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Comparative study on AMP deaminase in gill, muscle and blood of fish
Comp. Biochem. Physiol. Vol. 67B, pp. 533 to 540
0305-0491/80/1201-0533502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
COMPARATIVE STUDY ON AMP DEAMINASE IN GILL, MUSCLE AND BLOOD OF FISH J. P. RAFFIN and C. LERAY Laboratoire de Physiologie compar6e des R6gulations, C.N.R.S., B.P. 20 CR, 67037 Strasbourg-C6dex, France
(Received 14 February 1980) Abstract 1. High AMP deaminase activities were determined in the gill of one selachian, Scyliorhinus caniculus, and five teleosts, Anguilla anguilla, C yprinus carpio, Salmo gairdneri, Perca fluviatilis and Esox lucius. 2. The highest activity was generally found in skeletal white muscle, except in A. anguilla and S. caniculus. 3. In S. caniculus a very high AMP deaminase activity was found in the blood where it was shown to be tightly regulated by inorganic phosphate. 4. Seasonal variations were observed for AMP deaminase activity in gill and white muscle, but also for blood Hb and protein concentration in the three tissues examined.
INTRODUCTION A M P deaminase (AMP aminohydrolase, EC 3.5.4.6.) plays an important role in the regulation of adenylate energy charge (Chapman & Atkinson, 1973), total adenylate pool (Lowenstein, 1972) and purine nucleotide interconversion (Setlow et al., 1966). The activity of A M P deaminase of skeletal white muscle is very high in comparison with that of all other tissues, including red muscle and blood (Conway & Cooke, 1939; Raggi et al., 1969). Raggi et al. (1975) established a correlation between anaerobic metabolism and A M P deaminase activity in white muscle. It is well known that in muscle A M P deaminase is associated with fibrillar proteins and that inorganic phosphate, a natural inhibitor of the enzyme (Lee & Wang, 1968; Kaletha et al., 1976), induces the dissociation of the complex (Currie & Webster, 1962; Harris & Suelter, 1967; Ashby & Frieden, 1977; Shiraki et al., 1979). A M P deaminase is also present in fish tissues (Makarewicz & Zydowo, 1962; Makarewicz, 1963, 1969; Dingle & Hines, 1967; Walton & Cowey, 1977). The enzyme is present in high concentration in gill tissue where it is associated with a very active purine metabolism (Leray et al., 1979). We report here the activities and some properties of A M P deaminase in tissues of different fish species. The results are discussed with reference to seasonal variations and association of the enzyme with muscular fibrillar proteins. MATERIALS AND METHODS Materials The following five species of freshwater teleosts and one species of marine selachian were used: eel (Anguilla
* Abbreviations: DTT--oL-dithiothreitol; AMP--adenosine-5'-monophosphate; IMP--inosine-5'-monophosphate; MES---2[N-morpholino]ethane sulfonic acid; Hb--hemoglobin.
anguilla), rainbow trout (Salmo gairdneri), northern pike (Esox lucius), perch (Perca fluviatilis), carp (Cyprinus carpio) and dogfish (Scyliorhinus caniculus). The periods of investigation are given in Table 1. S. gairdneri were obtained from a local hatchery, S. caniculus were collected in the English Channel, all other species were collected in inland waters of Alsace. S. caniculus were maintained in aerated and filtered seawater at 10-13°C; all other species in aerated running freshwater at 12-13°C. The fish were kept at least 2 weeks in the laboratory before sampling. Preparation of the extracts The blood was taken from the caudal vessels with citrated heparin. Enzyme activity was determined on a sonicated (2 × 15 sec) blood extract. Gill arches were excised, gently blotted dry and the mucosa scrapped with a scalpel. A 10~o (w/v) suspension was made in 50 mM potassium phosphate pH 6.5, 0.1 mM DTT* or 50mM MES pH 6.5, 0.1 mM DTT, and the homogenization was carried out by two 30 sec sonications. The extract was centrifuged at 10,000 g for 5 min. A fragment of latero-dorsat muscle was excised just behind the head of the animal and treated as described for the gill. In order to prevent enzyme activation by proteolytic enzymes (Rattin & Leray, 1979), the AMP deaminase activity was measured immediately after preparation of the extract. Enzyme assay The AMP deaminase activity was determined at 265 nm (Kalckar, 1947), using a Jobin-Yvon spectrophotometer (Ultraspac). The reaction mixture (2 ml) contained 50 mM cacodylate pH 6.7, 0.1 mM AMP and 1-100 #1 extract. The amount of AMP converted to IMP was calculated using a A~mM of 8.1. One unit of AMP deaminase is defined as the amount of enzyme deaminating 1/~mol of AMP/rain at 25°C in our experimental conditions. The specific activity is expressed as Units per mg of protein. Determination of protein The protein content of the extracts was determined using an automated version of the method of Lowry et al. (1951). 533
March'78
5
4
5
5
3
508
736
179
246
513
836
185
146
204
216
358
383
± 30
± 37
± 20
± 6
± 88
±
± 32
± 20
± 29
± 23
± 57
(g)
Weight
±
±
57
90
55
88
85
±
±
±
±
±
66 ±
80
62 ±
112 ±
91
145 ±
(mg/ml)
Blood Hb
* P < 0.05. ** P < 0.01. The enzyme activities are expressed in mUnits/mg of Hb. For experimental details see Materials and Methods.
Scyliorhinus
November'77
May'79
Esox
Selachian
November'79
November'79
5
5
March'78
March'78
5
November'79
5
5
May'79
November'79
5
specimens
investigation
March'78
Number of
Period of
Salmo
Cyprinus
Perca
Anguilla
Teleosts
Species
x
6
24
4
12
I ~=
5
7
II
6
7 xx
II
Table 1. Weight, Hb concentration and blood A M P deaminase activity in 6 fish species(mean ± SE)
111.93
±20.93
7.07
± 0.62
11.25
±
± 0.67
3.19
105.30
± 0.18
3.87
-+ 0 . 0 3
± 0.16
0.90
1.89
± 0.43
3.47
± 0.18
± 0.92
5.07
0.83
± 0.48
3.66
AMP deaminase
x:: >
>
4~
May'79
Esox
-
91±4
104±3
132±6
n.d.
42.07±2.48
39.53±0.62
18.69±0.66
5.86±0.72
26.77±2.86
32.43±4.25
deaminase
A~
93±16
98+2
I13±2
126±5
145±2
133±2 ~:~:~
93±3
(mg/g ~eshweigh0
Protein
AMP
40.61±11.68
42.53± 0.99
43~48± 1.88
27.50+ 1.74
9.26± 0.91
59.44± 4.30
109.05±17.19
deaminase
MES extract
::
~
lO0
3.4±4.5
8.4±4.5
32.0±2.0
37.8±4.4
54.8±4.1 ~:
70.0±1.3
phosphate
by 0.5 r~
% inhibition
The enzyme activities are expressed in mUnits/mg of protein. For experimental conditions see Materials and Methods. In S. caniculus the A M P deaminase activities were corrected for contamination by blood.
n.d. = not detected. * P < 0.05. *** P < 0.001.
Scy~iorhinus
November'77
November'79
Sa~mo
Selachian
November'79
Cyprinu8
162±7
141±3 ::~:~:
November'79
November'79
104±4
(mg/g fresh weight)
Protein
May'79
investigation
Period of
Perca
Anguilla
Teleosts
Species
Phosphate extract
Table 2. Protein concentration and A M P deaminase activityin gillextracts of 6 fish species(mean ± SE)
m~
;D
>
536
J.P. RAFFINand C. LERAY
°°l 75
o
5O
"6
25
0
I
L
I
I
I
I
2
5
4
5
Phosphote,
mM
Fig. 1. Inhibition by inorganic phosphate of AMP deaminase in blood (O) and gill (*) of S. caniculus. Conditions same as in standard assay (see Materials and Methods).
Determination of Hb The concentration was measured on 20/A of blood or on 50 #1 of gill extract with a ferricyanide-cyanide reagent (test combination hemoglobin, Boehringer Mannheim).
Statistical treatment of data The results are expressed as the mean of the measurements + standard error. The comparison of the means have been tested by the variance ratio F of Fisher-Snedecor and Student's t-test.
Chemicals AMP, DTT and MES from Sigma Chemical Co. All other chemicals were from Merck, Darmstadt. RESULTS
Blood The weight of the fishes and the blood Hb concentration and A M P deaminase activity are given in Table 1. The highest Hb concentrations were found in A. anguilla. Seasonal variations were observed in A. anguilla and C. carpio. In A. anguilla, there was a decrease in blood Hb concentration between March and May with no further significant change between May and November. In contrast, in C. carpio there was an increase between March and November. The specific activity of A M P deaminase in blood was very high in S. caniculus. The activity was much lower in all other species studied and was the lowest in P. fluviatilis. In C. carpio there was an increase in A M P deaminase activity between March and November.
Gill The protein concentrations and A M P deaminase activities in phosphate and MES extracts of the gill
are given in Table 2. In the phosphate extract, the highest A M P deaminase activity was found in E. lucius and the lowest in S. caniculus, while in the MES extract the highest activity was found in A. anguilla and the lowest in P. fluviatilis. The addition of the phosphate extract to the enzyme assay mixture results in a final phosphate concentration of about 0.5 mM. This can explain the lower activity found in the phosphate extract relative to the MES extract. The percent inhibition of the enzyme by 0.5 m M phosphate is given in the right column of Table 2. The S. caniculus enzyme is almost completely inhibited by this phosphate concentration (Fig. 1). In this species, the branchial A M P deaminase is much more sensitive to inorganic phosphate than the blood enzyme. In A. anguilla we observed a difference in sensitivity to inorganic phosphate between May and November. In E. lucius and S. gairdneri the enzyme was much less sensitive to phosphate. Seasonal variations in gill protein concentration and A M P deaminase activity were observed in A. anguilla between May and November.
White muscle The protein concentrations and A M P deaminase activities in phosphate and MES extracts of white skeletal muscle are given in Table 3. In A. anguilla and C. carpio there was a decrease in branchial protein concentration between March and November. Except for A. anguilla and S. caniculus, the A M P deaminase activity was lower in MES than in phosphate extracts. This effect is probably related to the ability of inorganic phosphate to dissociate A M P deaminase from muscular fibrillar proteins (Currie &
March'78
-
-
57±3
78±2
n.d.
n.d.
159.48±31.07
|09.59±7.80
153.05±15.94
56.64±5.74
,':u'~,"
63±4
49±12
55±3
69±4
56±9
|00±6
69± 5
86±6
16.91±2.96 70.49± 9.59 :'""
54±7
78+4
'::"
~:
(mg/g fresh weight)
Protein
13.22±4.51
3.26±1.09
AMP deaminase
MES e x t r a c t
1.86±0.73
4.73±1.36
13.66±2.44
17.49i2.45
56.56±27.12
25.40±3.60
]9.34± I .34 ':'::"
5.84±0.97
I0.02±2.36
5.82±1.33
AMP deaminase
n.d. = not detected * P < 0.05. **P < 0.01. *** P < 0.001. The enzyme activities are expressed in m/Units/rag of protein. For experimental conditions see Materials and Methods.
Scyliorhinus
November'77
May'79
Esox
Selachian
November'79
:~:
61_+5 ~ : : :
90±5
March' 78
November' 79
77± 6
87±7
March' 78
November '79
51±5
9J±4
(mg/g fresh weight)
Protein
November'79
March'78
investigation
Period of
Salmo
Cyprinus
Perca
Anguilla
Teleosts
Species
Phosphate extract
O.II±O.03
0.16±0.02
0.38-+0.]9
0.49+0.12
O. 29±O. 03
0.40-+0. I l
].38±O.60
2.18±0.58
AMP d e a m i n a s e p h o s p h a t e
AMP deaminase MES
Table 3 Protein conc~ntrmion and A M P deaminase activityin white muscle extracts o f s i x fish species(mean ± SE) I
==
B
>
538
J.P. RAFFINand C. LERAY
Webster, 1962; Makarewicz, 1969). The ratio of the enzyme activities in MES and phosphate extracts was maximal in A. anguilla and minimal in E. lucius and S. gairdneri. This could be due to a better dissociation by phosphate of the complex AMP deaminase-myosin in E. lucius and S. gairdneri than in A. anguilla. In order to test this possibility, we measured the enzyme activities and the protein concentrations in the extracts before and after centrifugation at 10,000 g. As seen in Table 4, the low activity ratio observed in S. gairdneri can be explained by the fact that the enzyme is much better dissociated from myosin by phosphate than by MES buffer. This does not seem to be the case for A. anguilla where the activity is higher in MES than in phosphate extract. In S. caniculus no detectable activity was measured in the phosphate extract due probably to the high sensitivity of AMP deaminase in this species to inhibition by inorganic phosphate. In P. fluviatilis, and C. carpio an increase in muscle AMP deaminase activity was observed between March and November.
DISCUSSION
A. anguilla (March) and S. caniculus (November) were the same as that used in a previous study on the purine content of erythrocytes (Leray, 1979). The blood Hb concentrations found in P. fluviatilis and S. gairdneri are close to the values published by this author. However, in E. lucius, our values are lower. This could be explained by in increase in blood Hb concentration between May and November since the cited work was made in November. In C. carpio we observed a similar increase in Hb concentration between March and November. Seasonal variations were also observed for AMP deaminase activity in the gill (the specific activity decreases in A. anguilla) and in the white muscle (the specific activity increases in P. fluviatilis and C. carpio ). During this same period we observed a decrease in muscle protein concentration in A. anguilla and C. carpio, and an increase in gill protein concentration in A. anguilla. Thus, the changes observed for AMP deaminase specific activity seem to be the consequence of variations in protein concentration, at least in A. anguilla and C. carpio, while the enzyme level would be kept almost constant. As already reported by Makarewicz (1963), the gill contains high AMP deaminase activity. In his study, he reported that the activity was higher in S. gairdner than in A. anguilla. This contrasts with our results and can be explained by differences in the period of investigation and conditions of enzyme determination. In A. anguilla the gill enzyme is less sensitive to inhibition by phosphate in November than in May. This can be explained by the presence of different isoenzymes of AMP deaminase at these different periods. This hypothesis is corroborated by the fact that different isoenzymes patterns were demonstrated on phosphoceUulose during the annual cycle (unpublished results). The activity of muscle AMP deaminase is underestimated in our study. However, a delay of 1 or 2 hr between homogenization and centrifugation, which allows the dissociation of AMP deaminase from fibril-
lar proteins, would have resulted in a rapid activation of the enzyme by endogenous proteolytic enzymes (results not shown). In spite of that limitation, we observe that in P. fluviatilis, C. carpio, S. gairdneri and E. lucius the highest activity is found in white muscle. The theoretical activity of AMP deaminase in A. anguilla muscle, i.e. if all the enzyme would be dissociated from myosin, can be calculated from Table 4 by dividing the activity (U/ml) in MES buffer, before centrifugation, by the protein concentration after centrifugation. The specific activity of AMP deaminase would then be 70mU/mg protein. Thus, in A. anguilla, in November, the AMP deaminase activity is only 20Vo higher in the muscle than in the gill. In S. caniculus, a very high AMP deaminase activity was found in the blood. The specific activity in that tissue is of the same order as that found normally in white muscle. In particular, if we refer to the comparative study of Kruckeberg & Chilson (1973), this seems to be by far the highest activity found in the blood of various species. S. caniculus was the only selachian fish used in our study. It would be interesting to determine if such high AMP deaminase activities are found in the blood of other selachian species. However, an isolated test made on Raia clavata showed a very low activity in the blood of this species (result not shown). The enzyme of S. caniculus is submitted to a tight regulation by inorganic phosphate. In this species, the gill AMP deaminase is almost completely inhibited in the presence of 1 mM phosphate, while in S. gairdneri a 50 times higher concentration is necessary to achieve the same inhibition (Leray et al., 1979). In white muscle, the enzyme seems also to be strongly inhibited by phosphate as no activity could be detected in the phosphate extract. This also does not seem to be the case for all selachian species since a much lower sensitivity to inorganic phosphate was reported for the enzyme of Raia clavata (Makarewicz, 1969). We could find no correlation between the AMP deaminase activities in the three tissues investigated and the ecological features of the species used in our study. For example, in the gill, high AMP deaminase activities are found in A. anguilla, a species which tolerates hypoxic conditions. In C. carpio, a species showing the same tolerance to hypoxia, the activity is two times lower than in A. anguilla. In S. gairdneri, a species which requires higher oxygenated waters, the AMP deaminase activity is not very different from A. anguiUa. Moreover, no correlation was found concerning the sensitivity to phosphate. Similarly, no correlation was found between the blood AMP deaminase activities given in this study and the nucleotide contents previously reported (Leray, 1979). In white muscle, more accurate determination needs to be done due to an underestimation of AMP deaminase activity, in our study, as a consequence of only partial dissociation of the enzyme from myosin. It seems, however, that at least in gill and white muscle, AMP deaminase is a constitutive enzyme. So it would be interesting to determine, among species living in different environmental conditions, if there are variations in the regulation and the isoenzyme patterns of AMP deaminase. The hypothesis of a different role of AMP deaminase isoenzymes related to an adaptative capacity to aerobic or anaerobic metab-
Extract
Experimental details are given in Materials and M e t h o d s
6.36 ± 0.45
MES centrifuged
± 2.96
19.31
7.81 ± 0.77
phosphate
MES
20.34 ± 3.10
phosphate
centrifuged
4.25 ± 0.34
MES centrifuged
Salmo
7.07 ± 1.08
MES
5.36 ± 0.49
centrifuged
phosphate
(mg/ml)
9.60 ± 1.84
Protein
phosphate
Anguilla
I
i
± 0.143
O.119 ± 0.025
2.858 ± 0.334
0.852
2.616 ± 0.455
0.069 ± 0.020
0.297 ± 0.069
0.029 ± 0.005
0.308 ± 0.074
U /ml
AMP deaminase
19.78 ± 5.21
137.68 ± 14.38
113.45 ± 20.17
134.78 ± 17.94
17.09 ± 5.64
41.11 ± 7.64
5 . 7 5 ± 1.19
30.69 ± 3.49
activity
deaminase
Specific
~P
Table 4 Protein concentration and A M P deaminase activity in A. anguilla and S. gairdneri white muscle before and after centrifugation at 10,000 g for 5 min (mean + SE)
>
540
J.P. RAFFIN and C. LERAY
olism (Raggi et al., 1975) should be verified in different tissues and species. In summary, our experiments reveal that, at least in fish, the white muscle is not always the tissue containing the highest A M P deaminase activity. In particular, a very high activity is found in the blood of S. caniculus. These studies need to be extended to other freshwater and seawater fish and, in particular, other selachian species. Care must be taken to the occurrence of seasonal variations in A M P deaminase activity in some species.
REFERENCES
Asrmv B. & FRmDEN C. (1977) Interaction of AMP deaminase with myosin and its subfragments. J. biol. Chem. 252, 1869-1872. CHAPMAN A. G. & ATKINSON D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. CONWAV E. J. & COOKE R. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem. J. 33, 479-492. CURRIER. D. & WEBS~R H. L. (1962) Preparation of 5'-adenylic acid deaminase based on phosphate-induced dissociation of rat actomyosin--deaminase complexes. Biochim. biophys. Acta 64, 3040. DINGLE J. R. & HINES J. A. (1967) Extraction and some properties of adenosine 5'-monophosphate aminohydrolase from prerigor and postrigor muscle of cod. J. Fish. Res. Bd Can. 24, 1717-1730. HARRIS M. & SUELTER C. H. (1967) A simple chromatographic procedure for the preparation of rabbit-muscle myosin A free from AMP deaminase. Biochim biophys. Acta 133, 393--398. KALCKAR H. M. (1947) Differential spectrometry of purine compounds by means of specific enzymes~ II. Determination of adenine compounds. J. biol. Chem. 167, 445-459. KALETHA K., STANKIEW1CZ m., MAKAREWICZ W. & ZYOOWO M. (1976) The influence of phosphate, fluoride and potassium ions on the activity of AMP deaminase from human skeletal muscle. Int. J. Biochem. 7, 67-71. KRUCKEnERG W. C. & CHILSON O. P. (1973) Red blood cell AMP deaminase: levels of activity in hemolysates from twenty different vertebrate species. Comp. Biochem. Physiol. 46B, 653-660. LEE Y. P. & WaNG M. H. (1968) Studies on the nature of
the inhibitory action of inorganic phosphate, fluoride and detergents on 5'-adenylic acid deaminase activity and on the activation by adenosine triphosphate. J. biol. Chem. 243, 2260-2265. LERAV C. (1979) Patterns of purine nucleotides in fish erythrocytes. Comp. Biochem. Physiol. 64B, 77-82. LERAY C., RAFFIN J. P. & WINNINGER C. (1979) Aspects of purine metabolism in the gill epithelium of rainbow trout Salmo 9airdneri Richardson. Comp. Biochem. Physiol. 62B, 31-40. LOWENSTEIN J. M. (1972) Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Rev. 52, 382-414. LOWRV O H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAKAREW1CZ W. (1963) AMP aminohydrolase and glutaminase activities in the kidneys and gills of some freshwater vertebrates. Acta biochim, polon. 10, 363-369. MAKAREWlCZ W. (1969) AMP aminohydrolase in muscle of elasmobranch fish. Purification procedure and properties of the purified enzyme. Comp. Bioehem. Physiol. 29, 1-26. MAKAREWlCZ W. & ZVDOWO M. (1962). Comparative studies on some ammonia-producing enzymes in the excretory organs of vertebrates. Comp. Biochem. Physiol. 6, 269-275. RAFrlN J. P. & LERAVC. (1979) AMP deaminase in the gill of trout (Salmo gairdneri R.). Modalities of an activation by cellular proteolytic enzymes. Comp. Biochem. Physiol. 62B, 23-29. RAGGI A, BERGAMIN1C. & RONCA G. Isozymes of AMP deaminase in red and white skeletal muscles. FEBS Lett. 58, 19-23. RAGGI A., RONCA-TESTONI S. & RONCA G. (1969) Muscle AMP aminohydrolase. II. Distribution of AMP aminohydrolase, myokinase and creatine kinase activities in skeletal muscle. Biochim. biophys. Aeta 178, 619-622. SETLOW B~, BURGERR. & LOWENSTEINJ. M. (1966). Adenylate deaminase. I. The effects of adenosine and guanosine triphosphates on activity and the organ distribution of the regulated enzyme. J. biol. Chem. 241, 1244-1245. SHIRAKI H., OGAWA H., MATSUDA Y. & NAKAGAWA H. (1979) Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle. Biochim. biophys. Acta 566, 335-344~ WALTOn M. J. & COWEY C. B. (1977) Aspects of ammoniogenesis in rainbow trout, Salmo oairdneri. Comp. Biochem. Physiol. 57B, 143 149.
0305-0491/80/1201-0533502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
COMPARATIVE STUDY ON AMP DEAMINASE IN GILL, MUSCLE AND BLOOD OF FISH J. P. RAFFIN and C. LERAY Laboratoire de Physiologie compar6e des R6gulations, C.N.R.S., B.P. 20 CR, 67037 Strasbourg-C6dex, France
(Received 14 February 1980) Abstract 1. High AMP deaminase activities were determined in the gill of one selachian, Scyliorhinus caniculus, and five teleosts, Anguilla anguilla, C yprinus carpio, Salmo gairdneri, Perca fluviatilis and Esox lucius. 2. The highest activity was generally found in skeletal white muscle, except in A. anguilla and S. caniculus. 3. In S. caniculus a very high AMP deaminase activity was found in the blood where it was shown to be tightly regulated by inorganic phosphate. 4. Seasonal variations were observed for AMP deaminase activity in gill and white muscle, but also for blood Hb and protein concentration in the three tissues examined.
INTRODUCTION A M P deaminase (AMP aminohydrolase, EC 3.5.4.6.) plays an important role in the regulation of adenylate energy charge (Chapman & Atkinson, 1973), total adenylate pool (Lowenstein, 1972) and purine nucleotide interconversion (Setlow et al., 1966). The activity of A M P deaminase of skeletal white muscle is very high in comparison with that of all other tissues, including red muscle and blood (Conway & Cooke, 1939; Raggi et al., 1969). Raggi et al. (1975) established a correlation between anaerobic metabolism and A M P deaminase activity in white muscle. It is well known that in muscle A M P deaminase is associated with fibrillar proteins and that inorganic phosphate, a natural inhibitor of the enzyme (Lee & Wang, 1968; Kaletha et al., 1976), induces the dissociation of the complex (Currie & Webster, 1962; Harris & Suelter, 1967; Ashby & Frieden, 1977; Shiraki et al., 1979). A M P deaminase is also present in fish tissues (Makarewicz & Zydowo, 1962; Makarewicz, 1963, 1969; Dingle & Hines, 1967; Walton & Cowey, 1977). The enzyme is present in high concentration in gill tissue where it is associated with a very active purine metabolism (Leray et al., 1979). We report here the activities and some properties of A M P deaminase in tissues of different fish species. The results are discussed with reference to seasonal variations and association of the enzyme with muscular fibrillar proteins. MATERIALS AND METHODS Materials The following five species of freshwater teleosts and one species of marine selachian were used: eel (Anguilla
* Abbreviations: DTT--oL-dithiothreitol; AMP--adenosine-5'-monophosphate; IMP--inosine-5'-monophosphate; MES---2[N-morpholino]ethane sulfonic acid; Hb--hemoglobin.
anguilla), rainbow trout (Salmo gairdneri), northern pike (Esox lucius), perch (Perca fluviatilis), carp (Cyprinus carpio) and dogfish (Scyliorhinus caniculus). The periods of investigation are given in Table 1. S. gairdneri were obtained from a local hatchery, S. caniculus were collected in the English Channel, all other species were collected in inland waters of Alsace. S. caniculus were maintained in aerated and filtered seawater at 10-13°C; all other species in aerated running freshwater at 12-13°C. The fish were kept at least 2 weeks in the laboratory before sampling. Preparation of the extracts The blood was taken from the caudal vessels with citrated heparin. Enzyme activity was determined on a sonicated (2 × 15 sec) blood extract. Gill arches were excised, gently blotted dry and the mucosa scrapped with a scalpel. A 10~o (w/v) suspension was made in 50 mM potassium phosphate pH 6.5, 0.1 mM DTT* or 50mM MES pH 6.5, 0.1 mM DTT, and the homogenization was carried out by two 30 sec sonications. The extract was centrifuged at 10,000 g for 5 min. A fragment of latero-dorsat muscle was excised just behind the head of the animal and treated as described for the gill. In order to prevent enzyme activation by proteolytic enzymes (Rattin & Leray, 1979), the AMP deaminase activity was measured immediately after preparation of the extract. Enzyme assay The AMP deaminase activity was determined at 265 nm (Kalckar, 1947), using a Jobin-Yvon spectrophotometer (Ultraspac). The reaction mixture (2 ml) contained 50 mM cacodylate pH 6.7, 0.1 mM AMP and 1-100 #1 extract. The amount of AMP converted to IMP was calculated using a A~mM of 8.1. One unit of AMP deaminase is defined as the amount of enzyme deaminating 1/~mol of AMP/rain at 25°C in our experimental conditions. The specific activity is expressed as Units per mg of protein. Determination of protein The protein content of the extracts was determined using an automated version of the method of Lowry et al. (1951). 533
March'78
5
4
5
5
3
508
736
179
246
513
836
185
146
204
216
358
383
± 30
± 37
± 20
± 6
± 88
±
± 32
± 20
± 29
± 23
± 57
(g)
Weight
±
±
57
90
55
88
85
±
±
±
±
±
66 ±
80
62 ±
112 ±
91
145 ±
(mg/ml)
Blood Hb
* P < 0.05. ** P < 0.01. The enzyme activities are expressed in mUnits/mg of Hb. For experimental details see Materials and Methods.
Scyliorhinus
November'77
May'79
Esox
Selachian
November'79
November'79
5
5
March'78
March'78
5
November'79
5
5
May'79
November'79
5
specimens
investigation
March'78
Number of
Period of
Salmo
Cyprinus
Perca
Anguilla
Teleosts
Species
x
6
24
4
12
I ~=
5
7
II
6
7 xx
II
Table 1. Weight, Hb concentration and blood A M P deaminase activity in 6 fish species(mean ± SE)
111.93
±20.93
7.07
± 0.62
11.25
±
± 0.67
3.19
105.30
± 0.18
3.87
-+ 0 . 0 3
± 0.16
0.90
1.89
± 0.43
3.47
± 0.18
± 0.92
5.07
0.83
± 0.48
3.66
AMP deaminase
x:: >
>
4~
May'79
Esox
-
91±4
104±3
132±6
n.d.
42.07±2.48
39.53±0.62
18.69±0.66
5.86±0.72
26.77±2.86
32.43±4.25
deaminase
A~
93±16
98+2
I13±2
126±5
145±2
133±2 ~:~:~
93±3
(mg/g ~eshweigh0
Protein
AMP
40.61±11.68
42.53± 0.99
43~48± 1.88
27.50+ 1.74
9.26± 0.91
59.44± 4.30
109.05±17.19
deaminase
MES extract
::
~
lO0
3.4±4.5
8.4±4.5
32.0±2.0
37.8±4.4
54.8±4.1 ~:
70.0±1.3
phosphate
by 0.5 r~
% inhibition
The enzyme activities are expressed in mUnits/mg of protein. For experimental conditions see Materials and Methods. In S. caniculus the A M P deaminase activities were corrected for contamination by blood.
n.d. = not detected. * P < 0.05. *** P < 0.001.
Scy~iorhinus
November'77
November'79
Sa~mo
Selachian
November'79
Cyprinu8
162±7
141±3 ::~:~:
November'79
November'79
104±4
(mg/g fresh weight)
Protein
May'79
investigation
Period of
Perca
Anguilla
Teleosts
Species
Phosphate extract
Table 2. Protein concentration and A M P deaminase activityin gillextracts of 6 fish species(mean ± SE)
m~
;D
>
536
J.P. RAFFINand C. LERAY
°°l 75
o
5O
"6
25
0
I
L
I
I
I
I
2
5
4
5
Phosphote,
mM
Fig. 1. Inhibition by inorganic phosphate of AMP deaminase in blood (O) and gill (*) of S. caniculus. Conditions same as in standard assay (see Materials and Methods).
Determination of Hb The concentration was measured on 20/A of blood or on 50 #1 of gill extract with a ferricyanide-cyanide reagent (test combination hemoglobin, Boehringer Mannheim).
Statistical treatment of data The results are expressed as the mean of the measurements + standard error. The comparison of the means have been tested by the variance ratio F of Fisher-Snedecor and Student's t-test.
Chemicals AMP, DTT and MES from Sigma Chemical Co. All other chemicals were from Merck, Darmstadt. RESULTS
Blood The weight of the fishes and the blood Hb concentration and A M P deaminase activity are given in Table 1. The highest Hb concentrations were found in A. anguilla. Seasonal variations were observed in A. anguilla and C. carpio. In A. anguilla, there was a decrease in blood Hb concentration between March and May with no further significant change between May and November. In contrast, in C. carpio there was an increase between March and November. The specific activity of A M P deaminase in blood was very high in S. caniculus. The activity was much lower in all other species studied and was the lowest in P. fluviatilis. In C. carpio there was an increase in A M P deaminase activity between March and November.
Gill The protein concentrations and A M P deaminase activities in phosphate and MES extracts of the gill
are given in Table 2. In the phosphate extract, the highest A M P deaminase activity was found in E. lucius and the lowest in S. caniculus, while in the MES extract the highest activity was found in A. anguilla and the lowest in P. fluviatilis. The addition of the phosphate extract to the enzyme assay mixture results in a final phosphate concentration of about 0.5 mM. This can explain the lower activity found in the phosphate extract relative to the MES extract. The percent inhibition of the enzyme by 0.5 m M phosphate is given in the right column of Table 2. The S. caniculus enzyme is almost completely inhibited by this phosphate concentration (Fig. 1). In this species, the branchial A M P deaminase is much more sensitive to inorganic phosphate than the blood enzyme. In A. anguilla we observed a difference in sensitivity to inorganic phosphate between May and November. In E. lucius and S. gairdneri the enzyme was much less sensitive to phosphate. Seasonal variations in gill protein concentration and A M P deaminase activity were observed in A. anguilla between May and November.
White muscle The protein concentrations and A M P deaminase activities in phosphate and MES extracts of white skeletal muscle are given in Table 3. In A. anguilla and C. carpio there was a decrease in branchial protein concentration between March and November. Except for A. anguilla and S. caniculus, the A M P deaminase activity was lower in MES than in phosphate extracts. This effect is probably related to the ability of inorganic phosphate to dissociate A M P deaminase from muscular fibrillar proteins (Currie &
March'78
-
-
57±3
78±2
n.d.
n.d.
159.48±31.07
|09.59±7.80
153.05±15.94
56.64±5.74
,':u'~,"
63±4
49±12
55±3
69±4
56±9
|00±6
69± 5
86±6
16.91±2.96 70.49± 9.59 :'""
54±7
78+4
'::"
~:
(mg/g fresh weight)
Protein
13.22±4.51
3.26±1.09
AMP deaminase
MES e x t r a c t
1.86±0.73
4.73±1.36
13.66±2.44
17.49i2.45
56.56±27.12
25.40±3.60
]9.34± I .34 ':'::"
5.84±0.97
I0.02±2.36
5.82±1.33
AMP deaminase
n.d. = not detected * P < 0.05. **P < 0.01. *** P < 0.001. The enzyme activities are expressed in m/Units/rag of protein. For experimental conditions see Materials and Methods.
Scyliorhinus
November'77
May'79
Esox
Selachian
November'79
:~:
61_+5 ~ : : :
90±5
March' 78
November' 79
77± 6
87±7
March' 78
November '79
51±5
9J±4
(mg/g fresh weight)
Protein
November'79
March'78
investigation
Period of
Salmo
Cyprinus
Perca
Anguilla
Teleosts
Species
Phosphate extract
O.II±O.03
0.16±0.02
0.38-+0.]9
0.49+0.12
O. 29±O. 03
0.40-+0. I l
].38±O.60
2.18±0.58
AMP d e a m i n a s e p h o s p h a t e
AMP deaminase MES
Table 3 Protein conc~ntrmion and A M P deaminase activityin white muscle extracts o f s i x fish species(mean ± SE) I
==
B
>
538
J.P. RAFFINand C. LERAY
Webster, 1962; Makarewicz, 1969). The ratio of the enzyme activities in MES and phosphate extracts was maximal in A. anguilla and minimal in E. lucius and S. gairdneri. This could be due to a better dissociation by phosphate of the complex AMP deaminase-myosin in E. lucius and S. gairdneri than in A. anguilla. In order to test this possibility, we measured the enzyme activities and the protein concentrations in the extracts before and after centrifugation at 10,000 g. As seen in Table 4, the low activity ratio observed in S. gairdneri can be explained by the fact that the enzyme is much better dissociated from myosin by phosphate than by MES buffer. This does not seem to be the case for A. anguilla where the activity is higher in MES than in phosphate extract. In S. caniculus no detectable activity was measured in the phosphate extract due probably to the high sensitivity of AMP deaminase in this species to inhibition by inorganic phosphate. In P. fluviatilis, and C. carpio an increase in muscle AMP deaminase activity was observed between March and November.
DISCUSSION
A. anguilla (March) and S. caniculus (November) were the same as that used in a previous study on the purine content of erythrocytes (Leray, 1979). The blood Hb concentrations found in P. fluviatilis and S. gairdneri are close to the values published by this author. However, in E. lucius, our values are lower. This could be explained by in increase in blood Hb concentration between May and November since the cited work was made in November. In C. carpio we observed a similar increase in Hb concentration between March and November. Seasonal variations were also observed for AMP deaminase activity in the gill (the specific activity decreases in A. anguilla) and in the white muscle (the specific activity increases in P. fluviatilis and C. carpio ). During this same period we observed a decrease in muscle protein concentration in A. anguilla and C. carpio, and an increase in gill protein concentration in A. anguilla. Thus, the changes observed for AMP deaminase specific activity seem to be the consequence of variations in protein concentration, at least in A. anguilla and C. carpio, while the enzyme level would be kept almost constant. As already reported by Makarewicz (1963), the gill contains high AMP deaminase activity. In his study, he reported that the activity was higher in S. gairdner than in A. anguilla. This contrasts with our results and can be explained by differences in the period of investigation and conditions of enzyme determination. In A. anguilla the gill enzyme is less sensitive to inhibition by phosphate in November than in May. This can be explained by the presence of different isoenzymes of AMP deaminase at these different periods. This hypothesis is corroborated by the fact that different isoenzymes patterns were demonstrated on phosphoceUulose during the annual cycle (unpublished results). The activity of muscle AMP deaminase is underestimated in our study. However, a delay of 1 or 2 hr between homogenization and centrifugation, which allows the dissociation of AMP deaminase from fibril-
lar proteins, would have resulted in a rapid activation of the enzyme by endogenous proteolytic enzymes (results not shown). In spite of that limitation, we observe that in P. fluviatilis, C. carpio, S. gairdneri and E. lucius the highest activity is found in white muscle. The theoretical activity of AMP deaminase in A. anguilla muscle, i.e. if all the enzyme would be dissociated from myosin, can be calculated from Table 4 by dividing the activity (U/ml) in MES buffer, before centrifugation, by the protein concentration after centrifugation. The specific activity of AMP deaminase would then be 70mU/mg protein. Thus, in A. anguilla, in November, the AMP deaminase activity is only 20Vo higher in the muscle than in the gill. In S. caniculus, a very high AMP deaminase activity was found in the blood. The specific activity in that tissue is of the same order as that found normally in white muscle. In particular, if we refer to the comparative study of Kruckeberg & Chilson (1973), this seems to be by far the highest activity found in the blood of various species. S. caniculus was the only selachian fish used in our study. It would be interesting to determine if such high AMP deaminase activities are found in the blood of other selachian species. However, an isolated test made on Raia clavata showed a very low activity in the blood of this species (result not shown). The enzyme of S. caniculus is submitted to a tight regulation by inorganic phosphate. In this species, the gill AMP deaminase is almost completely inhibited in the presence of 1 mM phosphate, while in S. gairdneri a 50 times higher concentration is necessary to achieve the same inhibition (Leray et al., 1979). In white muscle, the enzyme seems also to be strongly inhibited by phosphate as no activity could be detected in the phosphate extract. This also does not seem to be the case for all selachian species since a much lower sensitivity to inorganic phosphate was reported for the enzyme of Raia clavata (Makarewicz, 1969). We could find no correlation between the AMP deaminase activities in the three tissues investigated and the ecological features of the species used in our study. For example, in the gill, high AMP deaminase activities are found in A. anguilla, a species which tolerates hypoxic conditions. In C. carpio, a species showing the same tolerance to hypoxia, the activity is two times lower than in A. anguilla. In S. gairdneri, a species which requires higher oxygenated waters, the AMP deaminase activity is not very different from A. anguiUa. Moreover, no correlation was found concerning the sensitivity to phosphate. Similarly, no correlation was found between the blood AMP deaminase activities given in this study and the nucleotide contents previously reported (Leray, 1979). In white muscle, more accurate determination needs to be done due to an underestimation of AMP deaminase activity, in our study, as a consequence of only partial dissociation of the enzyme from myosin. It seems, however, that at least in gill and white muscle, AMP deaminase is a constitutive enzyme. So it would be interesting to determine, among species living in different environmental conditions, if there are variations in the regulation and the isoenzyme patterns of AMP deaminase. The hypothesis of a different role of AMP deaminase isoenzymes related to an adaptative capacity to aerobic or anaerobic metab-
Extract
Experimental details are given in Materials and M e t h o d s
6.36 ± 0.45
MES centrifuged
± 2.96
19.31
7.81 ± 0.77
phosphate
MES
20.34 ± 3.10
phosphate
centrifuged
4.25 ± 0.34
MES centrifuged
Salmo
7.07 ± 1.08
MES
5.36 ± 0.49
centrifuged
phosphate
(mg/ml)
9.60 ± 1.84
Protein
phosphate
Anguilla
I
i
± 0.143
O.119 ± 0.025
2.858 ± 0.334
0.852
2.616 ± 0.455
0.069 ± 0.020
0.297 ± 0.069
0.029 ± 0.005
0.308 ± 0.074
U /ml
AMP deaminase
19.78 ± 5.21
137.68 ± 14.38
113.45 ± 20.17
134.78 ± 17.94
17.09 ± 5.64
41.11 ± 7.64
5 . 7 5 ± 1.19
30.69 ± 3.49
activity
deaminase
Specific
~P
Table 4 Protein concentration and A M P deaminase activity in A. anguilla and S. gairdneri white muscle before and after centrifugation at 10,000 g for 5 min (mean + SE)
>
540
J.P. RAFFIN and C. LERAY
olism (Raggi et al., 1975) should be verified in different tissues and species. In summary, our experiments reveal that, at least in fish, the white muscle is not always the tissue containing the highest A M P deaminase activity. In particular, a very high activity is found in the blood of S. caniculus. These studies need to be extended to other freshwater and seawater fish and, in particular, other selachian species. Care must be taken to the occurrence of seasonal variations in A M P deaminase activity in some species.
REFERENCES
Asrmv B. & FRmDEN C. (1977) Interaction of AMP deaminase with myosin and its subfragments. J. biol. Chem. 252, 1869-1872. CHAPMAN A. G. & ATKINSON D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. CONWAV E. J. & COOKE R. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem. J. 33, 479-492. CURRIER. D. & WEBS~R H. L. (1962) Preparation of 5'-adenylic acid deaminase based on phosphate-induced dissociation of rat actomyosin--deaminase complexes. Biochim. biophys. Acta 64, 3040. DINGLE J. R. & HINES J. A. (1967) Extraction and some properties of adenosine 5'-monophosphate aminohydrolase from prerigor and postrigor muscle of cod. J. Fish. Res. Bd Can. 24, 1717-1730. HARRIS M. & SUELTER C. H. (1967) A simple chromatographic procedure for the preparation of rabbit-muscle myosin A free from AMP deaminase. Biochim biophys. Acta 133, 393--398. KALCKAR H. M. (1947) Differential spectrometry of purine compounds by means of specific enzymes~ II. Determination of adenine compounds. J. biol. Chem. 167, 445-459. KALETHA K., STANKIEW1CZ m., MAKAREWICZ W. & ZYOOWO M. (1976) The influence of phosphate, fluoride and potassium ions on the activity of AMP deaminase from human skeletal muscle. Int. J. Biochem. 7, 67-71. KRUCKEnERG W. C. & CHILSON O. P. (1973) Red blood cell AMP deaminase: levels of activity in hemolysates from twenty different vertebrate species. Comp. Biochem. Physiol. 46B, 653-660. LEE Y. P. & WaNG M. H. (1968) Studies on the nature of
the inhibitory action of inorganic phosphate, fluoride and detergents on 5'-adenylic acid deaminase activity and on the activation by adenosine triphosphate. J. biol. Chem. 243, 2260-2265. LERAV C. (1979) Patterns of purine nucleotides in fish erythrocytes. Comp. Biochem. Physiol. 64B, 77-82. LERAY C., RAFFIN J. P. & WINNINGER C. (1979) Aspects of purine metabolism in the gill epithelium of rainbow trout Salmo 9airdneri Richardson. Comp. Biochem. Physiol. 62B, 31-40. LOWENSTEIN J. M. (1972) Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Rev. 52, 382-414. LOWRV O H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAKAREW1CZ W. (1963) AMP aminohydrolase and glutaminase activities in the kidneys and gills of some freshwater vertebrates. Acta biochim, polon. 10, 363-369. MAKAREWlCZ W. (1969) AMP aminohydrolase in muscle of elasmobranch fish. Purification procedure and properties of the purified enzyme. Comp. Bioehem. Physiol. 29, 1-26. MAKAREWlCZ W. & ZVDOWO M. (1962). Comparative studies on some ammonia-producing enzymes in the excretory organs of vertebrates. Comp. Biochem. Physiol. 6, 269-275. RAFrlN J. P. & LERAVC. (1979) AMP deaminase in the gill of trout (Salmo gairdneri R.). Modalities of an activation by cellular proteolytic enzymes. Comp. Biochem. Physiol. 62B, 23-29. RAGGI A, BERGAMIN1C. & RONCA G. Isozymes of AMP deaminase in red and white skeletal muscles. FEBS Lett. 58, 19-23. RAGGI A., RONCA-TESTONI S. & RONCA G. (1969) Muscle AMP aminohydrolase. II. Distribution of AMP aminohydrolase, myokinase and creatine kinase activities in skeletal muscle. Biochim. biophys. Aeta 178, 619-622. SETLOW B~, BURGERR. & LOWENSTEINJ. M. (1966). Adenylate deaminase. I. The effects of adenosine and guanosine triphosphates on activity and the organ distribution of the regulated enzyme. J. biol. Chem. 241, 1244-1245. SHIRAKI H., OGAWA H., MATSUDA Y. & NAKAGAWA H. (1979) Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle. Biochim. biophys. Acta 566, 335-344~ WALTOn M. J. & COWEY C. B. (1977) Aspects of ammoniogenesis in rainbow trout, Salmo oairdneri. Comp. Biochem. Physiol. 57B, 143 149.