biochem. 16, 189-192. ROCCA E. & GHIRETTI F. (1958) Purification and properties of D-glutamic acid oxidase from Octopus vulgaris Lam. Archs Biochem. Biophys. 77, 336-349. SODAK. (1968) Microdeterminationof D-amino acids and D-amino acid oxidase activity with 3-methyl-2benzothiazolone hydrazone hydrochloride. Analyt. Biochem. 25, 228-235. Key Word Index--D-Aspartate oxidase; Cephalopoda; Octopus; Eledone; Sepia; Allotheutis; lllex.
