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Multiple adenosine deaminases in the frog (Rana catesbeiana)
Comp. Bioehem. PhysioL, 1968, VoL 27, pp, 105 to 112. Pergamon Press. Printed in Great Britain
MULTIPLE
ADENOSINE
DEAMINASES
IN THE
FROG
(RANA CATESBEL4NA)* P A N G F. M A and JAMES R. F I S H E R Department of Chemistry; and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, U.S.A.
(Received26 February 1968) Abstract--1. Three classes of adenosine deaminases have been found in frog tissues. Types A, B and C have molecular weights in the range of 180,000, 100,000 and 35,000 respectively. Studies of tissues from a wide variety of vertebrates have shown that all of these enzyme types are widespread among vertebrates (Ma & Fisher, unpublished results). 2. Tissue specific differences in the frog result from different proportions of the three types of enzymes in the liver and duodenum. In the liver a type B enzyme accounts for most activity while in the duodenum a type C enzyme predominates. INTRODUCTION
TISSUE specific differences in adenosine deaminases (adenosine aminohydrolase E.C. 3.5.4.4) have been shown in the liver and duodenum of chickens and frogs (Fisher et al., 1965; Ma & Fisher, 1968). Brady & O'Donovan (1961, 1965) did not find significant differences in relative substrate specificities of adenosine deaminases from different tissues in the same animals, but differences were found between enzymes from the same tissues in the six mammalian species studied. However, differences between these enzymes from tissues of the calf have been described (Rockwell & Maguire, 1966; Cory et aL, 1967; Pfrogner, 1967). In a more recent study Ma & Fisher (1968) were able to show that the tissue specific differences in the chicken were due to the presence of a large (100,000 tool. wt.) enzyme in the liver which is absent in the duodenum. A smaller adenosine deaminase (30,000 mol. wt.) was present in both tissues and showed strikingly similar properties. This present study was undertaken to establish the basis for the tissue specific differences observed in the frog (Fisher et aL, 1965). The results obtained show that three classes of adenosine deaminases are present in both frog liver and duodenum. Also, electrophoretic studies of frog liver preparations have revealed the presence of multiple adenosine deaminases (Chilson, Bletterman & Cohen, personal communication). The tissue specific differences appear to be the result of different proportions of these enzymes in the two tissues. * Supported in part by Grant GM-11907 from the National Institutes of Health and by a contract with the Division of Biology and Medicine, U.S. Atomic Energy Commission. 105
106
PANe F. M_~ ANt) JAMES R. Fxsmm
MATERIALS AND METHODS Adenosine and dcoxyadenosine were obtained from Calbiochem, crystalline bovine serum albumin and cytochrome-c from Sigma Chemical Co., and chymotrypsinogen was bought from Mann Research Laboratories, Inc. Sephadex G-150 and blue dextran sulfate were purchased from Pharrnacia. Frogs (Ranacatesbeiana)were obtained from the Lemberger Co. Excised tissues were stored in a deep freeze at - 2 0 ° C . Appropriate amounts of the frozen tissues were thawed and homogenized in a pestle tissue grinder in glass distilled water at a concentration of 1 g tissue per 5 ml water. T h e procedure for the preparation of the enzyme was very much like that previously reported (Ma & Fisher, 1968). T h e homogenate was centrifuged at 18,000 g in a refrigerated centrifuge for 1 hr. T h e supernatant was poured through a fine nylon net into a graduated cylinder to remove the floating thin layer of lipid material. T h e supernatant was brought to 45 per cent saturation with solid ammonium sulfate. Slow addition and constant stirring are important. After stirring at 4°C for at least 2 hr, the solution was centrifuged at 18,000 g for 30 rain and the sediment was resnspended in a few drops of 0"05 M phosphate buffer (pI-I 7). Subsequently it was centrifuged again for 15 rain and the small volume of supernatant so obtained was stored in a deep freeze. Successive 5 per cent saturation cuts were made on the 45 per cent saturated supernatant up to 75 per cent saturation using solid ammonium sulfate. Precipitates were dissolved in 0"05 M phosphate buffer and stored in the same manner as above. All the fractions were checked for enzyme activity, and all of the fractions containing even the slightest enzyme activity were combined. T h e combined solution was centrifuged to remove some insoluble material and then chromatographed on a Sephadex G-150 column. T h e purpose of ammonium sulfate fractionation was to concentrate the enzyme and make sure that all the enzyme activity from the original homogenate was included. Sephadex G-150 was sieved so that the particle sizes were larger than 88 F, and was allowed to swell in 0"05 M phosphate buffer for 6 hr in a boiling water-bath. T h e swollen gel was cooled and a column (1.8 x 81 crn) was poured in the cold room and equilibrated overnight with flowing buffer. T h e column was standardized with blue dextran sulfate, bovine serum albumin, cytochr0me-c and chymotrypsinogen (Andrews, 1964). I n all experiments a 1"0-1-5 rrd sample was applied to the column and 2-ml fractions were collected. T h e elution pattern of enzyme activity was resolved into symmetrical peaks (see Fig. 1) with a DuPont 310 Curve Resolver. Gauasian distributions were assumed for aH components. T h e resolved curves represent es~nates of rather than accurately defined distributions of the three components. Enzyme activity was measured b y following the decrease in optical density at 265 nap (Kalckar, 1947). A unit of enzyme activity is defined as that amount of enzyme which catalyzes the hydrolysis of 1 Fmole of adenosine/rain under standard conditions; i.e. with 1"0 × 10 -4 M adenosine in 0"2 M phosphate buffer (pH 7"0) at 38°C. Measurements were made with a Model 2000 Gilford spectrophotometer in 1 cm pathlength cells containing 3"0 ml of substrate solution. Temperature was controlled by circulating water through the cell compartment. RESULTS
Multiple adenosine deaminases in frog liver A d e n o s i n e d e a m i n a s e s in f r o g l i v e r e x t r a c t s w e r e c o n c e n t r a t e d b y a m m o n i u m sulfate f r a c t i o n a t i o n a n d p a s s e d t h r o u g h a S e p h a d e x G - 1 5 0 c o l u m n . A t y p i c a l c h r o m a t o g r a p h i c p a t t e r n is s h o w n in F i g . 1. T h e s e r e s u l t s s u g g e s t t h a t t h r e e d i f f e r e n t t y p e s o f a d e n o s i n e d e a m i n a s e s are p r e s e n t . T h e p e a k o f a c t i v i t y at f r a c t i o n n u m b e r 65 s h o w s t h e p r e s e n c e o f o n e t y p e ( t y p e C) h a v i n g a m o l e c u l a r
M U L T I P L B AD]~2qOSINB DBAMINASE8 n q T H B F R O G
107
weight in the order of 35,000. The large peak of activity at fraction number 48 shows the presence of a second type (type B) having a molecular weight of about 100,000. The high relative substrate specificity (ratio of activity with deoxyadenosine to that with adenosine) see in early fractions (36-40) suggests the presence of a third type (type A) of adenosine deaminase with a molecular weight of about 180,000. The existence of type A adenosine deaminases has been confirmed in other organisms wherein they represent a much larger fraction of total enzymic activity (e.g. 50 per cent in the liver of the cooter turtle). The relative amount of each enzyme type can be estimated by resolving the elution pattern into its three components using a DuPont Curve Resolver. It was found that types A, B and C represent 7, 68 and 25 per cent of total activity respectively. The primary method of identifying these three adenosine deaminases was by means of their characteristic relative substrate specificities (RSS). The RSS value of type C enzyme was determined to be 1.36 using enzyme from the trailing edge of the last peak (Fig. 1).
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Fig. 1. A Sephadex G-150 chromatographic pattern of adenosine d ~ r , rtinase activity in a frog I/ver extract. T h e dotted lines show the resolved peaks obtained with a DuPont 310 Curve Resolver. Areas under each peak were obtained with the same instrument and reported as percent of total area. Relative substrate specificities (ratios of activity with 0"1 m M deoxyadenosine to t h a t w i t h 0"1 rnM adenosine) are shown for each fraction (2 nd). Void volume was determined with blue dextran.
108
PANOF. MA ANDJAMESR. FISHER
Since type B enzyme was mixed with type A or type C enzyme in all fractions, it was necessary to eliminate one of the contaminating types in order to determine the RSS of type B unambiguously. Preliminary experiments showed that type C enzyme is more heat labile than type B enzyme. Consequently, when frog liver extracts were heated to 68°C for 10 rain before ammonium sulfate fractionation, the extracts were almost free of the type C enzyme (Fig. 2). The heating step
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Fxo. 2. A Sephadex G-150 chromatographic pattern of adenosine deaminase
activity in a heated (68°C for 10 min) frog liver extract (See legend to Fig. 1). caused a 65 per cent loss in total activity. The RSS value of the type B enzyme was found to be 0.11 (Fig. 2). This value has been confirmed without heating by studies of tadpole liver (R. catesbeiana, stage 23). Type C enzyme is not present in this tissue (Fig. 3). So far, type A enzyme has not been obtained without major type B contamination. Consequently, its RSS value has only been estimated to be in the range of 0.5-0.8.
Tis~e ~ecifw dzfferences Fisher et al. (1965) reported tissue specific differences in the properties of adenosine deaminases from the liver and duodenum of frogs. The RSS values of adenosine deaminases in an ammonium sulfate fraction of frog liver and duodenum were found to be 0.34 and 1.06 respectively. Differences were also reported between adenosine deaminases in chicken liver and duodenum. Subsequently, Ma &
MULTIPLE ADENOSINE D E A M I N A S ~ I N THE FROG
109
Fisher (1968) showed that these differences in the chicken were due to the presence of type B enzyme (identified as liver enzyme I) and type C enzyme (identified as liver enzyme II) in the liver and only type C enzyme in the duodenum. It seemed reasonable to expect a similar situation in the frog in view of the presence of a large enzyme in frog liver. This possibility was tested by Sephadex G-150 column chromatography of frog duodenal extracts after ammonium sulfate fractionation
i
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FIG. 3. A Sephadex G-150 chromatograp~c pattern of adenosine deaminas¢ activity in a tadpole liver extract (See legend to Fig. 1),
(Fig. 4). These results showed a broad asymmetrical peak of adenosine deaminase activity. The RSS values for the various fractions suggested that all three types of enzymes found in frog liver were also present in frog duodenum. The complex peak of activity was resolved into three components with the DuPont 310 Curve Resolver, and it was found that type A, B and C enzymes represent 7, 10 and 83 per cent of total enzyme activity respectively. Previously it was shown that heating liver extracts at 68°C for 10 rain destroys most of the type C enzyme, much of the type A enzyme and some of the type B enzyme. This results in an enzyme preparation which is predominantly type B. An attempt was made to establish the presence of type B enzyme in duodenal extracts by this procedure. Heating resulted in a 90 per cent loss of total adenosine deaminase activity and a shift in RSS from 0.67 in the original duodenal extract to 0.13 after heating. These results indicate that a relatively heat-stable type B
110
P ~ o F. Ma ah'v JAMBSR. FmH~a
enzyme is present in frog duodenum which has an RSS value similar to the type B enzyme in fiver. Unfortunately, Sephadex chromatography of heated duodenal extracts has not been possible due to the low level of enzyme activity obtained. Exact comparisons of RSS values between the enzymes in liver and duodenum has been made with the type C adenosine deaminases. This fiver enzyme gives a value of 1"36 whereas the duodenal enzyme gives a value of 1.22. This indicates that these enzymes are similar but not identical. >, L2'
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FIo. 4. A Sephadex G-150 chromatographic pattern of adenosine deaminue activity in a frog duodenal extract (See legend to Fig. 1). These studies show that the large tissue specific differences between adenosine deaminases in frog fiver and duodenum are the result of differences in the type of enzyme which dominates; i.e. 68 per cent type B in the liver and 83 per cent type C in the duodenum. It appears that both tissues have all three types and that the same types in both tissues have similar relative substrate specificities. DISCUSSION Results presented in this paper show that frog tissues have three types of adenosine deamlnases. Similar studies with other amphibians, reptiles, fish and mammals have demonstrated the widespread occurrence of all three types of enzymes (Ma & Fisher, unpublished restdts). Consequently it seems reasonable to classify these enzymes into three groups. Types A, B and C enzymes are in the size ranges of 180,000, 100,000 and 35,000 tool. wt. respectively. Type C adenosine deaminases have been found in every vertebrate tissue studied (except the tadpole liver) including a variety of mammAl;an tissues (Brady & O'Sullivan, 1967; Cory
MULTIPLR ADI~NOSINE DBAMINASI~ IN TH~ FROG
111
et al., 1967; Pfrogner, 1967), chicken duodenum and liver (Hoagland & Fisher, 1967; Ma & Fisher, 1968), frog duodenum and liver and in the livers of a wide variety of fish and reptiles (Ma & Fisher, unpublished results). The larger adenosine deaminases are not ubiquitous; e.g. types A and B are missing in chicken duodenum (Hoagland & Fisher, 1967) and in the livers of most mammals (Ma & Fisher, unpublished results). A variety of tissue specific properties of adenosine dearainases have been described in mammalian tissues, including intestine (Brady & O'Connell, 1962; Chilson & Fisher, 1963), spleen (Pfrogner, 1967), heart (Rockwell & Maguire, 1966) and serum (Cory et al., 1967) of the calf. Since these tissues contain only type C enzymes, the differences are due to variations in the properties of these low molecular weight adenosine deaminases. In the chicken, Ma & Fisher (1968) showed that tissue specific differences are due to the presence of type B enzyme in the liver which is absent in the duodenum. Both tissues contain type C enzymes which are identical in every property tested. These properties included relative substrate specificities with five substrates, Michaelis constants, energies of activation and activation by p-mercuribenzoate. Results presented in this paper show that tissue specific differences between frog liver and duodenal adenosine deaminases are due to the difference in the proportions of the three types of enzymes present. In the liver type B enzyme dominates (68 per cent of total activity) and in the duodenum type C enzyme dominates (83 per cent of total activity). The relative substrate specificities of the corresponding types of enzymes in both tissues are quite similar. So far it has not been possible to isolate the enzymes from each tissue and make detailed comparisons. Ackno~oledgement--We would like to thank Dr. Earl Frieden and his coworkers for supplying and staging the tadpoles used in this study.
REFERENCES ANDREWSP. (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. j~. 91, 222-233. Bp~vY T. G. & O'CoNN~L W. (1962) A purification of adenosine deaminase from the superficial mucosa of calf intestine. Biochim. biophys. Acta 62, 216-229. BRADYT. G. & O'DONOV~ C. I. (1961) Survey of the distribution and nature of the mammalian adenosine deaminase. Biochem.y. 80, 17P. B~vY T. G. & O'DONOV~ C. I. (1965) A study of the tissue distribution of adenosine deaminase in six mammalian species. Comp. Biochem. Physiol. 14, 101-120. Bm~vYT. G. & O'SULLrV~ M. (1967) The effect of pH, urea and guanidine on adenosine deaminase isolated from bovine mucosa. Biochim. biophys. Acta 132, 127-137. CmLSONO. P. & FXSHZRJ. R. (1963) Some comparative studies of calf and chicken adenosine deaminase. Archs Biochem. Biophys. 102, 77-85. CORYJ. G., W E ~ A ~ G. & SVH~OLNIKR. J. (1967) Multiple forms of calf serum adenosine deaminase. Archs Biochem. Biophys. 118, 428--433. FmHE~J. R., MA P. F. & CmLSONO. P. (1965) Some tissue specific differences in adenosine deaminase. Comp. Biochem. Physiol. 16, 199-203. HOACLANVV. D., JR. & FISH~ J. R. (1967) Purification and properties of chicken duodenal adenosine deaminase, y. biol. Chem. 242, 4341-4351.
112
PANO F. MA AND JAM~ R. FISHBR
KALC~R H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes--III. Studies of the enzymes of purine metabolism. ~. biol. Chem. 167, 461--475. Ms P. F. & Faming J. R. (1968) Two different hepatic adenosine deaminases in the chicken. Biochim. biophys. .,qcta 159, 153-159. PlmOOlq~N. (1967) Adenosine deaminase fromcalfspleen--II. Chernicalend enzymological properties. ,drdu Biochem. Biophys. 119, 147-154. Rocgwm2. M. & Msotalm M. H. (1966) Studies on adenosine deaminase---I. Purification and properties of ox heart adenosine deaminase. Mol. Pharma¢ol. 2, 574-584.
MULTIPLE
ADENOSINE
DEAMINASES
IN THE
FROG
(RANA CATESBEL4NA)* P A N G F. M A and JAMES R. F I S H E R Department of Chemistry; and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, U.S.A.
(Received26 February 1968) Abstract--1. Three classes of adenosine deaminases have been found in frog tissues. Types A, B and C have molecular weights in the range of 180,000, 100,000 and 35,000 respectively. Studies of tissues from a wide variety of vertebrates have shown that all of these enzyme types are widespread among vertebrates (Ma & Fisher, unpublished results). 2. Tissue specific differences in the frog result from different proportions of the three types of enzymes in the liver and duodenum. In the liver a type B enzyme accounts for most activity while in the duodenum a type C enzyme predominates. INTRODUCTION
TISSUE specific differences in adenosine deaminases (adenosine aminohydrolase E.C. 3.5.4.4) have been shown in the liver and duodenum of chickens and frogs (Fisher et al., 1965; Ma & Fisher, 1968). Brady & O'Donovan (1961, 1965) did not find significant differences in relative substrate specificities of adenosine deaminases from different tissues in the same animals, but differences were found between enzymes from the same tissues in the six mammalian species studied. However, differences between these enzymes from tissues of the calf have been described (Rockwell & Maguire, 1966; Cory et aL, 1967; Pfrogner, 1967). In a more recent study Ma & Fisher (1968) were able to show that the tissue specific differences in the chicken were due to the presence of a large (100,000 tool. wt.) enzyme in the liver which is absent in the duodenum. A smaller adenosine deaminase (30,000 mol. wt.) was present in both tissues and showed strikingly similar properties. This present study was undertaken to establish the basis for the tissue specific differences observed in the frog (Fisher et aL, 1965). The results obtained show that three classes of adenosine deaminases are present in both frog liver and duodenum. Also, electrophoretic studies of frog liver preparations have revealed the presence of multiple adenosine deaminases (Chilson, Bletterman & Cohen, personal communication). The tissue specific differences appear to be the result of different proportions of these enzymes in the two tissues. * Supported in part by Grant GM-11907 from the National Institutes of Health and by a contract with the Division of Biology and Medicine, U.S. Atomic Energy Commission. 105
106
PANe F. M_~ ANt) JAMES R. Fxsmm
MATERIALS AND METHODS Adenosine and dcoxyadenosine were obtained from Calbiochem, crystalline bovine serum albumin and cytochrome-c from Sigma Chemical Co., and chymotrypsinogen was bought from Mann Research Laboratories, Inc. Sephadex G-150 and blue dextran sulfate were purchased from Pharrnacia. Frogs (Ranacatesbeiana)were obtained from the Lemberger Co. Excised tissues were stored in a deep freeze at - 2 0 ° C . Appropriate amounts of the frozen tissues were thawed and homogenized in a pestle tissue grinder in glass distilled water at a concentration of 1 g tissue per 5 ml water. T h e procedure for the preparation of the enzyme was very much like that previously reported (Ma & Fisher, 1968). T h e homogenate was centrifuged at 18,000 g in a refrigerated centrifuge for 1 hr. T h e supernatant was poured through a fine nylon net into a graduated cylinder to remove the floating thin layer of lipid material. T h e supernatant was brought to 45 per cent saturation with solid ammonium sulfate. Slow addition and constant stirring are important. After stirring at 4°C for at least 2 hr, the solution was centrifuged at 18,000 g for 30 rain and the sediment was resnspended in a few drops of 0"05 M phosphate buffer (pI-I 7). Subsequently it was centrifuged again for 15 rain and the small volume of supernatant so obtained was stored in a deep freeze. Successive 5 per cent saturation cuts were made on the 45 per cent saturated supernatant up to 75 per cent saturation using solid ammonium sulfate. Precipitates were dissolved in 0"05 M phosphate buffer and stored in the same manner as above. All the fractions were checked for enzyme activity, and all of the fractions containing even the slightest enzyme activity were combined. T h e combined solution was centrifuged to remove some insoluble material and then chromatographed on a Sephadex G-150 column. T h e purpose of ammonium sulfate fractionation was to concentrate the enzyme and make sure that all the enzyme activity from the original homogenate was included. Sephadex G-150 was sieved so that the particle sizes were larger than 88 F, and was allowed to swell in 0"05 M phosphate buffer for 6 hr in a boiling water-bath. T h e swollen gel was cooled and a column (1.8 x 81 crn) was poured in the cold room and equilibrated overnight with flowing buffer. T h e column was standardized with blue dextran sulfate, bovine serum albumin, cytochr0me-c and chymotrypsinogen (Andrews, 1964). I n all experiments a 1"0-1-5 rrd sample was applied to the column and 2-ml fractions were collected. T h e elution pattern of enzyme activity was resolved into symmetrical peaks (see Fig. 1) with a DuPont 310 Curve Resolver. Gauasian distributions were assumed for aH components. T h e resolved curves represent es~nates of rather than accurately defined distributions of the three components. Enzyme activity was measured b y following the decrease in optical density at 265 nap (Kalckar, 1947). A unit of enzyme activity is defined as that amount of enzyme which catalyzes the hydrolysis of 1 Fmole of adenosine/rain under standard conditions; i.e. with 1"0 × 10 -4 M adenosine in 0"2 M phosphate buffer (pH 7"0) at 38°C. Measurements were made with a Model 2000 Gilford spectrophotometer in 1 cm pathlength cells containing 3"0 ml of substrate solution. Temperature was controlled by circulating water through the cell compartment. RESULTS
Multiple adenosine deaminases in frog liver A d e n o s i n e d e a m i n a s e s in f r o g l i v e r e x t r a c t s w e r e c o n c e n t r a t e d b y a m m o n i u m sulfate f r a c t i o n a t i o n a n d p a s s e d t h r o u g h a S e p h a d e x G - 1 5 0 c o l u m n . A t y p i c a l c h r o m a t o g r a p h i c p a t t e r n is s h o w n in F i g . 1. T h e s e r e s u l t s s u g g e s t t h a t t h r e e d i f f e r e n t t y p e s o f a d e n o s i n e d e a m i n a s e s are p r e s e n t . T h e p e a k o f a c t i v i t y at f r a c t i o n n u m b e r 65 s h o w s t h e p r e s e n c e o f o n e t y p e ( t y p e C) h a v i n g a m o l e c u l a r
M U L T I P L B AD]~2qOSINB DBAMINASE8 n q T H B F R O G
107
weight in the order of 35,000. The large peak of activity at fraction number 48 shows the presence of a second type (type B) having a molecular weight of about 100,000. The high relative substrate specificity (ratio of activity with deoxyadenosine to that with adenosine) see in early fractions (36-40) suggests the presence of a third type (type A) of adenosine deaminase with a molecular weight of about 180,000. The existence of type A adenosine deaminases has been confirmed in other organisms wherein they represent a much larger fraction of total enzymic activity (e.g. 50 per cent in the liver of the cooter turtle). The relative amount of each enzyme type can be estimated by resolving the elution pattern into its three components using a DuPont Curve Resolver. It was found that types A, B and C represent 7, 68 and 25 per cent of total activity respectively. The primary method of identifying these three adenosine deaminases was by means of their characteristic relative substrate specificities (RSS). The RSS value of type C enzyme was determined to be 1.36 using enzyme from the trailing edge of the last peak (Fig. 1).
0
= 1.4
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void volume
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l!
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%~ .~ T ~ C '
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.N\ , 70.
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Fig. 1. A Sephadex G-150 chromatographic pattern of adenosine d ~ r , rtinase activity in a frog I/ver extract. T h e dotted lines show the resolved peaks obtained with a DuPont 310 Curve Resolver. Areas under each peak were obtained with the same instrument and reported as percent of total area. Relative substrate specificities (ratios of activity with 0"1 m M deoxyadenosine to t h a t w i t h 0"1 rnM adenosine) are shown for each fraction (2 nd). Void volume was determined with blue dextran.
108
PANOF. MA ANDJAMESR. FISHER
Since type B enzyme was mixed with type A or type C enzyme in all fractions, it was necessary to eliminate one of the contaminating types in order to determine the RSS of type B unambiguously. Preliminary experiments showed that type C enzyme is more heat labile than type B enzyme. Consequently, when frog liver extracts were heated to 68°C for 10 rain before ammonium sulfate fractionation, the extracts were almost free of the type C enzyme (Fig. 2). The heating step
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70
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50
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Fxo. 2. A Sephadex G-150 chromatographic pattern of adenosine deaminase
activity in a heated (68°C for 10 min) frog liver extract (See legend to Fig. 1). caused a 65 per cent loss in total activity. The RSS value of the type B enzyme was found to be 0.11 (Fig. 2). This value has been confirmed without heating by studies of tadpole liver (R. catesbeiana, stage 23). Type C enzyme is not present in this tissue (Fig. 3). So far, type A enzyme has not been obtained without major type B contamination. Consequently, its RSS value has only been estimated to be in the range of 0.5-0.8.
Tis~e ~ecifw dzfferences Fisher et al. (1965) reported tissue specific differences in the properties of adenosine deaminases from the liver and duodenum of frogs. The RSS values of adenosine deaminases in an ammonium sulfate fraction of frog liver and duodenum were found to be 0.34 and 1.06 respectively. Differences were also reported between adenosine deaminases in chicken liver and duodenum. Subsequently, Ma &
MULTIPLE ADENOSINE D E A M I N A S ~ I N THE FROG
109
Fisher (1968) showed that these differences in the chicken were due to the presence of type B enzyme (identified as liver enzyme I) and type C enzyme (identified as liver enzyme II) in the liver and only type C enzyme in the duodenum. It seemed reasonable to expect a similar situation in the frog in view of the presence of a large enzyme in frog liver. This possibility was tested by Sephadex G-150 column chromatography of frog duodenal extracts after ammonium sulfate fractionation
i
.~ 0,4
240
vold volume
4
200 x 160 ~n "E 120
g°
,/
" I
J,~" Nj 30 40
I I 50 60 Fraction
~ 70
I 80
FIG. 3. A Sephadex G-150 chromatograp~c pattern of adenosine deaminas¢ activity in a tadpole liver extract (See legend to Fig. 1),
(Fig. 4). These results showed a broad asymmetrical peak of adenosine deaminase activity. The RSS values for the various fractions suggested that all three types of enzymes found in frog liver were also present in frog duodenum. The complex peak of activity was resolved into three components with the DuPont 310 Curve Resolver, and it was found that type A, B and C enzymes represent 7, 10 and 83 per cent of total enzyme activity respectively. Previously it was shown that heating liver extracts at 68°C for 10 rain destroys most of the type C enzyme, much of the type A enzyme and some of the type B enzyme. This results in an enzyme preparation which is predominantly type B. An attempt was made to establish the presence of type B enzyme in duodenal extracts by this procedure. Heating resulted in a 90 per cent loss of total adenosine deaminase activity and a shift in RSS from 0.67 in the original duodenal extract to 0.13 after heating. These results indicate that a relatively heat-stable type B
110
P ~ o F. Ma ah'v JAMBSR. FmH~a
enzyme is present in frog duodenum which has an RSS value similar to the type B enzyme in fiver. Unfortunately, Sephadex chromatography of heated duodenal extracts has not been possible due to the low level of enzyme activity obtained. Exact comparisons of RSS values between the enzymes in liver and duodenum has been made with the type C adenosine deaminases. This fiver enzyme gives a value of 1"36 whereas the duodenal enzyme gives a value of 1.22. This indicates that these enzymes are similar but not identical. >, L2'
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FIo. 4. A Sephadex G-150 chromatographic pattern of adenosine deaminue activity in a frog duodenal extract (See legend to Fig. 1). These studies show that the large tissue specific differences between adenosine deaminases in frog fiver and duodenum are the result of differences in the type of enzyme which dominates; i.e. 68 per cent type B in the liver and 83 per cent type C in the duodenum. It appears that both tissues have all three types and that the same types in both tissues have similar relative substrate specificities. DISCUSSION Results presented in this paper show that frog tissues have three types of adenosine deamlnases. Similar studies with other amphibians, reptiles, fish and mammals have demonstrated the widespread occurrence of all three types of enzymes (Ma & Fisher, unpublished restdts). Consequently it seems reasonable to classify these enzymes into three groups. Types A, B and C enzymes are in the size ranges of 180,000, 100,000 and 35,000 tool. wt. respectively. Type C adenosine deaminases have been found in every vertebrate tissue studied (except the tadpole liver) including a variety of mammAl;an tissues (Brady & O'Sullivan, 1967; Cory
MULTIPLR ADI~NOSINE DBAMINASI~ IN TH~ FROG
111
et al., 1967; Pfrogner, 1967), chicken duodenum and liver (Hoagland & Fisher, 1967; Ma & Fisher, 1968), frog duodenum and liver and in the livers of a wide variety of fish and reptiles (Ma & Fisher, unpublished results). The larger adenosine deaminases are not ubiquitous; e.g. types A and B are missing in chicken duodenum (Hoagland & Fisher, 1967) and in the livers of most mammals (Ma & Fisher, unpublished results). A variety of tissue specific properties of adenosine dearainases have been described in mammalian tissues, including intestine (Brady & O'Connell, 1962; Chilson & Fisher, 1963), spleen (Pfrogner, 1967), heart (Rockwell & Maguire, 1966) and serum (Cory et al., 1967) of the calf. Since these tissues contain only type C enzymes, the differences are due to variations in the properties of these low molecular weight adenosine deaminases. In the chicken, Ma & Fisher (1968) showed that tissue specific differences are due to the presence of type B enzyme in the liver which is absent in the duodenum. Both tissues contain type C enzymes which are identical in every property tested. These properties included relative substrate specificities with five substrates, Michaelis constants, energies of activation and activation by p-mercuribenzoate. Results presented in this paper show that tissue specific differences between frog liver and duodenal adenosine deaminases are due to the difference in the proportions of the three types of enzymes present. In the liver type B enzyme dominates (68 per cent of total activity) and in the duodenum type C enzyme dominates (83 per cent of total activity). The relative substrate specificities of the corresponding types of enzymes in both tissues are quite similar. So far it has not been possible to isolate the enzymes from each tissue and make detailed comparisons. Ackno~oledgement--We would like to thank Dr. Earl Frieden and his coworkers for supplying and staging the tadpoles used in this study.
REFERENCES ANDREWSP. (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. j~. 91, 222-233. Bp~vY T. G. & O'CoNN~L W. (1962) A purification of adenosine deaminase from the superficial mucosa of calf intestine. Biochim. biophys. Acta 62, 216-229. BRADYT. G. & O'DONOV~ C. I. (1961) Survey of the distribution and nature of the mammalian adenosine deaminase. Biochem.y. 80, 17P. B~vY T. G. & O'DONOV~ C. I. (1965) A study of the tissue distribution of adenosine deaminase in six mammalian species. Comp. Biochem. Physiol. 14, 101-120. Bm~vYT. G. & O'SULLrV~ M. (1967) The effect of pH, urea and guanidine on adenosine deaminase isolated from bovine mucosa. Biochim. biophys. Acta 132, 127-137. CmLSONO. P. & FXSHZRJ. R. (1963) Some comparative studies of calf and chicken adenosine deaminase. Archs Biochem. Biophys. 102, 77-85. CORYJ. G., W E ~ A ~ G. & SVH~OLNIKR. J. (1967) Multiple forms of calf serum adenosine deaminase. Archs Biochem. Biophys. 118, 428--433. FmHE~J. R., MA P. F. & CmLSONO. P. (1965) Some tissue specific differences in adenosine deaminase. Comp. Biochem. Physiol. 16, 199-203. HOACLANVV. D., JR. & FISH~ J. R. (1967) Purification and properties of chicken duodenal adenosine deaminase, y. biol. Chem. 242, 4341-4351.
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PANO F. MA AND JAM~ R. FISHBR
KALC~R H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes--III. Studies of the enzymes of purine metabolism. ~. biol. Chem. 167, 461--475. Ms P. F. & Faming J. R. (1968) Two different hepatic adenosine deaminases in the chicken. Biochim. biophys. .,qcta 159, 153-159. PlmOOlq~N. (1967) Adenosine deaminase fromcalfspleen--II. Chernicalend enzymological properties. ,drdu Biochem. Biophys. 119, 147-154. Rocgwm2. M. & Msotalm M. H. (1966) Studies on adenosine deaminase---I. Purification and properties of ox heart adenosine deaminase. Mol. Pharma¢ol. 2, 574-584.