Studies on the identification and characterization of an aspartase activity in liver of elasmobranch fishes

Comp. Biochera. Physiol., 1972, Vol. 41B, pp. 905 to 919. Pergamon Press. Printed in Great Britain

S T U D I E S ON T H E I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF AN ASPARTASE A C T I V I T Y I N L I V E R OF E L A S M O B R A N C H F I S H E S LETIZIA CUTINELLI, CONCETTA PIETROPAOLO, SALVATORE VENUTA,* VINCENZO ZAPPIA and FRANCESCO SALVATORE Istituto di Chimica Biologica, I e II Cattedra, Facolt~ di Medicina e Chirurgia, Universith di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy; and Stazione Zoologica, Villa Comunale, 80121, Napoli, Italy

(Received 7 ffuly 1971) A b s t r a c t - - 1 . L-aspartate is actively deaminated by elasmobranch fish liver:

experiments with 15N-aspartate demonstrate the formation of 15N-ammonia. 2. The mechanism of the amino group detachment from L-aspartate has been investigated. Fumarate formation in stoichiometric amounts with ammonia, and experiments performed in the presence of et-oxoglutarate, L-glutamate, NAD.t Inosinic acid and other compounds support a direct mechanism of aspartate deamination rather than an indirect one. The enzyme activity has been visualized as an aspartate : ammonia lyase (aspartase), never found before in animal tissues. 3. The enzyme activity has been characterized, it has been found moderately stable and is associated with a homogeneous mitochondrial fraction. Attempts at its solubilization from the mitochondrial matrix with detergents, ultrasounds, etc. were unsuccessful. 4. The biological significance of this enzyme activity in elasmobranch fishes is discussed. INTRODUCTION TRANSAMINATION tO oxaloacetate represents the major pathway for the degradation of L-aspartate in Vertebrates (Greenberg, 1969). A n u m b e r of other reactions involve the coupling of L-aspartate with various compounds, i.e. citrulline, inosinic acid, etc. (Sallach & Fahien, 1969). T h e net result of these reactions is the transfer of the amino group from L-aspartate to various acceptors while the carbon chain remains as fumaric acid (Meister, 1965). L-aspartate can also bind the carbamyl group of carbamylphosphate to form carbamyl-L-aspartate, which is the precursor of dihydroorotic acid and ultimately of pyrirrddines (Reichard, 1959). T h r e e enzyme activities metabolizing L-aspartate by a lyase-type mechanism have also * Temporary address: Department of Molecular Biology, University of California, Berkeley, California 94720. t The following abbreviations will be used in the text: NAD(P), nicotinandde adenine dinucleotide (phosphate); ATP, adenosinetriphosphate; EDTA, ethylenediaminetetracetic acid; Tris, tris(hydroxymethyl)aminomethane; GTP, guanosinetriphosphate; IMP, inosinic acid; TEA, triethanolamine; SDS, sodium dodecylsulfate. 31

9O5

906

L. CUTINELLIet al.

been described in bacteria: c~- and fl-decarboxylases giving rise respectively to fl- and c~-alanine (Utter, 1961), and L-aspartate : ammonia lyase which produces NH 3 and fumarate (Greenberg, 1969). Finally, L-aspartate is involved in the biosynthesis of ornithine, threonine, lysine and methionine in several microorganisms (Rodwell, 1969). Figure 1 schematically reports the pathways involving the transfer of the amino group of L-aspartate to various compounds, which could eventually give rise to ammonia either directly or indirectly.

Aspartate ] /3-Afoni// ~ Glu,amote/

Purlnrieng (nitiroge~ I) (A-d~n'Igireaotid

Arginine

(guonidino

group)

Corbam daspart (-NHgroup)ate

FzG. 1. Schematic representation of the various metabolic pathways that convert the nitrogen atom of L-aspartic acid into the nitrogen of different compounds. Previous studies (Zappia et al., 1964, 1965a, b; Salvatore et al., 1965) have indicated an active deamination of L-aspartate in the tissues of elasmobranch and teleost fishes. Therefore, in order to elucidate the mechanism of such reaction(s), a study has been undertaken (Salvatore et aL, 1967a); also by the use of a new method for the estimation of fumaric acid in crude tissue extract (Zappia et aL, 1966a, b; Salvatore et al., 1967b). Evidence for the presence of an aspartase-like activity in the liver of elasmobranch fishes is presented in this paper. The enzyme activity, found to be associated with the mitochondrial fraction, has been partially characterized. Preliminary results have already been presented at the 158th Meeting of the American Chemical Society (Venuta et al., 1969).

EXPERIMENTAL Materials and methods

L-Aspartic acid was obtained from British Drug Houses; lSN-aspartic acid was purchased from Merck Sharp & Dohme of Canada, Ltd. ; hog kidney o-amino acid oxidase was obtained from Worthington; NAD and NADP from Boheringer. All other substances were pure

ASPARTASE I N ELASMOBRANCH FISHES

907

compounds obtained from the usual commercial sources. T h e stereochemical purity of L-aspartic acid and other L-amino acids was checked by means of D-amino acid oxidase by the method of Burton (1965). T h e deamination of aspartic acid was assayed following ammonia formation by Conway's microdiffusion technique, according to the modifications of Cedrangolo et aI. (1965); the enzyme activity was also measured following the formation of fumaric acid b y the method of Salvatore et al. (1967b). Protein was determined by the method of Lowry et al. (1951) using cristalline bovine serum albumin as standard. T h e analysis of XSN content was performed with a mass spectrometer (Italelettronica, Model Sp-21-F). Nitrogen gas was liberated from N H s by alkaline sodium hypobromite according to the method described by San Pietro (1957), as modified by Balestrieri (1968). T h e sonication of mitochondria was performed in a Branson Model S-75 sonicator, as indicated under Results. T h e mitochondria from Scyllium canicula liver were obtained according to the following general procedures, (i) and (ii). (i), 10% (w/v) liver homogenate in 0"25 M sucrose+0.002 M E D T A and 0.01 M T E A - H C I , p H 7.4, was centrifuged at 500g for 10 min; the supernatant was collected and centrifuged at 10,000g for 15 rain; the latter precipitate was washed twice with one-tenth of the initial volume of 0"25 M sucrose and used as indicated under Results. (ii), 10% (w/v) liver homogenate in 0.25 M sucrose+0.002 M E D T A + 0 . 0 1 M T E A - H C I + 0 . 0 0 1 M mercaptoethanol, p H 7 ' 3 , was centrifuged at 800 g for 20 rain. T h e supernatant was centrifuged again at 6500 g for 20 min; the latter precipitate was washed twice with one-tenth of the initial volume of 0.25 M sucrose, centrifuged at 15,000 g for 20 min and used as indicated under Results. T h e purity of the mitochondrial fraction was checked using a sucrose linear gradient (30-70%) according to the following procedure: a 50% (v/v) liver homogenate in 0.25 M s u c r o s e + 1 0 - S M E D T A + T r i s , pH7"3, was centrifuged at 12,000g for 15 min. T h e supernatant was again centrifuged at 6000 g for 15 rain. T h e precipitate, resuspended in 0"25 M sucrose+10 -8 M E D T A + T r i s , p H 7"3, was centrifuged at 12,000g for 15 rain. T h e precipitate was washed once with 0"25 M sucrose, then resuspended in one-tenth of the initial volume of 0"25 M sucrose and centrifuged at 20,000 g for 10 min. T h e precipitate obtained after this centrifugation and suspended in 0-25 M sucrose was stratified on a sucrose linear gradient (30-70%): the fractions obtained were checked for activity. Experimental procedure Elasmobranchs from the Mediterranean sea (S. canicula and Scyllium stellare) were used in most of the experiments; all other animals used are listed in Table 1. Freshly excised liver deprived from excess connective, gently blotted on filter paper and rinsed with cold buffer was immediately homogenized (30% v/v) in phosphate buffer (KH2PO,-KzHPO4) 0"1 M at p H 7"4, if not otherwise stated. T h e homogenization was performed in a glass Potter-Elvehjem apparatus with a Teflon pestle at 1000 rev/min for 2 rain at 2°C. Samples were prepared as indicated under the Tables and Figures in small Erlenmeyer flasks and placed in a Dubnoff shaking-incubator at 37°C. Sodium arsenite (3"3 × 10 -s M) was added to prevent ammonia incorporation b y glutamine synthetase (Krebs, 1935; Salvatore et al., 1965). Furthermore, the lack of addition of A T P and N A D H also prevented, in the systems employed, NH3 incorporation b y various enzymes: namely carbamylphosphate and glutamine-synthetase, glutamate dehydrogenase and NHa incorporation enzymes in purine nucleotides metabolism (see Takahashi et al., 1963). F o r NHz estimation, 1 ml of the incubation mixture was directly added to 2 ml of borate buffer prepared according to Reinhold & Chung (1961) in mierodiffusion bottles, as indicated b y Cedrangolo et al. (1965). F o r fumaric acid determination, 1"5 ml of the incubation mixture were added to 0"3 ml of trichloroacetic acid (20%) and centrifuged at 9000g for 15 rain. T o 1 ml of the supernatant 3 ml of pyridine and 4 ml of acetic anhydride were added following the directions of Salvatore et al. (1967b).

908

L. CUTINELLIet al. RESULTS

T h e extent of a m m o n i a formation from L-aspartate in the liver of various vertebrates along the evolutionary scale has been investigated. I n T a b l e 1 the results obtained f r o m these experiments are reported. Besides elasmobranch fish, m a n y vertebrate tissues, except mammalian, are active in deaminating L-aspartic acid: a m o n g avians, chicken showed a high rate of deamination. Elasmobranch fish liver has been selected for further studies because of the unique position of these organisms at the lowest level in the p h y l u m of vertebrates. TABLE 1--AMMONIA PRODUCTIONFROM L-ASPARTATEIN LIVEROF VERTEBRATES Animal

Activity *

Elasmobranch fish : S. canicula S. stellare

6'8 8"9 54.4

Amphibians : Rana esculenta Rana esculenta (tadpole) Triturus cristatus Triturus cristatus (tadpole)

Activity *

Reptiles : 8-1 8"4

Teleost fish : Gobius niger jozo Mugil cephalus Anguilla anguilla

Animal

7"0 2"3

Lacerta sicula Testudo graeca

Birds : Duck (Anas platyrhyncos) Chicken ( Gallus gallus) Pigeon (Columba livia) Mammals: Rat (Rattus rattus) Mouse (Mus musculus )

7"0 13"5 - 1.2 15"6 - 1"4 - 1"5

-0"1

3" 1

- 0"1

*/zmoles NH3/g wet tissue per 2 hr at 37°C. Samples without the addition of t-aspartate, as well as complete samples deproteinized at the start, were run as controls. Complete samples contained: 1 ml of 50% v/v homogenate in K , H P O i K H , P O , buffer 0"1 M, pH 7.0; sodium arsenite and MgC12:3"3 x 10 -s M; Laspartate: 10 -1 M. Final volume: 3 ml with H20. Incubation at 37°C for 2 hr in air. Data of typical experiments are presented in the Table. Results showing the deamination of XSN-labelled and unlabelled L-aspartic acid in different experimental conditions are shown in T a b l e 2. I t can be noted that, u n d e r the conditions of the assay, ammonia is formed f r o m L-aspartate in an a m o u n t of at least 4 tzmoles/g of tissue. W h e n oxygen, nitrogen or air at a reduced pressure were used, the rate of the reaction was approximately the same, thus indicating a non-oxidative deamination. F r o m the experiments with 15Naspartate it can be calculated that the a m m o n i a formed f r o m aspartate accounts for the enrichment of 15N in the a m m o n i a isolated after the incubation. Therefore, these data indicate that a m m o n i a definitely originates f r o m the nitrogen of the aspartate molecule: enhancement of endogenous substrates deamination by aspartate can be excluded. I n Fig. 2 the formation of N H 3 and that of fumarate f r o m L-aspartate, as a function of substrate concentration, are compared. While at 15 min the levels of

909

ASPARTASE IN ELASMOBRANCH FISHES T A B L E 2 - - D E A M I N A T I O N OF L-ASPARTATE I N AEROBIOSIS AND ANAEROBIOSIS

Gas phase

Enzymic source

Scyllium Scyllium Scyllium Scyllium

canicula canicula stellare stellare

Additions None L-Aspartate None L-Aspartate

None Nitrogen Oxygen Air Ammonia formed (/xg/g tissue) 72"4 171"0 84"0 163"2

73"2 162"6 82"4 161"4

74.4 168"6 86"2 166.2

70"8 161"4 85"8 165"0

lSN (%)* 0.37 4"85 0-36 3"85

* Analysed in the ammonia after microdiffusion. Complete samples contained: 1 ml of 50% v/v liver homogenate in K H 2 P O , K~HPO4 0"1 M, pH 7.0; sodium arsenite and MgC12:3"3 x 10 -s M ; L-aspartate (where added): 10 -x M. Final volume: 3 ml with water. Incubation at 37°C for 1 hr. The data reported in the last two columns are concerned with experiments performed with L-aspartate N15-1abelled (9-3% of 15N, measured on ammonia formed after Kjeldahl digestion). Controls consisted of a complete reaction mixture deproteinized at the start and a complete system without the enzyme. All the figures given represent the mean of five different experiments.

ammonia were found to be a little lower than those of fumarate, the reverse situation occurs (data not reported in the Figure) with a longer incubation time (30-90 min) indicating, in this case, a faster utilization of fumarate.

:55 so

E oe

25 20

E

E ~- 0 5 I

I

I

I

40

80

120

160

A s p a r t a t e conc.,

I

200

mM

FIG. 2. Ammonia and fumarate production from L-aspartate in the liver of S.

stellare: activity as a function of aspartate concentration. The assay system and the experimental conditions were as indicated in Table 2 except for the incubation time (15 rain in air) and enzyme concentration (750 mg of wet tissue/sample).

910

L. CUTINELLIet al.

Conventional kinetic experiments were carried out: the reaction rate was linear for at least 60 min with 0.1 M e-aspartate and 166 mg of tissue/sample; substrate and enzyme concentration which were more appropriate for measurement of the enzyme activity were found to be 10 -1 M L-aspartate and 500 mg of tissue/ 3 ml of incubation mixture; pH optimum was found at 7.0-7.2 in phosphate buffer and optimum temperature at 37-38°C. L-Aspartate could deaminate indirectly through glutamate, after transamination with endogenous ~-oxoglutarate, or previous condensation with inosinic acid and splitting of adenylosuccinate followed by adenylate deamination. In Table 3 are shown experiments where ~-oxoglutarate and inosinic acid in appropriate catalytic concentrations (Still et al., 1950; Hird & Marginson, 1966) failed to enhance aspartate deamination. Moreover, ammonia disappearance was observed when ~-oxoglutarate was added to L-aspartate, both in the liver of S. canicula and also S. stellare. T A B L E 3 - - A M M O N I A PRODUCTION FROM L-ASPARTATE I N PRESENCE OF ~-OXOGLUTARATE OR INOSINIC ACID

Ammonia formed ~g/g tissue) Additions None e-Aspartate c~-Oxoglutarate e-Aspartate + a-oxoglutarate Inosinie acid L-Aspartate+inosinic acid

S. canicula

S. stellare

56"6 + 3"1 200"1 + 13"3 54"0 + 4"6 150"6 + 12"0 60.6 + 3.7 204"6 + 18"0

88"2+ 6"0 244"4 + 18"1 87"0 + 5"8 144"6 + 10"6 91.2 + 6.6 207"6 + 14.1

Preparation of the samples and other experimental conditions were as indicated in Table 2. The gas phase was air. c~-Oxoglutarateor inosinic acid (when added) : 10 -3 M, neutralized at pH 7"0. In order to further verify the possibility of an indirect deamination of Laspartate through glutamate dehydrogenase, experiments described in Table 4 were carried out. In fact, McBean et al. (1966) reported that fish tissue homogenates required the addition of NAD to show glutamate-dehydrogenase activity to any good extent. Under our experimental conditions (see Table 4), the addition of NAD was confirmed to be critical for glutamate deamination, while no effect was detectable when aspartate deamination was measured. Even assuming that ~-oxoglutarate was not limiting in our preparation, if an indirect deamination of e-aspartate were operative, NAD had to enhance the deamination of the glutamate formed by transamination. However, since NAD did not show any appreciable effect, an indirect deamination mechanism through glutamate-dehydrogenase is not supported. Parallel experiments with NADP (data not reported in Table) also did

911

ASPARTASE IN ELASMOBRANCH FISHES TABLE ~ - A M M O N I A

FORMED I N PRESENCE OF VARIOUS SUBSTRATES AND COFACTORS IN LIVER OF

S. canicula

Ammonia formed (/~moles/g tissue) Substrates None NAD, 10 -3 M Glutamate, 10-1 M Glutamate, 10-3 M Glutamate, 10-t M+NAD, 10-a M Glutamate, 10 -3 M + NAD, 10-3 M

No aspartate

Aspartate, 10-1 M

-7.50 0.04 - 0.12 11"70 9.90

2"70 9"50 2"58 2"58 ---

The experimental conditions and the preparation of the samples were the same as indicated in Table 3, except for the incubation time which was 30 re_in. The figures reported in this Table represent the differences between ammonia formed in the presence of the substrate and ammonia formed in its absence (mean values of three experiments). not give any indication for the involvement of a NADP-dependent glutamatedehydrogenase. Another set of experiments was then performed using 15N-aspartate as substrate in the presence of an excess of glutamate. In these experiments the ratio of theoretic 15N°/o to the ZSN% measured in the ammonia formed was the same, both in the samples with aspartate alone and in those with an excess of L-glutamate. The theoretic 15N°/o was calculated by assuming, as shown in the experiments in Table 2, that ammonia formed in the presence of L-aspartate was completely derived from this amino acid. Therefore, this experiment also does not support a transdeamination mechanism for aspartate deamination. Various metabolic interrelationships would allow the amino group of Laspartate to change to nitrogen in several compounds, which eventually could produce ammonia in crude tissue extracts (see Fig. 1). Therefore, these compounds were tested in varying concentrations as ammonia producers in our system, both alone and in the presence of L-aspartate (Table 5). If aspartate deamination occurred according to any indirect mechanism, the addition of the first compound (L-aspartate) of the metabolic chain should not affect the second compound deamination in conditions of substrate excess (see Table 5). This never occurred, thus indicating the independent deamination of L-aspartate. In Table 6 are shown experiments on the effect of G T P and G T P plus inosinic acid on L-aspartate deamination. These experiments could test the possibility of an indirect deamination of aspartic acid by previous formation of adenylsuccinic acid, since G T P addition is critical for the synthesis of this latter compound (Meister, 1965). The data reported in Table 6 show that G T P inhibits the deamination of aspartate in fish tissues, and the addition of both G T P and IMP further decreases NH 3 production. These results permit to exclude an indirect

912

L . CUTINELLI et

al.

T A B L E 5 - - E F F E C T OF VARIOUS SUBSTRATES ON THE DEAMINATION OF L-ASPARTATE I N

HVER OF Scyllium* Ammonia formed (/zmoles/g tissue) Substrates

No aspartate

None L-c~-Alanine, 10 -1 M fl-Alanine, 10 -1 M L-Carbamylaspartate, 10 -1 M L-Carbamylaspartate, 10 -2 M L-Arginine, 10 -1 M L-Arginine, 10 -8 M

Aspartate, 10 -1 M

-0'04 0"12 0"18 0"40 0"04 -0"12

3"30 1"24 3"58 2-22 2"60 3"10 3"12

* Both S. stellare and S. canicula gave similar results. The figures represent the differences between ammonia formed in the presence of the substrate and ammonia formed in its absence (average of three experiments). Incubation in air for 30 min. For other experimental conditions and incubation mixtures see Table 3. d e a m i n a t i o n of aspartate t h r o u g h adenylosuccinate. A n inhibitory effect of G T P and I M P on bacterial aspartase is well k n o w n (Williams & Lartigue, 1969); the results p r e s e n t e d in T a b l e 6 indicate a similar behavior of the activity u n d e r study. T A B L E 6 - - E F F E C T OF INOSINIC ACID AND GUANOSINTRIPHOSPHATE ON ASPARTATE DEAMINATION IN LIVER OF Scyllium*

Ammonia formed (/zmoles/g tissue) Substrates None Inosinic acid, 10 -3 M Guanosintriphosphate, 10-8 NI Guanosintriphosphate, 10 -3 M + inosinic acid, 10 -8 M

No aspartate 0"20 - 0"50 0"86

Aspartate 10 -1 M 3"00 2"52 2"10 1 "80

* Both S. canicula and S. stellare gave similar results. The figures represent the differences between ammonia formed in the presence of the substrate and ammonia formed in its absence (average of three experiments). Incubation in air for 30 rain. For other experimental conditions and composition of the incubation mixtures see Table 3. I n order to determine the cellular localization of the e n z y m e activity, the liver h o m o g e n a t e was fractionated b y differential centrifugation. Nuclei, microsomial fraction, s u p e r n a t a n t at 2 0 , 0 0 0 g and at 105,000g, obtained according to the m e t h o d o f H o g e b o o m (1955), did not contain any appreciable e n z y m e activity,

913

ASPARTASE I N E L A S M O B R A N C H FISHES

while all the activity was recovered in the mitochondrial fraction prepared according to Sottocasa et al. (1967). Further studies were, therefore, performed on this fraction and characterization of the enzyme was undertaken. Figure 3 shows enzyme activity as a function of time: an almost linear relationship was found up to 90 rain.

E

40 ll

o ~0

E o

j

20

o

i

/

.a_ E <~

i

ll

I0

/ I 15

I 30

I,

I

I,

I

45

60

75

90

Time,

rain

FIG. 3. Ammonia production from L-aspartate in liver mitochondria from S. canicula: activity as a function of time. The liver mitochondria were obtained

according to the procedure described under Materials and Methods as (ii). The incubation mixture contained: 1 ml of mitochondrial preparation (precipitate of last centrifugation suspended in 0-25 M sucrose; for protein content see Table 7); Laspartate: 10 -1 M, MgCI~ and Na arsenite: 3"3 x 10 -3 M, K-phosphate buffer 0-05 M: 0"5 ml. Final volume: 3 ml. Incubation: 90 min in air at 37°C. Figure 4 shows enzyme activity as a function of enzyme concentration in the presence of two buffer systems and divalent cations. Phosphate buffer and Mg =+ were selected. T h e time-stability of the enzyme stored at 2°C was then studied and the results are presented in Fig. 5. Seventy per cent of its activity was recovered after the first 24 hr, then no further decrease was observed until the seventh day (55-60 per cent of the activity was recovered). A number of experiments were then performed in order to solubilize the enzyme activity from the mitochondrial structure. Several detergents, e.g. Teepol XL, Triton X100, Na-deoxycolate under various concentrations and experimental conditions, were unable to solubilize the enzyme activity. Also glycerol (50%) and osmotic shock performed with glycerol and phosphates failed to reach any result. Table 7 shows another set of experiments in which the solubilization was attempted by ultrasounds and SDS: both treatments were unsuccessful.

914

L. CUTINELLI

et al.

T A B L E 7 - - U L T R A S O U N D S AND SDS EFFECT ON THE DEAMINATION OF LoASPARTATE IN LIVER MITOCHONDRIA OF S. canicula

Sonication (sec)

Additions

Ammonia production (2~g/sample)

Protein (mg/sample)

Specific activity*

-30 60 60 60 60

---SDS -SDS

32.58 60.42 (A) 3.30 (B) 26"88 (A) 1-05 (B) 4.95 (A) 4.20 (B) 14"16 (A + B) 5.28 (A + B)

14.37 24.00 (A) 3.4 (B) 12.87 (A) 2"81 (B) 12.00 (A) 14"06 (B) 7-84 (A + B) 13"03 (A + B)

2.25 2.52 (A) 0.96 (B) 2"1 (A) 0-36 (B) 0"41 (A) 0"30(B) 1.80 (A + B) 0.40 (A + B)

*/~g NHa/mg protein per 90 min at 37°C. Liver mitochondria were prepared as described under Materials and Methods as procedure (i). The precipitate of the last centrifugation was suspended in 5 vol. of Na-phosphate buffer 0.075 M to obtain a concentration of approximately 20 mg of protein/ml. Sixty ml of this suspension were sonicated at 0°C for 30 sec (20 ml at the time) in the Branson S-75 sonicator. The sonicate was centrifuged at 20,000 g for 30 rain and the enzyme activity was assayed in the supernatant as well as in the precipitate. The latter was suspended in Na-phosphate buffer 0"075 M and sonicated again per 30 sec. The sonicate was centrifuged at 20,000 g for 30 min and the activity was estimated on the supernatant and precipitate. The latter was resuspended in 5 vol. of Na-phosphate buffer 0.075 M (protein concentration was N 20 mg/ml) and SDS was added (1 rag/10 mg of protein). The mitochondrial suspension and SDS were stirred for 10 min at 0°C, then centrifuged at 20,000 g for 20 rain and the enzyme activity was estimated on the supernatant and the precipitate. The samples were incubated for 90 min in air at 37°C, the incubation mixture was analogous to that used in Fig. 3. The data referred to as (A) are concerned with the precipitate obtained after various centrifugations, and resuspended in Na-phosphate buffer pH 7"4. The data referred to as (B) are concerned with the supernatant of the centrifugation. T h e tissue preparation was centrifuged on a linear sucrose gradient (30-70%) and the enzyme activity was found to be associated with a single, well-defined band at 50% sucrose concentration. This latter experiment shows that the mitochondrial fraction containing the enzyme activity is rather homogeneous. Figure 6 reports ammonia production from L-aspartate as a function of protein concentration in this preparation. DISCUSSION T h e enzyme aspartase (L-aspartate: ammonia lyase E.C. 4.3.1.1) has been reported to be present in several micro-organisms and in plants, but not in vertebrates (Greenberg, 1969). This enzyme reversibly catalyzes ammonia production from L-aspartate with a double bond formation into the carbon chain leading to fumaric acid. T h e name aspartase for this enzyme was proposed by Wolf (1929), while fractionation of the enzyme from Escherichia coli was carried out by Gale (1938). A n u m b e r of investigators have subsequently purified to various degrees the aspartase from a variety of bacterial species (Lichstein & Umbreit, 1947;

915

ASPARTASEIN ELASMOBRANCHFISHES i

I

i

•Mg+~.

i

A = Mg ++ K - P O 4 B u f f e r 50

o

40

o =

Mn++, Tris-HCL Buffer

~

3o

E

o

"E

2o

I I0

I 20

I,, 30

Protein,

mg

I 40

FIG. 4. Production of ammonia from L-aspartate in liver mitochondria of S. canicula: activity as a function of enzyme concentration. Mitochondria were prepared according to Sottocasa et al. (1967) and resuspended in sucrose 0"25 i . Incubation: 90 rain in air at 37°C. T h e incubation mixture was the same as indicated in Fig. 3 except that in some experiments MnCI= as well as Tris-HC1 were used. I tO0

~

I

I

I

I

I

I 5

I 6

mooo

9O

70

e.o -o ~4~0

~

°

6O i 5O

I i

I 2

I 3

I 4 Time,

7

days

FIG. 5. Stability of the enzyme activity deaminating L-aspartate in liver mitochondria of S. canicula. The activity measured soon after the mitochondrial preparation was considered = 100. Liver mitochondria were prepared as indicated under Materials and Methods as procedure (i) ; the last precipitate was resuspended in Na-phosphate buffer 0"075 M, p H 7.4. Incubation time: 90 rain in air at 37°C. For other experimental conditions see Fig. 3.

916

L. CUTINELLIet al. I

E o

40

:::t. :50 E ,2o 20 "E E

I

I

.f'J"

/

I0 / I 8

I 16

I 24

32

Protein, mg

FIG. 6. Ammonia production from L-aspartate in the 50% sucrose concentration sedimenting band of a mitocbondrial preparation span on a linear sucrose gradient (30-70%). The mitoehondrial fraction was prepared as described under Materials and Methods. The incubation mixture and other experimental conditions were as described in Fig. 3. Ellfolk, 1953a, b, 1954a, b; Williams & Mclntyre, 1955). A detailed review of the studies on aspartase has been written by Ellfolk (1956). The only report on the appearance of aspartase in vertebrates, namely during the ontogeny of the frog, was indicated by Kurata (1962), but the evidence is merely suggestive. The data reported in the present paper demonstrate that an aspartase-like activity is present in the liver of elasmobranch fishes, particularly of the genus Scyllium; the activity is localized in a fairly homogeneous mitochondrial fraction. One of the main problems to be solved was concerned with the mechanism of ammonia formation from L-aspartate in the system studied: i.e. whether it was a direct or an indirect deamination. In fact, in the system used, the stoichiometry between fumarate and ammonia could not be considered by itself as a demonstration of the presence of an aspartase activity; the formation of fumarate after intermolecular transfer of the amino group from L-aspartate to various compounds (argininosuccinic acid, adenylosuccinic acid, etc.) was still possible. Furthermore, the possibility of a transdeamination with the eventual conversion of oxaloacetic acid into fumaric acid should also be considered. However, the latter possibility can be definitely excluded after the results shown in Tables 3 and 4, which do not support the intermediate formation of glutamate in aspartate deamination. Under this respect the data obtained with 15N-aspartate in the presence of an excess of unlabelled glutamate appear significant. Experiments performed in the presence of inosinic acid (see Tables 3 and 6) also exclude ammonia formation from L-aspartate by previous involvement of its amino group in purine nucleotide biosynthesis. Furthermore, the intermediate formation of carbamylaspartate, a-alanine,

ASPARTASE I N ELASMOBRANCH FISHES

917

/3-alanine and arginine, during L-aspartate deamination, cart be excluded from the data presented in Table 5. Experiments reported in Table 2 support a non-oxidative mechanism of ammonia formation from L-aspartate. Moreover, no direct oxidative deamination of L-aspartate has been so far reported in the literature (Greenberg, 1969). The data reported in this paper, therefore, support the presence of an aspartate : ammonia lyase in fish tissues. However, attempts to isolate the enzyme have so far been unsuccessful and have merely indicated the presence of the enzyme activity in the mitochondrial fraction. A comparison with bacterial aspartase is difficult at this stage of experimentation. Table 6, however, shows in some respect a resemblance of the fish enzyme with bacterial aspartase, studied by Williams & Lartigue (1967). The finding of an aspartase in the liver of elasmobranch fishes also has an interesting beating because in the liver of these animals no detectable amounts of glutamic-oxaloacetic transaminase were found to be present (Buonocore, 1969, personal communication). Therefore, the enzyme aspartase could have an important physiological significance for the metabolism of L-aspartate in these organisms. Finally, bacterial aspartase seems to have regulatory properties (Williams & Lartigue, 1967), and this observation is of interest in the study of analogous properties in animal aspartase. The finding of an aspartase in fish, which are among the lowest vertebrates, together with the finding of Kurata (1962) indicating the appearance of the enzyme during early embryonic development in the frog, also adds further interest for the possibility of using this enzyme as a probe for studies on biochemical evolution. Acknowledgements--This work was supported by a grant from the "Consiglio Nazionale delle Ricerche", Roma, Italy. Our thanks are due to Mrs. M. Cottino for her skilled assistance in some of the experiments. REFERENCES

BALESTRIERIC. (1968) An improved procedure for the measurement of I~N content by mass spectrometry. Life Sci. 7, 269-274. BURTON K. (1965) D-amino acid oxidase from kidney. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. II, pp. 199-204. Academic Press, New York. CEDRANGOLOF., SALVATOREF., CIMINO F. & ZAPPIAV. (1965) An improved method for determining ammonia formed in enzyme reactions. Enzymologia 29, 143-154. ELLFOLKN. (1953a) Studies on aspartase---I. Quantitative separation of aspartase from bacterial cells, and its partial purification. Acta chem. scand. 7, 824-830. ELLFOLKN. (1953b) Studies on aspartase---II. On the chemical nature of aspartase. ~lcta chem. scand. 7, 1155-1163. ELLFOLKN. (1954a) Studies on aspartase--III. On the specificity of aspartase..Zlcta chem. scand. 8, 151-156. ELLFOLKN. (1954b) Studies on aspartase--IV. On the effect of pH on aspartase. ~lcta chem. scand. 8, 443--448. ELLFOLKN. (1956) Aspartase. A study on its nature and purification. Ann..Zlead. Sci. Fennicae, Set. A, II, 79, 1-74.

918

L. CUTINELLIet al.

GALE E. F. (1938) Factors influencing bacterial deamination--III. Aspartase I I : its occurrence in and extraction from Bacterium coli and its activation by adenosine and related compounds. Biochem. ft. 32, 1583-1599. Gm~'~BERG D. M. (1969) Carbon catabolism of amino acids. In Metabolic Pathways (Edited by GPaEFa'~BERGD. M.), 3rd edn., Vol. I I I , pp. 95-190. Academic Press, New York. HInD F. J. R. & M~mGINSON M. A. (1966) Oxidative deamination of glutamate and transdeamination through glutamate. Archs Biochem. Biophys. 115, 247-256. HOCEBOOM G. H. (1955) Fractionation of cell components of animal tissues. In Methods in Em:ymology (Edited by COLOWIeKS. P. & KAPLANN. O.), Vol. I, pp. 16-19. Academic Press, New York. K ~ B s H. A. (1935) Metabolism of amino acids--IV. The synthesis of glutamine from glutamic acid and ammonia, and the enzymic hydrolysis of glutamine in animal tissues. Biochem. J. 29, 1951-1969. KURATAY. (1962) On the appearance of hydrogenase, nitrate reductase and aspartase during the ontogeny of the frog. Expl Cell Res. 28, 424-429. LICHSTEINH. C. & U~mt~tT W. W. (1947) Biotin activation of certain deaminases. J. biol. Chem. 170, 423-424. LowRY O. H., ROSEBROUCHN. J., F ~ R A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. McBE~'~ R. L., NEPPEL M. J. & GOLDS~IN L. (1966) Glutamate dehydrogenase and ammonia production in the eel (Anguilla rostrata). Comp. Biochem. Physiol. 18, 909920. MEISTER A. (1965) Biochemistry of the Amino Acids, 2nd edn., Vol. II, pp. 593-1020. Academic Press, New York and London. REXCHARDP. (1959) Advances in Emrymology, Vol. 21, pp. 263-294. Interscience, London. REINHOLD J. G. & CHUNO C. C. (1961) Formation of artifactual ammonia in blood by action of alkali. Clin. Chem. 7, 54-69. RODWELL V. W. (1969) Biosynthesis of amino acids and related compounds. In Metabolic Pathways (Edited by GmmNBEaC D. M.), 3rd edn., Vol. III, pp. 238-315. Academic Press, New York and London. SALLACHH. J. & FAHIEN L. A. (1969) Nitrogen metabolism of amino acids. In Metabolic Pathways (Edited by GREENBERGD. M.), 3rd edn., Vol. I I I , pp. 1-94. Academic Press, New York and London. SALVATOREF., ZAPPIA V. & COSTA C. (1965) Comparative biochemistry of deamination of L-amino acids in elasmobranch and teleost fish. Comp. Biochem. Physiol. 16, 303-309. SALVATORBF., ZAPPIA V. & COSTA C. (1967a) On the presence of an aspartase-like activity in some vertebrates. Abstr. Seventh Int. Congr. Bioehem., Abstract IV (F-247), p. 810, Science Council of Japan, Ueno Park, Tokyo. SALVATOPmF., ZAPPIA V., PmamOPAOLO C. & COTTINO M. (1967b) An aspartase method by a modified Fiirth and Herrmann reaction for fumarate. En~ymologia 33, 169-178. SAN PIETRO A. (1957) The measurement of stable isotopes. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLAN N. O.), Vol. IV, pp. 473-488. Academic Press, New York. SOTTOCASAG. L., KUYLF,NSTIERNAB., ERNSTm~L. & ANDERSB. (1967) Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. X (Oxidation and phosphorylation, Edited by PULLMANM. E.), pp. 448--463. Academic Press, New York and London. STILL J. L., BUELL M. V. & GREEN D. E. (1950) Studies on the cyclopborase system--IX. Oxidation of L-alanine. Archs Biochem. 26, 413--419.

ASPARTASE IN ELASMOBRANCH FISHES

919

TAKAHASHIH., TANIGUCHIS. & EGAMI F. (1963) Inorganic nitrogen compounds: distribution and metabolism. In Comparative Biochemistry (Edited by FLORKINM. & MASON H. S.), Vol. V, pp. 91-202. Academic Press, London. UTTER M. F. (1961) Nonoxidative carboxylation and decarboxylation. In The Enzymes (Edited by BO~CERP. D., LARDYH. & MYP~CK K.), 2nd edn., Vol. 5, pp. 319-340. Academic Press, New York. VENUTA S., PIETROPAOLOC., CUTINELLIL. & SALVATOREF. (1969) Studies on characterization of an aspartase-like activity in liver mitochondria of Elasmobranchs. Abstr. 1S8th Meeting American Chemical Society (Biol-25), New York. WILLIAMS V. R. & LARTIGUE D. J. (1967) Quaternary structure and certain allosteric properties of aspartase..7, biol. Chem. 242, 2973-2978. WILLIAMSV. R. & LARTIGUED. J. (1969) Aspartase. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. X l I I (Citric acid cycle, Edited by LOWENSTEIN J. M.), pp. 354-361. Academic Press, New York. WILLIAMS V. R. & MclNTY~ R. T. (1955) Preparation and partial purification of the aspartase of Bacterium cadaveris. .7. biol. Chem. 217, 467--477. WOOLF B. (1929) Some enzymes in B. coli communis which act on fumaric acid. Biochem. 3t. 23, 472-482. ZAPPIA V., COSTAC. & SALVATOREF. (1965a) Deaminazione degli aminoacidi naturali in organi di pesci elasmobranchi e teleostei. Atti ,4cc. Naz. Lincei, Rend. Sc. fis. mat. nat., ser. VIII, 38, 540-544. ZAPPIAV., PIETROPAOLOC., COSTAC. & SALVATOREF. (1965b) Deamination of L-aspartate in vertebrate tissues. Abstr. 150th Meeting American Chemical Society (C-74), Atlantic City. ZAPPIAV., PIETROPAOLOC., COSTAC. & SALVATOP~F. (1966a) Aspartase estimation with a modified Fiirth-Herrmann reaction for fumarate, tlbstr. 152nd Meeting American Chemical Society, (C-49), New York. ZAPPIAV., SALVATOI~F., COSTAC. & RICCIOM. (1964) Oxidative deamination of L-amino acids in various tissues of Teleosts and Elasmobranchs. Abstr. Sixth Int. Congr. Biochem., I.U.B., Vol. 32 (V-B-58), p. 403, New York. ZAPPIA V., TlxmR M. & SALVATO~F. (1966b) A spectrophotometric method for fumaric acid estimation by Fiirth and Herrmann reaction. Atti Aec. Naz. Lincei, Rend. Sc. fis. mat. nat., ser. VIII, 41, 97-103.

Key Word Index--Aspartase; aspartase in Vertebrates; aspartase in elasmobranch fishes; ScyUium canicuIa; aspartate metabolism in elasmobranch fishes; elasmobranch fishes; ScyUium stellare; aspartate deamination.