- Email: [email protected]
Amino acid transamination in the freshwater clams Anodonta couperiana and Popenaias Buckleyi
Comp. Biochem. Physiol. Vol. 68B, pp. 119 to 123
0305-0491/81/0101-0119502.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
AMINO ACID TRANSAMINATION IN THE FRESHWATER CLAMS ANODONTA C O U P E R I A N A AND POPENAIAS BUCKLEYI C. N. FALANY* and F. E. FRIEDL Department of Biology, University of South Florida, Tampa, FL 33620, U.S.A.
(Received 6 May 1980) Abstract--l. The specificity of L-amino acid transamination with 2-oxoglutarate has been studied using hepat0pancreas homogenates from the freshwater bivalves Anodonta couperiana and Popenaias buckleyi. 2. With [2-~4C]oxoglutarate as the amino group acceptor, radiometrically determined transaminase activity with ALA was highest in both bivalves. ASP reacted 84~ as fast in A. couperiana and 58~o as fast in P. buckleyi (however, total ASP% activity appeared similar in both bivalves), Both bivalves also showed substantial activity with LEU, ILE and VAL and trace activity with TYR and ORN. 3. Both bivalves exhibit glutamate dehydrogenase activity, however, L-amino acid oxidase activity has been observed only in P. buckleyi. Against these differing profiles, transaminase specificity was essentially similar in both animals.
INTRODUCTION L-amino acid transaminases are widely distributed enzymes that play important roles in many metabolic pathways, However, there have been few studies of substrate specificity using amino acids other than alanine and aspartic acid in invertebrates. In the Mollusca, a broad specificity of transamination with 2-oxoglutarate has been previously demonstrated in a number of freshwater and terrestrial gastropods (Sollock, 1975; Sollock et al., 1979). An earlier study by Read (1962) showed trace levels of transaminase activity with several amino acids as substrates in the marine bivalves M ytilus edulis and M odiolus modiolus. F o r this report, the ~reshwater bivalves Anodonta couperiana Lea and Popenaias buckleyi (Lea) were investigated for the specificity of L-amino acid transamination with 2-oxoglutarate (2-OG). These two bivalves were chosen for study since both are known to contain glutamate dehydrogenase activity (Falany, 1979). Popenaias also contains L-amino acid oxidase activity (Rifkin, 1976; Rifkin et al., 1976) which could not be demonstrated in Anodonta couperiana (unpublished observation). In this study, the relationship of transamination to glutamate dehydrogenase and L-amino acid oxidase activities was examined in the context of function in deaminative ammonia-producing pathways.
MATERIALS AND METHODS
Animals Anodonta couperiana and Popenaias buckleyi were collected in Hillsborough County, Florida. They were maintained in aquaria containing well water and their natural
* Present address: The Toxicology Center, Department of Pharmacology, University of Iowa, Iowa City, IA 52240, U.S.A.
substratum. Animals were used within six weeks of collection.
Preparation of hepatopancreas homogenates Hepatopancreas homogenates from both clams were prepared identically. The animals were brushed clean and placed in frequently changed, aerated well water for at least 2 days before dissection. This water contained antibiotics (Penicillin, 16 units/ml; Streptomycin, 10,ug/ml) for some animals. No effects on the preparations or on the recovery of enzyme activities were noted in the presence of these antibiotics. The hepatopancreases were removed and placed in 20 ml of cold buffer (50raM Potassium phosphate-NaOH pH 7.1, with 1.0 mM ethylenediaminetetraacetate and 0.1 mM dithiothreitol). The pooled tissues were then weighed and placed in fresh cold buffer. Subsequently, tissue preparations were held at 0 or 4°C. The hepatopancreases were next diced with scissors and ultrasonically disrupted using a Bronwill Biosonik® It homogenizer (twice at 120 W for 30 sec). After sonication, the tissues were re-homogenized using four to five strokes of a motorized Teflon-glass homogenizer. The preparation was then re-sonicated as previously described. Next, the liquid portions were removed and brought to an estimated 20% (w/v) homogenate with buffer and centrifuged (2000 0, 15rain). The supernate was decanted and re-centrifuged (10,000 0, 15 min). Solid ammonium sulfate was then added to the supernate to achieve a 25% saturated solution of the salt. The pellet was recovered by centrifugation at 10,0000 for 15 min and designated the "0-25% fraction". Solid ammonium sulfate was again added to the last supernate to reach a 70~o saturation with the salt. After centrifugation as above, the pellet and its supernate were designated the "25-70% fraction" and the "70% supernate" respectively. The pellets were suspended in small volumes of buffer and all three preparations were dialyzed in the cold (at least 18 hr against three 500 ml volumes of buffer). For radiometric assays the fractions were used immediately upon removal from dialysis. Since it was found that the fractions could be frozen with little loss of activity, preparations were preserved in this way for spectrophotometric analyses. Protein concentrations were estimated by the dye binding method of Bradford (1976) using bovine serum albumin as a standard. 119
120
C.N. FALANY and F. E. FRIEDL
Spectrophotometric transaminase assays Absorption changes of pyridine nucleotide coenzyme were followed at 340nm using a Beckman Model DU spectrophotometer equipped with a 10" recorder and a logarithmic converting unit. Reaction temperatures were maintained at 27°C. Initial reaction velocities derived from evaluations of slopes of continuously recorded absorbance changes were expressed in micromols of pyridine nucleotide per mg assay protein per hour. The extinction coefficient used for NADH and NADPH was 6.27 x 106cm 2 per mole (Lowry & Passoneau, 1972). Addition of pyridoxal phosphate to ASP and ALA transaminase reactions did not increase reaction rates. Its considerable absorbance at 340 nm also hindered performance of the spectrophotometric assay. Accordingly, it was not routinely included in reaction mixtures. Aspartate transaminase reactions L-Aspartate Transaminase (EC 2.6.1.1) was assayed by following the decrease of NADH absorbance in a malate dehydrogenase coupled reaction. A standard reaction mixture contained 200/zmols potassium phosphate-NaOH buffer, pH 7.8 or 6.7; 0.27/zmols NADH; 0.1 ml digestive gland homogenate; 10 #mols potassium 2-oxoglutarate, pH 7.3; with exogenous malate dehydrogenase (Sigma) in a final volume of 2.4 ml. Endogenous activity of malate dehydrogenase was significant, but malate dehydrogenase was added and optimal coupling conditions were ascertained. Alanine transaminase reactions Alanine transaminase (EC 2.6.1.2) assays were performed by following the production of pyruvate as reflected in the decrease in NADH absorbance using a lactate dehydrogenase coupled reaction. Standard rections contained 200#mols potassium phosphate NaOH buffer, pH 7.8; 0.27/zmols L-alanine; and 10:0#mols of potassium 2-oxoglutarate, pH 7.4; with exogenously added lactate dehydrogenase (Sigma Type II) in a final volume of 2.4 ml. In neither clam could endogenous activity of lactate dehydrogenase be detected, thus lactate dehydrogenase was added in amounts ascertained to give optimal coupling. Radiometric studies of transaminase specificity Radiometric transaminase specificity reactions were based on the production of [14C]glutamate when a nonlabeled e-amino acid was used as the amino group donor and [U-2-14C]oxoglutaric acid (New England Nuclear)
was the acceptor molecule (Read, 1962; Sallach & Fahien, 1969; Sollock, 1975; Sollock et al., 1979). The [U-2-14C]oxoglutaric acid was purified by thin-layer chromatography prior to use. All radiometric assays were run in siliconized (Sigmacote ®) 12 x 75 mm tubes at 27°C. A standard reaction mixture contained 4.4/~mols potassium phosphate buffer, pH 7.8 or 6.7; 15 pmols of hepatopancreas homogenate; 0.5#mol donor amino acid; and 0.5/~mol [2-14C]oxoglutarate (approx 30,000 cpm) in a final volume of 50 #I. Reactions were stopped by immersion in ice water and then freezing at -20°C. Controls without donor amino acid or hepatopancreas homogenate were routinely included. The enzyme-free group of controls had ALA and ASP as usual donors supplemented by trials of other amino acids in several experiments. Each donor amino acid was tested at least in triplicate; two samples were removed from each reaction mixture for counting. Pyridoxal phosphate was not added to the radiometric reactions to avoid the possibility of non-enzymatic transamination (Sallach & Fahien, 1969). Thin layer chromatography on Sigmacell® microcrystalline cellulose was used to separate labeled glutamate from labeled 2-oxoglutarate. The solvent system was the upper phase of a mixture of n-butanol-formic acid-deionizeddistilled water (4:1:5, v/v) which was allowed to age 12 hr prior to use (Carles et al., 1958). Fresh solvent was prepared daily. In this thin layer chromatographic system, 2-oxoglutarate and glutamate had R I values of 0.73 and 0.37 respectively. Glutamate counts were recovered by scraping the glutamate band into a scintillation vial. Five milliliters of Triton X-100 scintillation cocktail were added and the vials were then allowed to stand overnight at 4°C before counting.
RESULTS
Transaminase fractionation and stability In the course of this investigation, the hepatopancreas homogenates of P. buckleyi were routinely fractionated into three sub-preparations with a m m o n i u m sulfate. All of the alanine transaminase activity was present in the 25-70% cut (Table 1). Only the aspartate transaminase activity was divided, with 25% of the total units of activity present in the 70% supernate and the remainder in the 25-70% cut.
Table 1. Ammonium sulfate fractionation of hepatopancreas homogenates of Anodonta couperiana and Popenaias buckleyi Fraction
0-25% Anodonta
Alanine Transaminase
Aspartate Transaminase
Protein
A.
1.20
U.
0.266
A.
2.55
U.
0.309
%
5.7
A, Activity in #mols/hr/mg protein. U, Units in/~mols/hr. ~,,,, percent w/v. , No activity detected.
25-70%
Popenaias
Anodonta 3.98 61.51
4.67 75.03
4.7
87.7
70% S u p e r n a t e
Popenaias 3.68 279.5
4.07
Anodonta
Popenaias
.
.
.
.
.
.
.
.
--
105.1
347.6
--
115.66
93.6
6.6
1.7
Transamination in A. couperiana and P. buckleyi Table
2. Transaminase
pH maxima preparations
Preparation
for
121
hepatopancreas
ASP
ALA
P openaias 25-70% Fraction
7.75
7.75
Popenaias 70% Supernate
6.75
....
7.1-8.4
7.78
Anodonta
25-70% Fraction
The division of the ASP transaminase activity of P.
buckleyi into two fractions suggests that it is isozymic. Not only are the solubilities different but there are separate and distinct pH maxima. The form in the 70~o supernate is relatively unstable, 82~ of the activity being lost upon freeze-thawing. Its pH maximum is 6.75 in MES buffer (Table 2). Since the ALA transaminase also has a single pH maximum at pH 7.75 (Table 2), it appears that at least three enzymes are present. One form active only with ASP with a pH maximum at 6.75 and two forms having the more basic pH maximum active with ALA and ASP respectively. Transaminase activity in Anodonta was divided differently by ammonium sulfate fractionation (Table 1). Neither ALA or ASP activity was present in the 70~o supernate. Trace levels of both enzymes were detected in the 0-25~ fraction but for both this represented less than half of one percent of the total. Their presence in the 0-25~ fraction can probably be attributed to contamination from the other fractions. The major-
ity of both activities was present in the 25-70~ fraction. ALA activity had a peak at pH 7.8, however, ASP activity showed a broad maximum ranging from 7.1 to 8.4 (Table 2). This may reflect the presence of more than one form of the A. couperiana ASP transaminase, perhaps similar to the situation in P.
buckleyi. Transaminase substrate specificities Since ALA and ASP transaminases from both clams showed high levels of activity at pH 7.8, all of the radiometric studies of L-amino acid substrate specificity in the 25-70~o fractions were done at this pH. The relative rates determined in these assays are shown in Figs 1 and 2, and are expressed as a percent of ALA activity assigned as 100. Qualitatively and quantitatively, the relative specificities and rates found for A. couperiana and P. buckleyi were quite similar. ALA in both cases, gave the highest activity with ASP reactivity being 84.4~ that of ALA in A.
% Alanine Activity 50
% Alanlne Activity 50
Ioo
Ioo l
ALA
ALA
ASP
ASP
I
LEU ILE
I
LEU
I
ILE
VAL
VAL
SER
SER
GLY
GLY
THR
THR
HIS
HIS
TYR
TYR
ORN
ORN
CIT
CIT
ARG
ARG
LYS
LYS
TRP
TRP
MET
MET PHE
PHE
No Activity
Fig. I. Relative L-amino acid transamination rates obtained from radioactive reactions using Popenaias buckleyi hepatopancreas 25-70~ fraction. The alanine transaminase rate is considered as 100~oactivity.
No Activity
Fig. 2. Relative L-amino acid transamination rates obtained from radioactive reactions using Anodonta couperiana hepatopancreas 25-70~ fraction. The alanine transaminase rate is considered as 100~oactivity.
C. N. FALANYand F. E. FRIEDL
122
couperiana and 58% in P. buckleyi. The 70% supernate of P. buckleyi showed substantial ASP activity as expected. Utilizing the spectrophotometric assays, total ASP transaminase activity was greater in both bivalves, by 22% and 66% respectivity for A. couperiana and P. buckleyi (Table 1). The lower relative activities measured for ASP transaminases in the radiometric assays may be caused by the inhibitory effects of the buildup of oxaloacetate (Boyd, 1961). The ASP activity in the 70% supernate represented 25% of the total ASP activity in P. buckleyi as assayed spectrophotometrically (Table 1). If this extra ASP transaminase activity is taken into account in calculating the radiometric reaction rates, the sum of the ASP reactivity in both fractions is about 75% that of ALA. The relative reaction rates in P. buckleyi are thus similar to the relative levels found in A. couperiana, even though the enzymes fractionated differently. In both clams, notable activity with LEU, ILL, and VAL was present along with consistent low levels of activity with ORN and TYR. Thus only 7 of 17 L-amino acids tested gave evidence of possible involvement in the formation of glutamate, and of these, only 5 showed appreciable reactivity. DISCUSSION
In this investigation great similarities were seen in the numbers of L-amino acids transaminated and the rates of reaction with 2-oxoglutarate in both A. couperiana and P. buckleyi. Highest activity was found with alanine and aspartic acid, both present as significant members of amino acid pools (Duchateau et al., 1951). Less activity was exhibited by leucine, isoleucine and valine, commonly felt to be among "essential amino acids" of animal nutrition (Prosser, 1973). Evidence for trace reactivity with tyrosine, and the nonprotein amino acid ornithine, was also found. All of these have previously been reported as transaminase substrates in other molluscan systems (Read, 1962; Sollock et al., 1979).
Table 3 displays a comparison of the level of transamination of individual amino acids in a number of gastropods and marine bivalves. In general, however. the overall number of amino acids transaminated by the freshwater bivalves A. couperiana and P. buckleyi would appear to be lower than that observed for gastropods and marine bivalves, but as observed in other molluscs, alanine and aspartic acid are the substrates giving the highest relative transaminase activities. Aspartate transaminase activity in P. buckleyi is most likely present in isozymic forms as evidenced by different solubilities and pH maxima. Corresponding multiple forms were not observed in A. couperiana by the procedures employed. Radiometrically determined apparent transaminase activities for the freshwater bivalves are also seen to be moderately lower than those found in gastropods (Table 3). A. couperiana and P. buckleyi, however, both have fairly high relative levels of Leu, Ile and Val activity, significantly above those reported for other molluscs (Table 3). The relationship of the transaminases to the general process of transdeamination is not clear. Both A. couperiana and P. buckleyi are known to have glutamate dehydrogenase (Falany, 1979). If this process is indeed important in the catabolism of amino acids in these animals, the possible number of active substrates appears limited. However, since alanine and aspartic acid generally represent major components of amino acid pools, as well as being the most active substrates in transamination, catabolism of some amino acids could be channelled through them. Another route of amino acid degradation is through the action of amino acid oxidases. Although L-amino acid oxidases are known to be present in various molluscs (Campbell, 1972), A. couperiana and P. buckleyi differ in possessing this activity. In A. couperiana no L-amino acid oxidase could be detected in gill, mantle or hepatopancreas (unpublished observation), whereas it is present in P. buckleyi (Rifkin, 1976; Rifkin et al., 1976). A comparison of transaminase and oxidase activities of P. buckleyi indicate that only a possible two of the amino acids transaminated, Leu
Table 3. Comparison of relative transamination rates in molluscs with alanine activity as 100~,,,
ALA ASP LEU ILE VAL SER GLY THR HIS TYR ORN CI T ARG LYS TRP MET PHE
Popenaias buckle~
Anodonta couperianaa
i00.0 (0.79) 75.0 29.5 45.5 33.4 0.0 0.0 0.0 0.0 6.9 3,7 O. 0 0.0 O. 0 O. 0 0.0 O. 0
100.0 (0.81) 84.4 26.9 39.3 24.5 0.0 0.0 0.0 0.0 2.8 3.0 O. 0 0.0 O. 0 0.0 O. 0 O. 0
~ edulis ~ +++ +4-+ +/+/+/+/+/+/+/+/NT NT +/+/+/-
Modiolus modiolus 2 4-I-+ ~ +/+/NT NT +/NT NT NT NT
NT NT
Otala lactea ~
Strophocheilus oblon~ I
i00.0 (3.05) 100.0 (0.22) 104.9 i, 069.2 i. 1 5.6 i. 9 3.7 3.7 4.8 0,5 NT 0,3 NT 0.0 NT 0.2 NT 13.5 7.4 2.8 27.0 NT NT 2.5 4.1 0.3 NT 0.8 NT O. 0 NT O. 7 5.2
NT, Not tested. Activity, #mols alanine transaminated/hr/mg protein, in parentheses. 1Sollock (1975). 2Read (1962).
Viviparous xiviparous 1
Biomphalaria $1abratus ]--
i00.0 (3.98) 97.9 1.9 3.1 1.2 0.5 0.2 0.3 0.5 7.0 0.3 NT 0.3 0.3 0.9 1.4 1.0
i00.0 (5.77) 42.3 0.3 4.8 0.0 NT NT NT NT 0.4 1.0 NT 1.0 NT NT NT 0.2
Transamination in A. couperiana and P. buckleyi % ALA Transamlnase Activity ( r ~ ) 0
50
IOO
SER
P. buckleyi could exist for a specialized function that is still enigmatic.
i
REFERENCES
ALA ~mm I
ASP
LEU ILE VAL
123
!
I
I I
,...._
GLY THR HIS TYR ARG
LYS TRP MET
PHE No Activity
5~ % LEU L-AAO Activity ( l ) Fig. 3. Comparison of L-amino acid substrate specificities of the Popenaias buckleyi 25-70~ fraction hepatopancreas transaminase activity and the L-amino acid oxidase (L-AAO) activity localized in the gill (Rifkin, 1976).
and Tyr, also contribute to significant levels of oxidase activity (Fig. 3). In general, the specificities of transamination and oxidation in P. buckleyi do not match, but exhibit unique patterns. Additionally, A. couperiana, not possessing an oxidase, still has a pattern of transamination closely resembling that of P. buckleyi. The lack of an increase in the number of transaminated amino acids in A. couperiana suggests that L-amino acid oxidation does not play an essential role in mainline amino acid metabolism. It is doubtful that the seemingly similar metabolisms of P. buckleyi and A. couperiana would be so different in this basic metabolic area. The L-amino acid oxidase activity in
BOYD J. W. (1961) The intracellular distribution, latency, and electrophoretic mobility of L-glutamate-oxaloacetate transaminase from rat liver. Biochem. J. 81, 434-441. BRADFORDM. 0976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-253. CARLESJ., SCHNEIDERA. & LACOSTEA. M. (1958) Contribution a l'etude chromatographique des principaux acides organiques. Bull. Soc. Chim. Biol. 40, 221-232. DUCHArEAU G,, SARLET H., CAMIEN M. & FLORKIN M. (1951) Acides amines non proteiniques des tissus chez les mollusques lamellibranches et chez les vers. Comparaison des formes marines et des formes dulcicoles. Archs int. Physiol. 60, 124-125. FALANYC. N. (1979) L-amino acid transaminase and glutamate dehydrogenase activities in the hepatopancreata of the freshwater bivalves Anodonta couperiana Lea and Popenaias buckleyi (Lea). M.A. Thesis, University of South Florida, Tampa, Florida. LOWRY O. H. & PASSONNEAUJ. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York. PROSSER C. L. (1973) Nutrition. In Comparative Animal Physiology (Edited by PROSSER C. L.) pp. 111-132. Saunders, Philadelphia. READ K. R. H. (1962) Transamination in certain tissue homogenates of the bivalved molluscs Mytilus edulis L. and Modiolus modiolus L. Comp. Biochem. Physiol. 7, 15-22. RIFKIN D. (1976) Oxidative deamination in gill tissue homogenates of the freshwater mussel Popenaias buckleyi (Lea). M.A. Thesis, University of South Florida, Tampa, Florida. RIFKIN D. E., KNOX G. F. & FRIEDL F. E. (1976) Oxidative deamination in gill tissues of the freshwater mussel Popenaias buckleyi (Lea). Am. Zool. 16, 221. SALLACHH. J. t~¢ FAHIENL. A. 1969. Nitrogen Metabolism. In Metabolic Pathways, Vol. III. (Edited by GREENBERG D. M.) pp. 1-94. Academic Press, New York. SOLLOCKR. L. (1975) Amino acid metabolism in gastropod molluscs. Ph.D. Dissertation, Rice University, Houston, Texas. SOLLOCK R. L., VORHABENJ. E. & CAMPBELLJ. W. (1979) Transaminase reactions and glutamate dehydrogenase in gastropod hepatopancreas. J. comp. Physiol. B 129, 129-135.
0305-0491/81/0101-0119502.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
AMINO ACID TRANSAMINATION IN THE FRESHWATER CLAMS ANODONTA C O U P E R I A N A AND POPENAIAS BUCKLEYI C. N. FALANY* and F. E. FRIEDL Department of Biology, University of South Florida, Tampa, FL 33620, U.S.A.
(Received 6 May 1980) Abstract--l. The specificity of L-amino acid transamination with 2-oxoglutarate has been studied using hepat0pancreas homogenates from the freshwater bivalves Anodonta couperiana and Popenaias buckleyi. 2. With [2-~4C]oxoglutarate as the amino group acceptor, radiometrically determined transaminase activity with ALA was highest in both bivalves. ASP reacted 84~ as fast in A. couperiana and 58~o as fast in P. buckleyi (however, total ASP% activity appeared similar in both bivalves), Both bivalves also showed substantial activity with LEU, ILE and VAL and trace activity with TYR and ORN. 3. Both bivalves exhibit glutamate dehydrogenase activity, however, L-amino acid oxidase activity has been observed only in P. buckleyi. Against these differing profiles, transaminase specificity was essentially similar in both animals.
INTRODUCTION L-amino acid transaminases are widely distributed enzymes that play important roles in many metabolic pathways, However, there have been few studies of substrate specificity using amino acids other than alanine and aspartic acid in invertebrates. In the Mollusca, a broad specificity of transamination with 2-oxoglutarate has been previously demonstrated in a number of freshwater and terrestrial gastropods (Sollock, 1975; Sollock et al., 1979). An earlier study by Read (1962) showed trace levels of transaminase activity with several amino acids as substrates in the marine bivalves M ytilus edulis and M odiolus modiolus. F o r this report, the ~reshwater bivalves Anodonta couperiana Lea and Popenaias buckleyi (Lea) were investigated for the specificity of L-amino acid transamination with 2-oxoglutarate (2-OG). These two bivalves were chosen for study since both are known to contain glutamate dehydrogenase activity (Falany, 1979). Popenaias also contains L-amino acid oxidase activity (Rifkin, 1976; Rifkin et al., 1976) which could not be demonstrated in Anodonta couperiana (unpublished observation). In this study, the relationship of transamination to glutamate dehydrogenase and L-amino acid oxidase activities was examined in the context of function in deaminative ammonia-producing pathways.
MATERIALS AND METHODS
Animals Anodonta couperiana and Popenaias buckleyi were collected in Hillsborough County, Florida. They were maintained in aquaria containing well water and their natural
* Present address: The Toxicology Center, Department of Pharmacology, University of Iowa, Iowa City, IA 52240, U.S.A.
substratum. Animals were used within six weeks of collection.
Preparation of hepatopancreas homogenates Hepatopancreas homogenates from both clams were prepared identically. The animals were brushed clean and placed in frequently changed, aerated well water for at least 2 days before dissection. This water contained antibiotics (Penicillin, 16 units/ml; Streptomycin, 10,ug/ml) for some animals. No effects on the preparations or on the recovery of enzyme activities were noted in the presence of these antibiotics. The hepatopancreases were removed and placed in 20 ml of cold buffer (50raM Potassium phosphate-NaOH pH 7.1, with 1.0 mM ethylenediaminetetraacetate and 0.1 mM dithiothreitol). The pooled tissues were then weighed and placed in fresh cold buffer. Subsequently, tissue preparations were held at 0 or 4°C. The hepatopancreases were next diced with scissors and ultrasonically disrupted using a Bronwill Biosonik® It homogenizer (twice at 120 W for 30 sec). After sonication, the tissues were re-homogenized using four to five strokes of a motorized Teflon-glass homogenizer. The preparation was then re-sonicated as previously described. Next, the liquid portions were removed and brought to an estimated 20% (w/v) homogenate with buffer and centrifuged (2000 0, 15rain). The supernate was decanted and re-centrifuged (10,000 0, 15 min). Solid ammonium sulfate was then added to the supernate to achieve a 25% saturated solution of the salt. The pellet was recovered by centrifugation at 10,0000 for 15 min and designated the "0-25% fraction". Solid ammonium sulfate was again added to the last supernate to reach a 70~o saturation with the salt. After centrifugation as above, the pellet and its supernate were designated the "25-70% fraction" and the "70% supernate" respectively. The pellets were suspended in small volumes of buffer and all three preparations were dialyzed in the cold (at least 18 hr against three 500 ml volumes of buffer). For radiometric assays the fractions were used immediately upon removal from dialysis. Since it was found that the fractions could be frozen with little loss of activity, preparations were preserved in this way for spectrophotometric analyses. Protein concentrations were estimated by the dye binding method of Bradford (1976) using bovine serum albumin as a standard. 119
120
C.N. FALANY and F. E. FRIEDL
Spectrophotometric transaminase assays Absorption changes of pyridine nucleotide coenzyme were followed at 340nm using a Beckman Model DU spectrophotometer equipped with a 10" recorder and a logarithmic converting unit. Reaction temperatures were maintained at 27°C. Initial reaction velocities derived from evaluations of slopes of continuously recorded absorbance changes were expressed in micromols of pyridine nucleotide per mg assay protein per hour. The extinction coefficient used for NADH and NADPH was 6.27 x 106cm 2 per mole (Lowry & Passoneau, 1972). Addition of pyridoxal phosphate to ASP and ALA transaminase reactions did not increase reaction rates. Its considerable absorbance at 340 nm also hindered performance of the spectrophotometric assay. Accordingly, it was not routinely included in reaction mixtures. Aspartate transaminase reactions L-Aspartate Transaminase (EC 2.6.1.1) was assayed by following the decrease of NADH absorbance in a malate dehydrogenase coupled reaction. A standard reaction mixture contained 200/zmols potassium phosphate-NaOH buffer, pH 7.8 or 6.7; 0.27/zmols NADH; 0.1 ml digestive gland homogenate; 10 #mols potassium 2-oxoglutarate, pH 7.3; with exogenous malate dehydrogenase (Sigma) in a final volume of 2.4 ml. Endogenous activity of malate dehydrogenase was significant, but malate dehydrogenase was added and optimal coupling conditions were ascertained. Alanine transaminase reactions Alanine transaminase (EC 2.6.1.2) assays were performed by following the production of pyruvate as reflected in the decrease in NADH absorbance using a lactate dehydrogenase coupled reaction. Standard rections contained 200#mols potassium phosphate NaOH buffer, pH 7.8; 0.27/zmols L-alanine; and 10:0#mols of potassium 2-oxoglutarate, pH 7.4; with exogenously added lactate dehydrogenase (Sigma Type II) in a final volume of 2.4 ml. In neither clam could endogenous activity of lactate dehydrogenase be detected, thus lactate dehydrogenase was added in amounts ascertained to give optimal coupling. Radiometric studies of transaminase specificity Radiometric transaminase specificity reactions were based on the production of [14C]glutamate when a nonlabeled e-amino acid was used as the amino group donor and [U-2-14C]oxoglutaric acid (New England Nuclear)
was the acceptor molecule (Read, 1962; Sallach & Fahien, 1969; Sollock, 1975; Sollock et al., 1979). The [U-2-14C]oxoglutaric acid was purified by thin-layer chromatography prior to use. All radiometric assays were run in siliconized (Sigmacote ®) 12 x 75 mm tubes at 27°C. A standard reaction mixture contained 4.4/~mols potassium phosphate buffer, pH 7.8 or 6.7; 15 pmols of hepatopancreas homogenate; 0.5#mol donor amino acid; and 0.5/~mol [2-14C]oxoglutarate (approx 30,000 cpm) in a final volume of 50 #I. Reactions were stopped by immersion in ice water and then freezing at -20°C. Controls without donor amino acid or hepatopancreas homogenate were routinely included. The enzyme-free group of controls had ALA and ASP as usual donors supplemented by trials of other amino acids in several experiments. Each donor amino acid was tested at least in triplicate; two samples were removed from each reaction mixture for counting. Pyridoxal phosphate was not added to the radiometric reactions to avoid the possibility of non-enzymatic transamination (Sallach & Fahien, 1969). Thin layer chromatography on Sigmacell® microcrystalline cellulose was used to separate labeled glutamate from labeled 2-oxoglutarate. The solvent system was the upper phase of a mixture of n-butanol-formic acid-deionizeddistilled water (4:1:5, v/v) which was allowed to age 12 hr prior to use (Carles et al., 1958). Fresh solvent was prepared daily. In this thin layer chromatographic system, 2-oxoglutarate and glutamate had R I values of 0.73 and 0.37 respectively. Glutamate counts were recovered by scraping the glutamate band into a scintillation vial. Five milliliters of Triton X-100 scintillation cocktail were added and the vials were then allowed to stand overnight at 4°C before counting.
RESULTS
Transaminase fractionation and stability In the course of this investigation, the hepatopancreas homogenates of P. buckleyi were routinely fractionated into three sub-preparations with a m m o n i u m sulfate. All of the alanine transaminase activity was present in the 25-70% cut (Table 1). Only the aspartate transaminase activity was divided, with 25% of the total units of activity present in the 70% supernate and the remainder in the 25-70% cut.
Table 1. Ammonium sulfate fractionation of hepatopancreas homogenates of Anodonta couperiana and Popenaias buckleyi Fraction
0-25% Anodonta
Alanine Transaminase
Aspartate Transaminase
Protein
A.
1.20
U.
0.266
A.
2.55
U.
0.309
%
5.7
A, Activity in #mols/hr/mg protein. U, Units in/~mols/hr. ~,,,, percent w/v. , No activity detected.
25-70%
Popenaias
Anodonta 3.98 61.51
4.67 75.03
4.7
87.7
70% S u p e r n a t e
Popenaias 3.68 279.5
4.07
Anodonta
Popenaias
.
.
.
.
.
.
.
.
--
105.1
347.6
--
115.66
93.6
6.6
1.7
Transamination in A. couperiana and P. buckleyi Table
2. Transaminase
pH maxima preparations
Preparation
for
121
hepatopancreas
ASP
ALA
P openaias 25-70% Fraction
7.75
7.75
Popenaias 70% Supernate
6.75
....
7.1-8.4
7.78
Anodonta
25-70% Fraction
The division of the ASP transaminase activity of P.
buckleyi into two fractions suggests that it is isozymic. Not only are the solubilities different but there are separate and distinct pH maxima. The form in the 70~o supernate is relatively unstable, 82~ of the activity being lost upon freeze-thawing. Its pH maximum is 6.75 in MES buffer (Table 2). Since the ALA transaminase also has a single pH maximum at pH 7.75 (Table 2), it appears that at least three enzymes are present. One form active only with ASP with a pH maximum at 6.75 and two forms having the more basic pH maximum active with ALA and ASP respectively. Transaminase activity in Anodonta was divided differently by ammonium sulfate fractionation (Table 1). Neither ALA or ASP activity was present in the 70~o supernate. Trace levels of both enzymes were detected in the 0-25~ fraction but for both this represented less than half of one percent of the total. Their presence in the 0-25~ fraction can probably be attributed to contamination from the other fractions. The major-
ity of both activities was present in the 25-70~ fraction. ALA activity had a peak at pH 7.8, however, ASP activity showed a broad maximum ranging from 7.1 to 8.4 (Table 2). This may reflect the presence of more than one form of the A. couperiana ASP transaminase, perhaps similar to the situation in P.
buckleyi. Transaminase substrate specificities Since ALA and ASP transaminases from both clams showed high levels of activity at pH 7.8, all of the radiometric studies of L-amino acid substrate specificity in the 25-70~o fractions were done at this pH. The relative rates determined in these assays are shown in Figs 1 and 2, and are expressed as a percent of ALA activity assigned as 100. Qualitatively and quantitatively, the relative specificities and rates found for A. couperiana and P. buckleyi were quite similar. ALA in both cases, gave the highest activity with ASP reactivity being 84.4~ that of ALA in A.
% Alanine Activity 50
% Alanlne Activity 50
Ioo
Ioo l
ALA
ALA
ASP
ASP
I
LEU ILE
I
LEU
I
ILE
VAL
VAL
SER
SER
GLY
GLY
THR
THR
HIS
HIS
TYR
TYR
ORN
ORN
CIT
CIT
ARG
ARG
LYS
LYS
TRP
TRP
MET
MET PHE
PHE
No Activity
Fig. I. Relative L-amino acid transamination rates obtained from radioactive reactions using Popenaias buckleyi hepatopancreas 25-70~ fraction. The alanine transaminase rate is considered as 100~oactivity.
No Activity
Fig. 2. Relative L-amino acid transamination rates obtained from radioactive reactions using Anodonta couperiana hepatopancreas 25-70~ fraction. The alanine transaminase rate is considered as 100~oactivity.
C. N. FALANYand F. E. FRIEDL
122
couperiana and 58% in P. buckleyi. The 70% supernate of P. buckleyi showed substantial ASP activity as expected. Utilizing the spectrophotometric assays, total ASP transaminase activity was greater in both bivalves, by 22% and 66% respectivity for A. couperiana and P. buckleyi (Table 1). The lower relative activities measured for ASP transaminases in the radiometric assays may be caused by the inhibitory effects of the buildup of oxaloacetate (Boyd, 1961). The ASP activity in the 70% supernate represented 25% of the total ASP activity in P. buckleyi as assayed spectrophotometrically (Table 1). If this extra ASP transaminase activity is taken into account in calculating the radiometric reaction rates, the sum of the ASP reactivity in both fractions is about 75% that of ALA. The relative reaction rates in P. buckleyi are thus similar to the relative levels found in A. couperiana, even though the enzymes fractionated differently. In both clams, notable activity with LEU, ILL, and VAL was present along with consistent low levels of activity with ORN and TYR. Thus only 7 of 17 L-amino acids tested gave evidence of possible involvement in the formation of glutamate, and of these, only 5 showed appreciable reactivity. DISCUSSION
In this investigation great similarities were seen in the numbers of L-amino acids transaminated and the rates of reaction with 2-oxoglutarate in both A. couperiana and P. buckleyi. Highest activity was found with alanine and aspartic acid, both present as significant members of amino acid pools (Duchateau et al., 1951). Less activity was exhibited by leucine, isoleucine and valine, commonly felt to be among "essential amino acids" of animal nutrition (Prosser, 1973). Evidence for trace reactivity with tyrosine, and the nonprotein amino acid ornithine, was also found. All of these have previously been reported as transaminase substrates in other molluscan systems (Read, 1962; Sollock et al., 1979).
Table 3 displays a comparison of the level of transamination of individual amino acids in a number of gastropods and marine bivalves. In general, however. the overall number of amino acids transaminated by the freshwater bivalves A. couperiana and P. buckleyi would appear to be lower than that observed for gastropods and marine bivalves, but as observed in other molluscs, alanine and aspartic acid are the substrates giving the highest relative transaminase activities. Aspartate transaminase activity in P. buckleyi is most likely present in isozymic forms as evidenced by different solubilities and pH maxima. Corresponding multiple forms were not observed in A. couperiana by the procedures employed. Radiometrically determined apparent transaminase activities for the freshwater bivalves are also seen to be moderately lower than those found in gastropods (Table 3). A. couperiana and P. buckleyi, however, both have fairly high relative levels of Leu, Ile and Val activity, significantly above those reported for other molluscs (Table 3). The relationship of the transaminases to the general process of transdeamination is not clear. Both A. couperiana and P. buckleyi are known to have glutamate dehydrogenase (Falany, 1979). If this process is indeed important in the catabolism of amino acids in these animals, the possible number of active substrates appears limited. However, since alanine and aspartic acid generally represent major components of amino acid pools, as well as being the most active substrates in transamination, catabolism of some amino acids could be channelled through them. Another route of amino acid degradation is through the action of amino acid oxidases. Although L-amino acid oxidases are known to be present in various molluscs (Campbell, 1972), A. couperiana and P. buckleyi differ in possessing this activity. In A. couperiana no L-amino acid oxidase could be detected in gill, mantle or hepatopancreas (unpublished observation), whereas it is present in P. buckleyi (Rifkin, 1976; Rifkin et al., 1976). A comparison of transaminase and oxidase activities of P. buckleyi indicate that only a possible two of the amino acids transaminated, Leu
Table 3. Comparison of relative transamination rates in molluscs with alanine activity as 100~,,,
ALA ASP LEU ILE VAL SER GLY THR HIS TYR ORN CI T ARG LYS TRP MET PHE
Popenaias buckle~
Anodonta couperianaa
i00.0 (0.79) 75.0 29.5 45.5 33.4 0.0 0.0 0.0 0.0 6.9 3,7 O. 0 0.0 O. 0 O. 0 0.0 O. 0
100.0 (0.81) 84.4 26.9 39.3 24.5 0.0 0.0 0.0 0.0 2.8 3.0 O. 0 0.0 O. 0 0.0 O. 0 O. 0
~ edulis ~ +++ +4-+ +/+/+/+/+/+/+/+/NT NT +/+/+/-
Modiolus modiolus 2 4-I-+ ~ +/+/NT NT +/NT NT NT NT
NT NT
Otala lactea ~
Strophocheilus oblon~ I
i00.0 (3.05) 100.0 (0.22) 104.9 i, 069.2 i. 1 5.6 i. 9 3.7 3.7 4.8 0,5 NT 0,3 NT 0.0 NT 0.2 NT 13.5 7.4 2.8 27.0 NT NT 2.5 4.1 0.3 NT 0.8 NT O. 0 NT O. 7 5.2
NT, Not tested. Activity, #mols alanine transaminated/hr/mg protein, in parentheses. 1Sollock (1975). 2Read (1962).
Viviparous xiviparous 1
Biomphalaria $1abratus ]--
i00.0 (3.98) 97.9 1.9 3.1 1.2 0.5 0.2 0.3 0.5 7.0 0.3 NT 0.3 0.3 0.9 1.4 1.0
i00.0 (5.77) 42.3 0.3 4.8 0.0 NT NT NT NT 0.4 1.0 NT 1.0 NT NT NT 0.2
Transamination in A. couperiana and P. buckleyi % ALA Transamlnase Activity ( r ~ ) 0
50
IOO
SER
P. buckleyi could exist for a specialized function that is still enigmatic.
i
REFERENCES
ALA ~mm I
ASP
LEU ILE VAL
123
!
I
I I
,...._
GLY THR HIS TYR ARG
LYS TRP MET
PHE No Activity
5~ % LEU L-AAO Activity ( l ) Fig. 3. Comparison of L-amino acid substrate specificities of the Popenaias buckleyi 25-70~ fraction hepatopancreas transaminase activity and the L-amino acid oxidase (L-AAO) activity localized in the gill (Rifkin, 1976).
and Tyr, also contribute to significant levels of oxidase activity (Fig. 3). In general, the specificities of transamination and oxidation in P. buckleyi do not match, but exhibit unique patterns. Additionally, A. couperiana, not possessing an oxidase, still has a pattern of transamination closely resembling that of P. buckleyi. The lack of an increase in the number of transaminated amino acids in A. couperiana suggests that L-amino acid oxidation does not play an essential role in mainline amino acid metabolism. It is doubtful that the seemingly similar metabolisms of P. buckleyi and A. couperiana would be so different in this basic metabolic area. The L-amino acid oxidase activity in
BOYD J. W. (1961) The intracellular distribution, latency, and electrophoretic mobility of L-glutamate-oxaloacetate transaminase from rat liver. Biochem. J. 81, 434-441. BRADFORDM. 0976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-253. CARLESJ., SCHNEIDERA. & LACOSTEA. M. (1958) Contribution a l'etude chromatographique des principaux acides organiques. Bull. Soc. Chim. Biol. 40, 221-232. DUCHArEAU G,, SARLET H., CAMIEN M. & FLORKIN M. (1951) Acides amines non proteiniques des tissus chez les mollusques lamellibranches et chez les vers. Comparaison des formes marines et des formes dulcicoles. Archs int. Physiol. 60, 124-125. FALANYC. N. (1979) L-amino acid transaminase and glutamate dehydrogenase activities in the hepatopancreata of the freshwater bivalves Anodonta couperiana Lea and Popenaias buckleyi (Lea). M.A. Thesis, University of South Florida, Tampa, Florida. LOWRY O. H. & PASSONNEAUJ. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York. PROSSER C. L. (1973) Nutrition. In Comparative Animal Physiology (Edited by PROSSER C. L.) pp. 111-132. Saunders, Philadelphia. READ K. R. H. (1962) Transamination in certain tissue homogenates of the bivalved molluscs Mytilus edulis L. and Modiolus modiolus L. Comp. Biochem. Physiol. 7, 15-22. RIFKIN D. (1976) Oxidative deamination in gill tissue homogenates of the freshwater mussel Popenaias buckleyi (Lea). M.A. Thesis, University of South Florida, Tampa, Florida. RIFKIN D. E., KNOX G. F. & FRIEDL F. E. (1976) Oxidative deamination in gill tissues of the freshwater mussel Popenaias buckleyi (Lea). Am. Zool. 16, 221. SALLACHH. J. t~¢ FAHIENL. A. 1969. Nitrogen Metabolism. In Metabolic Pathways, Vol. III. (Edited by GREENBERG D. M.) pp. 1-94. Academic Press, New York. SOLLOCKR. L. (1975) Amino acid metabolism in gastropod molluscs. Ph.D. Dissertation, Rice University, Houston, Texas. SOLLOCK R. L., VORHABENJ. E. & CAMPBELLJ. W. (1979) Transaminase reactions and glutamate dehydrogenase in gastropod hepatopancreas. J. comp. Physiol. B 129, 129-135.