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Adenosine aminohydrolase from the clam, Meretrix meretrix lusoria (Gmelin)
Comp. Biochem. Physiol., 1966, Vol. 17, pp. 271 to 284. Pergamon Press Ltd. Printed in Great Britain
ADENOSINE AMINOHYDROLASE FROM THE CLAM, M E R E T R I X M E R E T R I X L USORIA (GMELIN) TOYOO AIKAWA Biological Institute, Chiba University, Chiba, Japan (Received 11 ffune 1965)
Abstract--1. An adenosine aminohydrolase was extracted from the-mid-gut gland of the clam, Meretrix meretrix lusoria (Gmelin), in a highly purified form which, though heat-labile, could he stored over a month at 0°C without losing the specific activity amounting to 750-fold. 2. The preparation catalysed the reaction of the first order, with the activation energy of 14,600 cal under the experimental conditions, showing the velocity constant of 13-18 x 10 -3 rain -1. 3. A sharp optimum was found at pH 5'0. The Michaelis-Menten constant was 6.9 x 10-u M at 26°C at the optimal pH. 4. This enzyme specifically decomposes adenosine and deoxyadenosine. Mercury, either metallic or organic, and fluoride inhibit the enzymatic reaction, though many common agents are not found effective. INTRODUCTION AMONG the enzymes of nucleic acid decomposition, the most specified are those in higher vertebrates and microorganisms that catalyse degradation of purinecontaining nucleotides to uric acid; their nature, physiological significance and distribution in the cells and tissues have been described in detail (Chargaff & Davidson, 1955 ; Florkin & Mason, 1960; Greenberg, 1961). Enzymes with functions similar to the ones mentioned above have been found in invertebrates, and discussed largely from the physiological or systematic point of view (Lennox, 1941 ; Florkin, 1945 ; Wagner & Mitchell, 1948; Ishida, 1955, 1956; Roush & Betz, 1956; Aikawa, 1959; Florkin & Mason, 1960). But only few enzymes have been obtained in purified forms. As to the so-called adenosine deaminase, first studied by GySrgy & R6thler (1927) in the autolysing rabbit tissues, a number of investigators have described its specificity and distribution in various vertebrate tissues (Schmidt, 1928; Conway & Cooke, 1939; Brady, 1942; Kalckar, 1947b, 1947c) and in microorganisms (Kaplan et al., 1952; Powell & Hunter, 1956). The author had already noted that the homogenated mid-gut gland of the clam, M e r e t r i x m e r e t r i x lusoria, shows an obvious activity of catalysing production of ammonia from adenosine (Aikawa, 1959). The organ seemed to contain a kind of enzyme which is similar in general function to, but different in detailed characters from the vertebrate adenosine deaminase. For this reason, the author has been attempting to purify the enzyme and has been partly successful in obtaining the material which, though not crystallized yet, is satisfactory for carrying out the work which includes kinetic and 271
272
ZoYoo AIKAWA
c o m p a r a t i v e studies. A c c o r d i n g to t h e d e c i s i o n of t h e E n z y m e C o m m i s s i o n t h e t e r m a d e n o s i n e d e a m i n a s e s h o u l d be r e p l a c e d b y a d e n o s i n e a m i n o h y d r o l a s e , an d this d e c i s i o n is f o l l o w e d in t h e p r e s e n t p a p e r ( D i x o n & W e b b , 1964). MATERIALS AND METHODS
1. Enzyme assay Adenosine aminohydrolase catalyses the reaction: Adenosine + H 2 0 ~ NH3 + Inosine. T h e activity of this enzyme can be determined on a spectrophotometer according to Kalckar's method (1947a, 1947b, 1947c). In many cases the enzyme and substrate were mixed in a quartz vessel of the temperature-controlled spectrophotometer within which the reaction occurred: a diluted enzyme solution (0'5 ml) was added to 3"0 ml of the solution containing 5 x 10 -5 M of adenosine and 0"1 M of citrate-phosphate buffer at p H 5"0, at 26°C. T h e optical density of the reaction mixture was determined at intervals at the wavelength of 265 m/z, and its decrease from 0-58 to 0"48/min was defined as representing 10 units of the enzyme activity. In case the direct spectrophotometry was not practicable, e.g. if a higher temperature or a more concentrated substrate or a crude form of enzyme was taken into account, the reaction mixture should be prepared in a test tube. In such a case 0.5 ml of enzyme solution was added to a mixture of 1 ml of the substrate solution plus 2'5 ml of 0"1 M buffer solution. At any desired moment the reaction could be stopped by the addition of 1 ml of 1 N sulphuric acid which brought the pH of the mixture down to 1'9. Then, if necessary, the mixture was centrifuged. After being adequately diluted with water, the optical density of the sample was determined at 265 m/z. A modification of Lang's method (1958) was applied for determining protein-nitrogen. T h e protein content of the enz)ane solution was determined directly by the copper-Folin method of Lowry et al. (1951), and also spectrophotometrically after the formu.la of Kalckar (1947c) : protein mg/ml ----l'45E~s0- 0"74E26o. For the standard protein solutions the bovine serum albumin fraction V was used. Adenosine and other substrates were manufactured by the Nutritional Biochemical Corporation, the Sigma Chemical Company, the Zellstofffabrik Waldhof, and the Tokyo Kagaku Kasei Kogyo Company. Deionized and distilled water was used. T h e spectrophotometer used was the Hitachi E P U - 2 type. T h e Toyo Rika G A - S glass-electrode pH meter was used.
2. Purification procedure Preparation of the enzyme solution. T h e mid-gut gland tissues were collected from fresh individuals of the clam, Meretrix meretrix lusoria, and homogenized with 9 vol of chilled water for 3 min in a Waring blendor with stainless steel edges. During the purification procedures which will be described presently the temperature was always kept near 0°C. After standing for 30-60 min the homogenate was centrifuged for 1 hr at 17,000 g, and the supernatant was collected to be employed as the starting enzyme solution (supernatant-1). Partial removal of impurities by lowering pH. T h e above solution (supernatant-1) usually was from pH 6"3-7-0. By adding drops of 0-5 M acetic acid solution the pH was lowered to 5"0. This produced a precipitate which was discarded 1 hr later by centrifugation. A clear yellowish supernatant was obtained. T h e enzyme activity was well preserved in it (supernatant-2). The first ammonium sulphate fractionation. For salting out, 30'4 g of finely powdered ammonium sulphate was added to 100 ml of the above supematant (supernatant-2) while being constantly stirred. During this course pH was kept exactly at 7'0 by adding 5 N
ADENOSINEAMINOHYDROLASEFROM THE CLAM
273
solutions of ammonia and sulphuric acid, and the temperature was kept at 0°C. After standing for 2 hr, the resultant precipitate was discarded. T o 100 ml of the supernatant 19"7 g of the powdered ammonium sulphate was added in the manner as before, and it was left to stand for 2 hr at pH 7"0 at 0°C and then the precipitate was produced. This precipitate was dissolved in chilled water of which the volume amounted to four-fifths of the original solution (supernatant-2). The first ammonium sulphate fraction thus made was divided into portions, each about 20 ml, placed in cellulose bags (2'5 cm dia.), and then dialysed against deionized water (with specific resistance over 5 M ~ . c m , chilled below 5°C) for 80 hr. The water was changed three times during the first 15 hr and thereafter once at every twelfth hour. A great amount of precipitate was formed within the bags during dialysis, and this was excluded by centrifugation. Most of the aminohydrolase activity was retained in the supernatant liquid, of which pH was 4-4-4'8 (supematant-3). A good result was obtained if the TABLE I--PuRIFICATION OF ADENOSINEAMINOHYDROLASEFROM THE CLAM MID-GUT GLAND
Procedure
Initial extract Supernatant freed from precipitation at pH 5-0 1st ammonium sulphate fraction (after dialysis) 2nd ammonium sulphate fraction (after dialysis) C7-gel fraction (negative adsorption) Ca-phosphate gel eluate (after dialysis)
Total protein
Units in total Units per mg preparation protein Purification Yield
mg 6,460
13,000
2'01
1
2,020
12,700
6-3
3"14
186 "4
9,016
48 "4
24" 1
25"5
5,467
214"0
106"5
42
12"7
5,194
410"0
204
40
1"8
2,720
1,510"0
751
20"9
(100) 97.6
69" 5
Source: 33 g wet wt of the clam mid-gut gland. concentration of the protein in the supematant, while being dialysed, was reduced to less than 0"1 per cent. The quantity of the precipitate is very variable, presumably owing to the varying amount of contaminating proteins in the tissues. The second ammonium sulphatefractionation. To 100 ml of the above preparation (supernatant-3) 43"3 g of the powdered ammonium sulphate was added in the same manner as before, and, after 2 hr the precipitate was discarded. T h e n 6"1 g of ammonium sulphate was added to 100 ml of the supernatant, and the precipitate was collected and dissolved in water of which the volume was three-tenths of the initial supernatant (supernatant-3). The precipitate was portioned and placed in cellulose bags, and dialysed against deionized water for 80 hr at 5°C. T h e precipitate, though slight, was centrifuged off. The supematant, with pH 4"5-4"8, retained a strong activity of the enzyme (supernatant-4). Fractionation by adsorbents. Alumina-C7 (Willst~itter & Kraut, 1923) and calcium phosphate gel (Keilin & Hartree, 1938) were effective to increase the yield of the well purified preparations (Table 1).
274
ToYoo AIKAWA
(1) Negative adsorption. To the product of the previous fractionation (supernatant-4) one-tenth at a volume of 0"1 M acetate buffer of pH 5'0, was added. Alumina Cy gel, equal in weight to that of the total protein in the supernatant, was added under constant agitation, at 0°C. After 10 min the gel was removed by centrifugation. The supernatant was found to be very active. When the result was not satisfactory, the treatment was repeated. (2) Positive adsorption. To the above product, calcium phosphate gel weighing four times the total protein, was added. After the adjustment of pH to 5"6, the suspension was stirred for 10 min at 0°C. Nearly all of the enzyme was adsorbed into the gel. This was collected by centrifugation and the gel was washed with chilled acetate buffer (0"01 NI, pH 5-0), and then eluted with 0-1 M sodium phosphate buffer, pH 6"5, amounting to 0"4 times the volume of supernatant-4, within 15 rain after the procedure at 0°C. The eluate was dialysed against deionized water at 5 °C, for 15 hr with a change. The product was stored in an ice box at 0°C. Table 1 summarizes a result typical of the procedure of purification described above. RESULTS In the following are described certain properties and kinetic behavior of the adenosine aminohydrolase extracted and purified from the m i d - g u t gland tissues of the clam. T h i s invertebrate enzyme appears different in detailed characters from the vertebrate samples. All the preparations for characterizing the present enzyme were of the specific activity of 7.0 or more (optical density change/rain at 265 m~/ protein m g in reaction mixture). T h e initial velocity in this work represents the change of optical density (--AEz6s)/min, unless otherwise noticed. Stability. T h e purified enzyme was not resistant to heat; it was inactivated at 30°C in 20 min, and at 40°C, in 5 min. At 26°C the enzyme lost half of its activity in 2 hr, as shown in Fig. 1, and in 150 min the activity decreased to 42 per cent
. I00 -6 "E
~_ so
< 40
I 30 T i m e of p r e i n c u b a t i o n
I 60 at 26°C,
I 90
I 120 rain
FIG. 1. Inactivation by preincubation of enzyme solution at 26°C. of the initial. Freeze-drying or deep-freezing also resulted in an immediate loss of the activity. However, it was found that the enzymatic activity of the crude solution was not lost in 10 min at 50°C. Dissolved in water alone, and kept at 0°C, the enzyme remained intact for 1 m o n t h ( p H 4-7). On the one hand, the enzyme solution (15/~g of protein/ml) in tris buffer or citrate-phosphate buffer (0.05 M) retained most of the initial activity for 6 days at 0°C, if kept at p H 7-2. As shown in Fig. 2, the enzyme in the buffer solution was stable only near neutrality whereas it lost m u c h of its activity in other ranges, especially in alkaline state.
ADENOSINE AMINOHYDROLASE FROM T H E CLAM
275
Effect of temperature on reaction velocity. T h e apparent activation energy of the enzyme was approximately 14,600 cal as plotted after Arrhenius' equation (Fig. 3), In this experiment, the reaction was started by adding 0.5 ml of the ~0
I00
so o
.c ~, 4c ~6
20
3
4
5
6
7
8
9
pH
Fxo. 2. Effect of pH on the stability of the aminohydrolase, 15 pg protein per ml of 0"05 M buffer, stored 6 days at 0°C. e , citrate-phosphate buffer; ©, tris buffer.
w <] "2 o -I.~ > '2
~6-zc E
3.0'
310
t
52
I
I 34
I
I 36
I
x 104
FIG. 3. Effect of temperature on the initial velocity of the aminohydrolase. T, absolute temperature. enzyme solution to the preincubated substrate solution (1 ml of 2.5 × 10 -4 M adenosine mixed with 2.5 ml of 0.1 M citrate-phosphate buffer, pH 5-0). After 2 min of incubation at the desired temperature, the reaction was stopped by the rapid addition of I ml of 1 N sulphuric acid. T h e decrease of the optical density at 265 m/~ was determined and the reaction velocity was calculated. OptimalpH. Different from the enzyme from other sources, the clam adenosine aminohydrolase in the citrate-phosphate buffer (0.1 M) showed a narrow range of optimal pH with a peak at 5-0. T h e activity fell one their side, especially on the
276
TOYOO AIKAWA
acid side of the optimum. In the acetate buffer the optimum shifted to pH 4"6, and in citrate or succinate it came to pH 5.4 (Fig. 4). Enzyme concentration and reaction velocity. T h e decomposing reaction of adenosine by the present enzyme was of the first order. As shown in Fig. 5, the plots of log ( a / a - x) against t were on a straight line, where a stands for the initial 40
30
g
.E
3
4
5
6
7
pH
FIG. 4. Effect of pH on the aminohydrolase activity in different buffers. Reaction mixture contained 5 x 10 -5 M adenosine in 0"1 M buffer and 3'6 units of enzyme. Temperature was 26°C. 0 , citrate-phosphate; O, citrate; • , acetate; II, succinate. 0"3
o ~ 0"2
_J 0-1
I
I
I
I
Z Time,
3
I
4
5
rain
FIG. 5. Plots of log (a/a-x) versus time in rain. Reaction mixture contained 5 x 10 -5 M adenosine in citrate-phosphate buffer, pH 5"0, and 7"2 units of the enzyme. Temperature, 26°C. substrate concentration and x the decrease of the concentration of the substrate due to conversion in time t. T h e velocity constant, k, under the experimental conditions, was calculated as 13-18 x 10 .2 min -1 when 7.2 units of enzyme was in
ADENOSINE
AMINOHY'DROLASE
FROM THE CLAM
277
the reaction mixture. If the substrate was constant in concentration, the relation between enzyme concentration and reaction velocity was rectilinear, as shown in Fig. 6. Substrate concentration and reaction velodty. T h e initial reaction velocity was determined at 26°C and p H 5"0 (0.1 M citrate-phosphate buffer), with different
y
<1 i-c "~ 0.E m o
0.6
0.4
,/ I 3
I 4
I 5
Prolein,
I 6
I 7
I S
t~cj/rnl,
FIC. 6. Effect of enzyme concentration on the reaction velocity. Reaction mixture contained 5 x 10 -5 M adenosine, 0"1 M citrate-phosphate buffer, pH 5.0. Temperature, 26°C. 04
0.3
-I> 0.2
0.1
/,,J
I0
2.0
30
40
50
S
FIG. 7. Effect of substrate concentration on the initial reaction velocity, at 26°C. Reaction mixture contained 0"1 M citrate-phosphate buffer, pH 5-0, 7"5 units of the enzyme, and substrate. Initial velocity (V) is expressed as relative value of change in adenosine per rain. Substrate concentration (S) is expressed in micromoles per rnl. concentration of the substrate. T h e Michaelis-Menten constant, Km, calculated after Lineweaver & Burk (1934), was 6.9 × 10 -5 M (Fig. 7). Substrate specifidty. T h e enzyme did not attack adenine, adenosine-5'-monophosphate, deoxyadenosine-5'-monophosphate, guanine, 8-azaguanine, guanosine, guanosine-monophosphate, cytosine, cytidine and cytidine-monophosphate. It
278
ToYoo AIKAWA
hydrolysed adenosine-3'-monophosphate very slowly; the reaction rate was only about 4 per cent of that for adenosine. Deoxyadenosine was decomposed by the
oy 100
8o
E
.~ 60
40
2O
I
I
I
I
5
I0
Fluoride concentration,
/~M/rnt
FIG. 8. Inhibition by different concentrations of fluoride on the reaction velocity, at 26°C. Reaction mixture contained 0"1 M citrate-phosphate buffer, pH 5'0, 6"6 units of the enzyme, and fluoride which final concentration is expressed on abscissa in micromoles per ml. _1> 0.5
~
0.4
o ~
0.2:
0.t
I
I0
I
I
20
30
I 40
I 50
I
T
FIG. 9. Effect of substrate concentration on the inhibitory action of fluoride. Initial velocity (V) is expressed as -AE~sdrnin , and the substrate concentration (S) is expressed as micromoles/ml. Citrate-phosphate buffer, 0"1 M and pH 5"0, was used. Temperature was 26°C. 0, without inhibitor; ©, with 2 x 10 -~ M fluoride. present enzyme at the rate as fast as, or even faster than that in adenosine, under the same conditions. All these substrates were investigated in 0.1 M citratephosphate buffer at three different p H ' s , 4.3, 5-0 and 7.0, using the spectrop h o t o m e t e r and the methods of other authors (Kalckar, 1947a, 1947b, 1947c; Roush & Norris, 1950; W a n g et al., 1950). Effects of anions. A m o n g the anions, CI-, Br-, I - , N O ~ - , SO42-, C H a C O 0 - , citrate and succinate showed scarcely no effect on the enzyme reaction. Pyrophosphate was slightly inhibitory. Fluoride was very inhibitory. In this case the per
279
ADENOSINE AMINOHYDROLASE FROM THE CLAM
c e n t i n h i b i t i o n was p r o p o r t i o n a l to t h e c o n c e n t r a t i o n of t h e i n h i b i t o r (Fig. 8). T h e effect of t h e s u b s t r a t e c o n c e n t r a t i o n o n t h e i n h i b i t i o n b y f l u o r i d e (2 × 10 -3 M in t h e final c o n c e n t r a t i o n o f F - ) was s t u d i e d s p e c t r o p h o t o m e t r i c a l l y , a n d t h e g r a p h i c a l t r e a t m e n t of t h e results after L i n e w e a v e r & B u r k (1934), s h o w e d t h a t t h e i n h i b i t i o n was n o n - c o m p e t i t i v e ( F i g . 9). Effects of heavy metals. A s m u c h as 93 p e r c e n t of t h e e n z y m e a c t i v i t y was i n h i b i t e d b y H g ~+ ion of 10 -3 M in t h e final c o n c e n t r a t i o n . L i t t l e effect was s h o w n b y N a +, K +, C a 2+, a n d M g 2+. Ba 2+ s h o w e d no significant effect. Z i n c ion at 1 x 10 -3 M was n o t i n h i b i t o r y . H o w e v e r , C o S+ at 1 × 10 -3 M i n h i b i t e d 6-5 p e r c e n t o f t h e activity. TABLE 2--EFFECTS
oF DIFFERENT ADENOSINE
Agents
Na + K+ M g ~+ Ca ~+ Co z+ B a 8+
Zn 2+ Hg ~+ FFFBrINOB-
Final concentration (M)
Inhibition (%)
1 x 10 -2 1 × 10 -2 1 x 10 -2 1 x 10 -~ 1 x 10 -s 1 x 10 -s 1 x 10 -3 1 x 10 -3 1 x 10 -3 5 x 10 -3 1 x 10 -3 1 x 10 -3 1 x 10 -3 1 x 10 -3
(1"7) (4) (1-4) (5) 6"5 1 (4"8) 92.9 19 43 61"9 (1) (1"5) (4"8)
AGENTS
ON
THE ACTIVITY OF THE CLAM
AMINOHYDROLASE
Agents
SO4 2Pyrophosphate Versene Versene PCMB Monoiodoaeetie acid Iodosobenzoate Ferricyanide H~O2 Ascorbie acid Hydroquinone Cysteine Oxalic acid Na2S~O4
Final concentration (M) 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x
10 -3 10 -3 10 -8 10 -z 10 -s 10 -3 10 -3 10 -z 10 -2 10 -4 10 -3 10 -3 10 -a 10 -~
Inhibition (%) (5) 10-3 1 12"3 33"7 (5) (7) (11) (3) 0 (6"2) (8) (4) (5)
Reaction mixture consisted of 5 x 10 -n M adenosine, 0.1 M citrate-phosphate buffer, p H 5"0, and 6-3 units of enzyme, at 26°C. Figures in parentheses represent acceleration, also in per cent.
Effects of sulphhydryl group binders and other agents. V e r s e n e ( e t h y l e n e d i a m i n e tetra/icetic a c i d t e t r a s o d i u m salt) (1 × 10 -~ M ) i n h i b i t e d 12.3 p e r c e n t o f t h e e n z y m a t i c activity. T h e existence of 1 x 10 -5 M P C M B ( p - e h l o r o m e r c u r i c b e n z o i c acid) in t h e r e a c t i o n m i x t u r e r e d u c e d t h e v e l o c i t y to 43.7 p e r c e n t o f t h e c o n t r o l ; h o w e v e r , m o n o i o d o a c e t i c a c i d o r i o d o s o b e n z o a t e was n o t i n h i b i t o r y , o r even accelerating. T h e r e d u c i n g a g e n t s s u c h as oxalic acid, s o d i u m h y d r o s u l p h a t e , h y d r o q u i n o n e a n d c y s t e i n e effected r e s p e c t i v e l y 4, 5, 6-2 a n d 8 p e r c e n t o f t h e i n c r e a s e o f activity, b u t a s c o r b i c a c i d h a d no effect. H y d r o g e n p e r o x i d e was n o t effective. P o t a s s i u m f e r r i c y a n i d e (1 x 10 4 M ) effeeted 11 p e r c e n t acceleration. T a b l e 2 s u m m a r i z e s t h e s e results.
280
ToYoo AIKAWA
DISCUSSION The crude extract from the clam mid-gut gland maintained the activity of adenosine aminohydrolase even after being heated at 50°C for 10 min or longer. When purified, the enzyme appears far less resistant to heating, deep freezing and freeze-drying, being readily inactivated. However, if stored as an aqueous solution at 0°C, the purified enzyme remained intact for one month, at pH 4"7. Treated with buffers the enzyme behaved differently; it was best preserved nearly at neutrality as examined after 6 days of storage in an ice box. The apparent activation energy was calculated approximately as 14,600 cal. As the purified enzyme is liable to be inactivated by heat, the data obtained at temperatures higher than 30°C might somewhat have been affected. Nevertheless they were adopted in the calculation, for only a slight error might be caused by heating in a very short period (2 min) of reaction. The present enzyme shows a sharply peaked optimum at pH 5.0 or thereabout. In this respect it differs much from any kind of adenosine aminohydrolases from other sources. The adenosine aminohydrolases from the rabbit and other vertebrates are most effective near the neutral state, and are comparatively active over a wide range of pH (Schmidt, 1928 ; Conway & Cooke, 1939 ; Brady, 1942; Kalckar, 1947c). The enzyme in the blowfly larva has an optimum at pH 8.3, according to Lennox (1941). The activity of the enzyme from Drosophila larva was determined at pH 6.5 (Wagner & Mitchell, 1948). The adenosine aminohydrolase in the ammonium sulphate fraction of the lobster hepatopancreas was studied at pH 7-0 (Roush & Betz, 1956). The maximum activity of the enzyme in Bacillus cereus spore is shown at pH 8"7 (Powell & Hunter, 1956), and the non-specific adenosine aminohydrolase from taka-diastase has an optimum at pH 6.8 (Kaplan et al., 1952). Previously the author reported that the homogenate of the mid-gut gland of the clam showed the aminohydrolase activity over a wide range of pH 3-8 (Aikawa, 1959). That the purified enzyme has a much more restricted range of activity is interesting from the physiological viewpoint. A similar tendency is observed in the same enzyme in the clam gill (Umemori, unpublished). On the other hand, the activity of the adenosine aminohydrolase in the adductor and foot muscles of the clam is sharpely intensified at pH 7-5-8.0 (Aikawa, 1959). Therefore, these adenosine aminohydrolases in various tissues of the clam, should be studied further in purified form. Under the present experimental conditions, a rectilinear proportionality is found between the initial velocity and the enzyme concentration, and the reaction producing inosine and ammonia from adenosine must be of the first order, and it was found that one mole of inosine is produced from one mole of adenosine. The production of inosine was confirmed by the increase of optical density change at 240 m~. The highly specific clam enzyme is in contrast with the adenosine aminohydrolase from taka-diastase which is non-specific (Kaplan et al., 1952). The amino groups of free adenine and other purine and pyrimidine compounds are resistant to the clam enzyme. Deoxyadenosine is the only substrate which can be hydrolysed
ADENOSINE A M I N O H Y D R O L A S E FROM THE CLAM
281
equally as adenosine, and it is probable that both nucleosides are attacked by the same enzyme, as already suggested by Brady (1942) and Schaedel et al. (1947). Roush & Betz (1956) found that an ammonium sulphate fraction of the lobster hepatopancreas has enzymatic activities sufficient to decompose adenosine and deoxyadenosine. They consider that two different enzymes are contained in this fraction, since they obtained two different values of specific activity, 0.66 for adenosine and 0.28 for deoxyadenosine. According to Brady (1942), the blood extracts of the rabbit, fowl, rat, and man, and also some tissue extracts of the rabbit hydrolyse deoxyadenosine at half the rate of adenosine, while the enzyme from the calf mucosa hydrolyses both substrates at an equal rate. He concludes from the experiment, in which both substrates are mixed, that these activities do not belong to separate enzymes. Considering Brady's result, the activities of the lobster hepatopancreas for adenosine and deoxyadenosine might be exerted by a single enzyme. Schaedel et al. (1947) suggested that the high specificity of the intestinal aminohydrolase is not only due to a perfect fit of the purine ring to the enzyme but to many other structural restrictions of the substrate. According to Byrne (1954) the highly purified adenosine aminohydrolase from beef spleen can hydrolize amino groups of several nucleotides with the optimum at pH 5.2. As shown in Table 2, fluoride ion is strongly inhibitory to the clam enzyme. This inhibition is proportional to fluoride concentration and is noncompetitive, though a mechanism in more detail is unknown. The activity is extensively inhibited by Hg 2+, while most other metal ions have little or no effect. Versene and pyrophosphate inhibit more than 10 per cent of the activity. These results suggest that the metals may not be required for the enzyme. Among the sulphhydryl binding agents only PCMB, the mercuric compound, is obviously inhibitory. The enzyme may be very sensitive to Hg in the PCMB molecule, for its chemical behavior toward this substance is like that toward the metallic Hg. The present results are rather similar to those of the calf enzyme which in general is scarcely affected by common inhibitors (Brady, 1942). Wagner & Mitchell (1948) observed a proportionality between the growth rate and the adenosine aminohydrolase activity in the larva of Drosophila melanogaster. In the chick embryo, the enzyme was formed by an adaptive induction (Gordon & Roder, 1953). Stern et al. (1952), investigated vertebrate tissues and found adenosine aminohydrolase and guanine aminohydrolase (guanase) in cytoplasm but not in nuclei. Thus they consider that these enzymes are not essential for the universal pattern of nuclei metabolism. Furthermore, they found that the adenosine aminohydrolase activity was forty-fold higher in the cytoplasm of the adult tissue than in that of the fetal calf intestinal mucosa. In view of these reports, it seems likely that the adenosine aminohydrolase in general is very changeable in its activity (or amount), either in the clam tissues or in other animal tissues. In addition, constancy in nature and amount of contaminated proteins may not be expected in enzyme solutions from the clam mid-gut gland, and it may be considered that the enzyme purity at different purification steps will be affected by many factors which vary with seasons, age and others. Further studies are needed on seasonal changes of the
282
ToYoo AIKAWA
enzymatic activity in relation to changes in other metabolic enzymes and also on amino acid constituents of relevant proteins. Adenosine has a strong vasodepressor effect, according to Drury & SzentGySrgyi (1929), Ewing et al. (1949), and Clarke et al. (1952). They discuss that the effect of adenosine could be temporary owing to the decomposing activity of the enzyme, while some other analogs retain the vasodepressor effect longer since they are resistant to the enzyme. Assuming from the work of Roush & Betz (1956) on crustaceans, the adenosine aminohydrolase may also play a part in pharmacological protection in the clam. It is also suggested that the enzyme participates in offering amino groups for the synthesis of amino compounds and in changing the permeability of cells (Conway & Cooke, 1939). Despite the existence of adenosine aminohydrolase, adenosine is effectively incorporated into adenosine monophosphate in the biosynthesis in the vertebrate, according to many authors (Caputto, 1951; Kornberg & Pricer, 1951 ; Lowy et al., 1952), and the enzyme action in metabolism is not elucidated yet. Recently, Umemori found in the homogenate of the clam mid-gut gland a high phosphatase activity toward adenosine-5'-monophosphate, inosine-5'-monophosphate, and a guanylic acid (unpublished). This phosphatase has two optimal peaks at pH 4.0-4.2 and 8-5-9.0, and always shows a depression near the neutrality. The activity is considerably higher on the alkaline side than on the acid. Further studies on these nucleotidases in more purified forms are required to make clear the role of the adenosine aminohydrolase in the pathway of adenylate metabolism, in the clam mid-gut gland and other tissues. SUMMARY 1. Adenosine aminohydrolase was extracted from the mid-gut gland of the clam, Meretrix meretrix lusoria, and purified by several fractionation methods. Preparation with high specific activity was obtained. 2. The purified enzyme is not resistant to heat, but can be stored unchanged at 0°C if dissolved in deionized and distilled water. Preserved in buffer solution of pH 7.2 at 0°C, it remained almost unchanged for 6 days. 3. The apparent activation energy of the enzyme activity was approximately 14,600 cal. Under usual conditions the reaction of the enzymatic decomposition of adenosine was of the first order, and the velocity constant, k, was of 13.18 x 10 -2 min -1. Change of reaction velocity was proportional to that of the enzyme concentration. 4. Optimal pH of the enzyme in citrate-phosphate buffer was 5-0 which was a sharply elevated peak. 5. At pH 5.0 and 26°C, the Michaelis-Menten constant, Kin, was 6-9 x 10 -a M. 6. The enzyme in purified form is highly specific. While it decomposes both adenosine and deoxyadenosine equally, it is not effective on most purines and pyrimidines, their nucleosides and nucleotides.
ADENOSINE AMINOHYDROLASE FROM THE CLAM
283
7. Effects of anions, metals, s u l p h h y d r y l binders and some o t h e r agents were examined. S t r o n g inhibitory effects were s h o w n b y fluoride and b y metallic or organic m e r c u r y . 8. T h e properties and metabolic significance of the e n z y m e were discussed in c o m p a r i s o n with that f r o m other sources. Acknowledgements--The author expresses his cordial thanks to Professor Shuzo Ishida and Professor Kiyoshi Aoki for their encouragement and criticisms. He is also grateful to Miss Yoko Umemori of the Chiba University for her help in many ways. This work was supported in part by the Grant-in-Aid for Fundamental Scientific Research of the Ministry of Education of Japan. REFERENCES AIKAWA T. (1959) Deamination of adenosine and adenylic acids in the clam, Meretrix meretrix lusoria (Gmelin). Sci. Rep. T6hoku Univ. (Biol.) 25, 73-80. BRADYT. (1942) Adenosine deaminase. Biochem..7. 36, 478-484. BYRNE W. L. (1954) The purification of an adenylic acid deaminase. Abstr. 126th National Meeting Am. Chem. Soc., N.Y., p. 73c (1955) In The Nucleic Acids (Edited by CHARGAFF E. & DAVmSON J. N.), Vol. 1, p. 597. Academic Press, N.Y. CAPUTTO R. (1951) The enzymic synthesis of adenylic acid; adenosinekinase. 07. Biol. Chem. 189, 801-814. CHARGAFFE. 86 DAVIDSONJ. N. (1955) The Nucleic Acids, Vol. 1, p. 595, and Vol. 2, p. 423. Academic Press, N.Y. CLARKED. A., DAVOLLJ., PHILIPS F. S. & BROWNG. B. (1952) Enzymatic deamination and vasodepressor effects of adenosine analogs..7. Pharm. Exp. Therap. 106, 291-302. CONWAY E. J. & COOKE R. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem. 07. 33, 479-492. DIXON M. & WEBS E. C. (1964) Enzymes 2nd Ed. p. 756. Longmans, London. DRURY A. N. & SZENT-GYSROYIA. (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. 07. Physiol. 68, 213-237. EWING P. L., SCHLENKF. & EMERSONG. A. (1949) Comparison of smooth muscle effects of crotonoside (isoguanosine) and adenosine. 07. Pharm. Exp. Therap. 97, 379-383. FLORKIN M. (1945) L'evolution du m6tabolisme des substances azot6es chez les animaux. Actualitds biochimiques, No. 3. Paris et Lifige. FLORKIN M. & MASON H. S. (1960) Comparative Biochemistry, Vol. 2, p. 178. Academic Press, N.Y. GORDON M. W. & RODER M. (1953) Adaptive enzyme formation in the chick embryo. 07. Biol. Chem. 200, 859-866. GR~ENBERQ D. M. (1961) Metabolic Pathways, Vol. 2, p. 419. Academic Press, N.Y. GY{SRGYP. & R{STHLERH. (1927) l~ber Bedingungen der autolytischen Ammoniakbildung in Geweben. III. Mitteilung. Die Beziehungen des Gewebsammoniaks zum Purinstoffwechsel. Biochem. Z. 187, 194--219. ISHIDA S. (1955) Metabolic patterns in bivalves. VII. Some aspects of the purine metabolism in the clam, Meretrix meretrix lusoria (Gmelin). ~. Coll. Arts Sci. Chiba Univ. 1, 270-272. ISmDA S. (1956) Metabolic patterns in bivalves. V I I I . Distribution of the guaninedeaminating activity in the soft parts of some marine bivalves. Bull. Mar. Biol. Sta. Asamushi 8, 9-18. KALCK~a~H. M. (1947a) Differential spectrophotometry of purine compounds by means of specific enzymes. I. Determination of hydroxypurine compounds, ft. Biol. Chem. 167, 429-443.
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ToYoo AIKAWA
KALCKARH. M. (1947b) Differential spectrophotometry of purine compounds by means of specific enzymes. I I. Determination of adenine compounds. J. Biol. Chem. 167, 4 4 5 4 5 9 . KALCKAR H. M. (1947c) Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167, 461-475. KAPLAN N. O., COLOWICKS. P. • CIOTTI M. M. (1952) Enzymatic deamination of adenosine derivatives. J. Biol. Chem. 194, 579-591. KEILIN D. & HARTREE E. F. (1938) On the mechanism of the decomposition of hydrogen peroxide by catalase. Proc. R. Soc. London (Ser. B) 124, 397-405. KORNBERG A. & PRICER W. E. JR. (1951) Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J. Biol. Chem. 193, 481-495. LANG C. A. (1958) Simple microdetermination of Kjeldahl nitrogen in biological materials. Anal. Chem. 30, 1692-1694. LENNOX F. G. (1941) The physiology and toxicology of blowflies. IX. T h e enzymes responsible for ammonia production by larvae of Lucillia cuprina. Aust. Council Sci. Industr. Res. Pamphlet No. 109, 37-64. Cited from Chem. Abstr. 36, 3559 (1942). LINEWEAVER H. & BURK D. (1934) T h e determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56, 658-666. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. T. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. LowY B. A., DAVOLL J. & BROWN G. B. (1952) The utilization of purine nucleosides for nucleic acid synthesis in the rat. J. Biol. Chem. 197, 591-600. POWELL J. F. & HUNTER J. R. (1956) Adenosine deaminase and ribosidase in spores of Bacillus cereus. Biochem. J. 62, 381-387. RousH A. H. & BETZ R. B. (1956) The adenosine deaminase of crustaceans. Biochim. biophys. Acta 19, 579-580. ROUSH A. & NORRIS E. R. (1950) Deamination of 8-azaguanine by guanase. Arch. Biochem. 29, 124-129. SCrIAEDEL M. L., WALDVOGEL M. J. & SCHLENK F. (1947) T h e specificity of adenosine deaminase and purine nucleosidase. J. Biol. Chem. 171, 135-141. SCHMIDT G. (1928) fiber fermentative Desaminierung im Muskel. Z. Physiol. Chem. 179, 243-282. STERN H., ALLFREYV., MIRSKYA. E. & SAETRENH. (1952) Some enzymes of isolated nuclei. -7. Gen. Physiol. 35, 559-578. WAGNER R. P. & MITCHELL H. K. (1948) An enzymatic assay for studying the nutrition of Drosophila melanogaster. Arch. Biochem. 17, 87-96. WANG T. V., SABLE H. Z. & LAMPEN O. (1950) Enzymatic deamination of cytosine nucleosides..7. Biol. Chem. 184, 17-28. WILLSTATTER R. & KRAUT H. (1923) Ober ein Tonerde-Gel yon der Formel AI(OH)3. II. Mitteilung tiber Hydrate und Hydrogele. Ber. Dtsch. Chem. Ges. 56, 1117-1121.
ADENOSINE AMINOHYDROLASE FROM THE CLAM, M E R E T R I X M E R E T R I X L USORIA (GMELIN) TOYOO AIKAWA Biological Institute, Chiba University, Chiba, Japan (Received 11 ffune 1965)
Abstract--1. An adenosine aminohydrolase was extracted from the-mid-gut gland of the clam, Meretrix meretrix lusoria (Gmelin), in a highly purified form which, though heat-labile, could he stored over a month at 0°C without losing the specific activity amounting to 750-fold. 2. The preparation catalysed the reaction of the first order, with the activation energy of 14,600 cal under the experimental conditions, showing the velocity constant of 13-18 x 10 -3 rain -1. 3. A sharp optimum was found at pH 5'0. The Michaelis-Menten constant was 6.9 x 10-u M at 26°C at the optimal pH. 4. This enzyme specifically decomposes adenosine and deoxyadenosine. Mercury, either metallic or organic, and fluoride inhibit the enzymatic reaction, though many common agents are not found effective. INTRODUCTION AMONG the enzymes of nucleic acid decomposition, the most specified are those in higher vertebrates and microorganisms that catalyse degradation of purinecontaining nucleotides to uric acid; their nature, physiological significance and distribution in the cells and tissues have been described in detail (Chargaff & Davidson, 1955 ; Florkin & Mason, 1960; Greenberg, 1961). Enzymes with functions similar to the ones mentioned above have been found in invertebrates, and discussed largely from the physiological or systematic point of view (Lennox, 1941 ; Florkin, 1945 ; Wagner & Mitchell, 1948; Ishida, 1955, 1956; Roush & Betz, 1956; Aikawa, 1959; Florkin & Mason, 1960). But only few enzymes have been obtained in purified forms. As to the so-called adenosine deaminase, first studied by GySrgy & R6thler (1927) in the autolysing rabbit tissues, a number of investigators have described its specificity and distribution in various vertebrate tissues (Schmidt, 1928; Conway & Cooke, 1939; Brady, 1942; Kalckar, 1947b, 1947c) and in microorganisms (Kaplan et al., 1952; Powell & Hunter, 1956). The author had already noted that the homogenated mid-gut gland of the clam, M e r e t r i x m e r e t r i x lusoria, shows an obvious activity of catalysing production of ammonia from adenosine (Aikawa, 1959). The organ seemed to contain a kind of enzyme which is similar in general function to, but different in detailed characters from the vertebrate adenosine deaminase. For this reason, the author has been attempting to purify the enzyme and has been partly successful in obtaining the material which, though not crystallized yet, is satisfactory for carrying out the work which includes kinetic and 271
272
ZoYoo AIKAWA
c o m p a r a t i v e studies. A c c o r d i n g to t h e d e c i s i o n of t h e E n z y m e C o m m i s s i o n t h e t e r m a d e n o s i n e d e a m i n a s e s h o u l d be r e p l a c e d b y a d e n o s i n e a m i n o h y d r o l a s e , an d this d e c i s i o n is f o l l o w e d in t h e p r e s e n t p a p e r ( D i x o n & W e b b , 1964). MATERIALS AND METHODS
1. Enzyme assay Adenosine aminohydrolase catalyses the reaction: Adenosine + H 2 0 ~ NH3 + Inosine. T h e activity of this enzyme can be determined on a spectrophotometer according to Kalckar's method (1947a, 1947b, 1947c). In many cases the enzyme and substrate were mixed in a quartz vessel of the temperature-controlled spectrophotometer within which the reaction occurred: a diluted enzyme solution (0'5 ml) was added to 3"0 ml of the solution containing 5 x 10 -5 M of adenosine and 0"1 M of citrate-phosphate buffer at p H 5"0, at 26°C. T h e optical density of the reaction mixture was determined at intervals at the wavelength of 265 m/z, and its decrease from 0-58 to 0"48/min was defined as representing 10 units of the enzyme activity. In case the direct spectrophotometry was not practicable, e.g. if a higher temperature or a more concentrated substrate or a crude form of enzyme was taken into account, the reaction mixture should be prepared in a test tube. In such a case 0.5 ml of enzyme solution was added to a mixture of 1 ml of the substrate solution plus 2'5 ml of 0"1 M buffer solution. At any desired moment the reaction could be stopped by the addition of 1 ml of 1 N sulphuric acid which brought the pH of the mixture down to 1'9. Then, if necessary, the mixture was centrifuged. After being adequately diluted with water, the optical density of the sample was determined at 265 m/z. A modification of Lang's method (1958) was applied for determining protein-nitrogen. T h e protein content of the enz)ane solution was determined directly by the copper-Folin method of Lowry et al. (1951), and also spectrophotometrically after the formu.la of Kalckar (1947c) : protein mg/ml ----l'45E~s0- 0"74E26o. For the standard protein solutions the bovine serum albumin fraction V was used. Adenosine and other substrates were manufactured by the Nutritional Biochemical Corporation, the Sigma Chemical Company, the Zellstofffabrik Waldhof, and the Tokyo Kagaku Kasei Kogyo Company. Deionized and distilled water was used. T h e spectrophotometer used was the Hitachi E P U - 2 type. T h e Toyo Rika G A - S glass-electrode pH meter was used.
2. Purification procedure Preparation of the enzyme solution. T h e mid-gut gland tissues were collected from fresh individuals of the clam, Meretrix meretrix lusoria, and homogenized with 9 vol of chilled water for 3 min in a Waring blendor with stainless steel edges. During the purification procedures which will be described presently the temperature was always kept near 0°C. After standing for 30-60 min the homogenate was centrifuged for 1 hr at 17,000 g, and the supernatant was collected to be employed as the starting enzyme solution (supernatant-1). Partial removal of impurities by lowering pH. T h e above solution (supernatant-1) usually was from pH 6"3-7-0. By adding drops of 0-5 M acetic acid solution the pH was lowered to 5"0. This produced a precipitate which was discarded 1 hr later by centrifugation. A clear yellowish supernatant was obtained. T h e enzyme activity was well preserved in it (supernatant-2). The first ammonium sulphate fractionation. For salting out, 30'4 g of finely powdered ammonium sulphate was added to 100 ml of the above supematant (supernatant-2) while being constantly stirred. During this course pH was kept exactly at 7'0 by adding 5 N
ADENOSINEAMINOHYDROLASEFROM THE CLAM
273
solutions of ammonia and sulphuric acid, and the temperature was kept at 0°C. After standing for 2 hr, the resultant precipitate was discarded. T o 100 ml of the supernatant 19"7 g of the powdered ammonium sulphate was added in the manner as before, and it was left to stand for 2 hr at pH 7"0 at 0°C and then the precipitate was produced. This precipitate was dissolved in chilled water of which the volume amounted to four-fifths of the original solution (supernatant-2). The first ammonium sulphate fraction thus made was divided into portions, each about 20 ml, placed in cellulose bags (2'5 cm dia.), and then dialysed against deionized water (with specific resistance over 5 M ~ . c m , chilled below 5°C) for 80 hr. The water was changed three times during the first 15 hr and thereafter once at every twelfth hour. A great amount of precipitate was formed within the bags during dialysis, and this was excluded by centrifugation. Most of the aminohydrolase activity was retained in the supernatant liquid, of which pH was 4-4-4'8 (supematant-3). A good result was obtained if the TABLE I--PuRIFICATION OF ADENOSINEAMINOHYDROLASEFROM THE CLAM MID-GUT GLAND
Procedure
Initial extract Supernatant freed from precipitation at pH 5-0 1st ammonium sulphate fraction (after dialysis) 2nd ammonium sulphate fraction (after dialysis) C7-gel fraction (negative adsorption) Ca-phosphate gel eluate (after dialysis)
Total protein
Units in total Units per mg preparation protein Purification Yield
mg 6,460
13,000
2'01
1
2,020
12,700
6-3
3"14
186 "4
9,016
48 "4
24" 1
25"5
5,467
214"0
106"5
42
12"7
5,194
410"0
204
40
1"8
2,720
1,510"0
751
20"9
(100) 97.6
69" 5
Source: 33 g wet wt of the clam mid-gut gland. concentration of the protein in the supematant, while being dialysed, was reduced to less than 0"1 per cent. The quantity of the precipitate is very variable, presumably owing to the varying amount of contaminating proteins in the tissues. The second ammonium sulphatefractionation. To 100 ml of the above preparation (supernatant-3) 43"3 g of the powdered ammonium sulphate was added in the same manner as before, and, after 2 hr the precipitate was discarded. T h e n 6"1 g of ammonium sulphate was added to 100 ml of the supernatant, and the precipitate was collected and dissolved in water of which the volume was three-tenths of the initial supernatant (supernatant-3). The precipitate was portioned and placed in cellulose bags, and dialysed against deionized water for 80 hr at 5°C. T h e precipitate, though slight, was centrifuged off. The supematant, with pH 4"5-4"8, retained a strong activity of the enzyme (supernatant-4). Fractionation by adsorbents. Alumina-C7 (Willst~itter & Kraut, 1923) and calcium phosphate gel (Keilin & Hartree, 1938) were effective to increase the yield of the well purified preparations (Table 1).
274
ToYoo AIKAWA
(1) Negative adsorption. To the product of the previous fractionation (supernatant-4) one-tenth at a volume of 0"1 M acetate buffer of pH 5'0, was added. Alumina Cy gel, equal in weight to that of the total protein in the supernatant, was added under constant agitation, at 0°C. After 10 min the gel was removed by centrifugation. The supernatant was found to be very active. When the result was not satisfactory, the treatment was repeated. (2) Positive adsorption. To the above product, calcium phosphate gel weighing four times the total protein, was added. After the adjustment of pH to 5"6, the suspension was stirred for 10 min at 0°C. Nearly all of the enzyme was adsorbed into the gel. This was collected by centrifugation and the gel was washed with chilled acetate buffer (0"01 NI, pH 5-0), and then eluted with 0-1 M sodium phosphate buffer, pH 6"5, amounting to 0"4 times the volume of supernatant-4, within 15 rain after the procedure at 0°C. The eluate was dialysed against deionized water at 5 °C, for 15 hr with a change. The product was stored in an ice box at 0°C. Table 1 summarizes a result typical of the procedure of purification described above. RESULTS In the following are described certain properties and kinetic behavior of the adenosine aminohydrolase extracted and purified from the m i d - g u t gland tissues of the clam. T h i s invertebrate enzyme appears different in detailed characters from the vertebrate samples. All the preparations for characterizing the present enzyme were of the specific activity of 7.0 or more (optical density change/rain at 265 m~/ protein m g in reaction mixture). T h e initial velocity in this work represents the change of optical density (--AEz6s)/min, unless otherwise noticed. Stability. T h e purified enzyme was not resistant to heat; it was inactivated at 30°C in 20 min, and at 40°C, in 5 min. At 26°C the enzyme lost half of its activity in 2 hr, as shown in Fig. 1, and in 150 min the activity decreased to 42 per cent
. I00 -6 "E
~_ so
< 40
I 30 T i m e of p r e i n c u b a t i o n
I 60 at 26°C,
I 90
I 120 rain
FIG. 1. Inactivation by preincubation of enzyme solution at 26°C. of the initial. Freeze-drying or deep-freezing also resulted in an immediate loss of the activity. However, it was found that the enzymatic activity of the crude solution was not lost in 10 min at 50°C. Dissolved in water alone, and kept at 0°C, the enzyme remained intact for 1 m o n t h ( p H 4-7). On the one hand, the enzyme solution (15/~g of protein/ml) in tris buffer or citrate-phosphate buffer (0.05 M) retained most of the initial activity for 6 days at 0°C, if kept at p H 7-2. As shown in Fig. 2, the enzyme in the buffer solution was stable only near neutrality whereas it lost m u c h of its activity in other ranges, especially in alkaline state.
ADENOSINE AMINOHYDROLASE FROM T H E CLAM
275
Effect of temperature on reaction velocity. T h e apparent activation energy of the enzyme was approximately 14,600 cal as plotted after Arrhenius' equation (Fig. 3), In this experiment, the reaction was started by adding 0.5 ml of the ~0
I00
so o
.c ~, 4c ~6
20
3
4
5
6
7
8
9
pH
Fxo. 2. Effect of pH on the stability of the aminohydrolase, 15 pg protein per ml of 0"05 M buffer, stored 6 days at 0°C. e , citrate-phosphate buffer; ©, tris buffer.
w <] "2 o -I.~ > '2
~6-zc E
3.0'
310
t
52
I
I 34
I
I 36
I
x 104
FIG. 3. Effect of temperature on the initial velocity of the aminohydrolase. T, absolute temperature. enzyme solution to the preincubated substrate solution (1 ml of 2.5 × 10 -4 M adenosine mixed with 2.5 ml of 0.1 M citrate-phosphate buffer, pH 5-0). After 2 min of incubation at the desired temperature, the reaction was stopped by the rapid addition of I ml of 1 N sulphuric acid. T h e decrease of the optical density at 265 m/~ was determined and the reaction velocity was calculated. OptimalpH. Different from the enzyme from other sources, the clam adenosine aminohydrolase in the citrate-phosphate buffer (0.1 M) showed a narrow range of optimal pH with a peak at 5-0. T h e activity fell one their side, especially on the
276
TOYOO AIKAWA
acid side of the optimum. In the acetate buffer the optimum shifted to pH 4"6, and in citrate or succinate it came to pH 5.4 (Fig. 4). Enzyme concentration and reaction velocity. T h e decomposing reaction of adenosine by the present enzyme was of the first order. As shown in Fig. 5, the plots of log ( a / a - x) against t were on a straight line, where a stands for the initial 40
30
g
.E
3
4
5
6
7
pH
FIG. 4. Effect of pH on the aminohydrolase activity in different buffers. Reaction mixture contained 5 x 10 -5 M adenosine in 0"1 M buffer and 3'6 units of enzyme. Temperature was 26°C. 0 , citrate-phosphate; O, citrate; • , acetate; II, succinate. 0"3
o ~ 0"2
_J 0-1
I
I
I
I
Z Time,
3
I
4
5
rain
FIG. 5. Plots of log (a/a-x) versus time in rain. Reaction mixture contained 5 x 10 -5 M adenosine in citrate-phosphate buffer, pH 5"0, and 7"2 units of the enzyme. Temperature, 26°C. substrate concentration and x the decrease of the concentration of the substrate due to conversion in time t. T h e velocity constant, k, under the experimental conditions, was calculated as 13-18 x 10 .2 min -1 when 7.2 units of enzyme was in
ADENOSINE
AMINOHY'DROLASE
FROM THE CLAM
277
the reaction mixture. If the substrate was constant in concentration, the relation between enzyme concentration and reaction velocity was rectilinear, as shown in Fig. 6. Substrate concentration and reaction velodty. T h e initial reaction velocity was determined at 26°C and p H 5"0 (0.1 M citrate-phosphate buffer), with different
y
<1 i-c "~ 0.E m o
0.6
0.4
,/ I 3
I 4
I 5
Prolein,
I 6
I 7
I S
t~cj/rnl,
FIC. 6. Effect of enzyme concentration on the reaction velocity. Reaction mixture contained 5 x 10 -5 M adenosine, 0"1 M citrate-phosphate buffer, pH 5.0. Temperature, 26°C. 04
0.3
-I> 0.2
0.1
/,,J
I0
2.0
30
40
50
S
FIG. 7. Effect of substrate concentration on the initial reaction velocity, at 26°C. Reaction mixture contained 0"1 M citrate-phosphate buffer, pH 5-0, 7"5 units of the enzyme, and substrate. Initial velocity (V) is expressed as relative value of change in adenosine per rain. Substrate concentration (S) is expressed in micromoles per rnl. concentration of the substrate. T h e Michaelis-Menten constant, Km, calculated after Lineweaver & Burk (1934), was 6.9 × 10 -5 M (Fig. 7). Substrate specifidty. T h e enzyme did not attack adenine, adenosine-5'-monophosphate, deoxyadenosine-5'-monophosphate, guanine, 8-azaguanine, guanosine, guanosine-monophosphate, cytosine, cytidine and cytidine-monophosphate. It
278
ToYoo AIKAWA
hydrolysed adenosine-3'-monophosphate very slowly; the reaction rate was only about 4 per cent of that for adenosine. Deoxyadenosine was decomposed by the
oy 100
8o
E
.~ 60
40
2O
I
I
I
I
5
I0
Fluoride concentration,
/~M/rnt
FIG. 8. Inhibition by different concentrations of fluoride on the reaction velocity, at 26°C. Reaction mixture contained 0"1 M citrate-phosphate buffer, pH 5'0, 6"6 units of the enzyme, and fluoride which final concentration is expressed on abscissa in micromoles per ml. _1> 0.5
~
0.4
o ~
0.2:
0.t
I
I0
I
I
20
30
I 40
I 50
I
T
FIG. 9. Effect of substrate concentration on the inhibitory action of fluoride. Initial velocity (V) is expressed as -AE~sdrnin , and the substrate concentration (S) is expressed as micromoles/ml. Citrate-phosphate buffer, 0"1 M and pH 5"0, was used. Temperature was 26°C. 0, without inhibitor; ©, with 2 x 10 -~ M fluoride. present enzyme at the rate as fast as, or even faster than that in adenosine, under the same conditions. All these substrates were investigated in 0.1 M citratephosphate buffer at three different p H ' s , 4.3, 5-0 and 7.0, using the spectrop h o t o m e t e r and the methods of other authors (Kalckar, 1947a, 1947b, 1947c; Roush & Norris, 1950; W a n g et al., 1950). Effects of anions. A m o n g the anions, CI-, Br-, I - , N O ~ - , SO42-, C H a C O 0 - , citrate and succinate showed scarcely no effect on the enzyme reaction. Pyrophosphate was slightly inhibitory. Fluoride was very inhibitory. In this case the per
279
ADENOSINE AMINOHYDROLASE FROM THE CLAM
c e n t i n h i b i t i o n was p r o p o r t i o n a l to t h e c o n c e n t r a t i o n of t h e i n h i b i t o r (Fig. 8). T h e effect of t h e s u b s t r a t e c o n c e n t r a t i o n o n t h e i n h i b i t i o n b y f l u o r i d e (2 × 10 -3 M in t h e final c o n c e n t r a t i o n o f F - ) was s t u d i e d s p e c t r o p h o t o m e t r i c a l l y , a n d t h e g r a p h i c a l t r e a t m e n t of t h e results after L i n e w e a v e r & B u r k (1934), s h o w e d t h a t t h e i n h i b i t i o n was n o n - c o m p e t i t i v e ( F i g . 9). Effects of heavy metals. A s m u c h as 93 p e r c e n t of t h e e n z y m e a c t i v i t y was i n h i b i t e d b y H g ~+ ion of 10 -3 M in t h e final c o n c e n t r a t i o n . L i t t l e effect was s h o w n b y N a +, K +, C a 2+, a n d M g 2+. Ba 2+ s h o w e d no significant effect. Z i n c ion at 1 x 10 -3 M was n o t i n h i b i t o r y . H o w e v e r , C o S+ at 1 × 10 -3 M i n h i b i t e d 6-5 p e r c e n t o f t h e activity. TABLE 2--EFFECTS
oF DIFFERENT ADENOSINE
Agents
Na + K+ M g ~+ Ca ~+ Co z+ B a 8+
Zn 2+ Hg ~+ FFFBrINOB-
Final concentration (M)
Inhibition (%)
1 x 10 -2 1 × 10 -2 1 x 10 -2 1 x 10 -~ 1 x 10 -s 1 x 10 -s 1 x 10 -3 1 x 10 -3 1 x 10 -3 5 x 10 -3 1 x 10 -3 1 x 10 -3 1 x 10 -3 1 x 10 -3
(1"7) (4) (1-4) (5) 6"5 1 (4"8) 92.9 19 43 61"9 (1) (1"5) (4"8)
AGENTS
ON
THE ACTIVITY OF THE CLAM
AMINOHYDROLASE
Agents
SO4 2Pyrophosphate Versene Versene PCMB Monoiodoaeetie acid Iodosobenzoate Ferricyanide H~O2 Ascorbie acid Hydroquinone Cysteine Oxalic acid Na2S~O4
Final concentration (M) 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x
10 -3 10 -3 10 -8 10 -z 10 -s 10 -3 10 -3 10 -z 10 -2 10 -4 10 -3 10 -3 10 -a 10 -~
Inhibition (%) (5) 10-3 1 12"3 33"7 (5) (7) (11) (3) 0 (6"2) (8) (4) (5)
Reaction mixture consisted of 5 x 10 -n M adenosine, 0.1 M citrate-phosphate buffer, p H 5"0, and 6-3 units of enzyme, at 26°C. Figures in parentheses represent acceleration, also in per cent.
Effects of sulphhydryl group binders and other agents. V e r s e n e ( e t h y l e n e d i a m i n e tetra/icetic a c i d t e t r a s o d i u m salt) (1 × 10 -~ M ) i n h i b i t e d 12.3 p e r c e n t o f t h e e n z y m a t i c activity. T h e existence of 1 x 10 -5 M P C M B ( p - e h l o r o m e r c u r i c b e n z o i c acid) in t h e r e a c t i o n m i x t u r e r e d u c e d t h e v e l o c i t y to 43.7 p e r c e n t o f t h e c o n t r o l ; h o w e v e r , m o n o i o d o a c e t i c a c i d o r i o d o s o b e n z o a t e was n o t i n h i b i t o r y , o r even accelerating. T h e r e d u c i n g a g e n t s s u c h as oxalic acid, s o d i u m h y d r o s u l p h a t e , h y d r o q u i n o n e a n d c y s t e i n e effected r e s p e c t i v e l y 4, 5, 6-2 a n d 8 p e r c e n t o f t h e i n c r e a s e o f activity, b u t a s c o r b i c a c i d h a d no effect. H y d r o g e n p e r o x i d e was n o t effective. P o t a s s i u m f e r r i c y a n i d e (1 x 10 4 M ) effeeted 11 p e r c e n t acceleration. T a b l e 2 s u m m a r i z e s t h e s e results.
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DISCUSSION The crude extract from the clam mid-gut gland maintained the activity of adenosine aminohydrolase even after being heated at 50°C for 10 min or longer. When purified, the enzyme appears far less resistant to heating, deep freezing and freeze-drying, being readily inactivated. However, if stored as an aqueous solution at 0°C, the purified enzyme remained intact for one month, at pH 4"7. Treated with buffers the enzyme behaved differently; it was best preserved nearly at neutrality as examined after 6 days of storage in an ice box. The apparent activation energy was calculated approximately as 14,600 cal. As the purified enzyme is liable to be inactivated by heat, the data obtained at temperatures higher than 30°C might somewhat have been affected. Nevertheless they were adopted in the calculation, for only a slight error might be caused by heating in a very short period (2 min) of reaction. The present enzyme shows a sharply peaked optimum at pH 5.0 or thereabout. In this respect it differs much from any kind of adenosine aminohydrolases from other sources. The adenosine aminohydrolases from the rabbit and other vertebrates are most effective near the neutral state, and are comparatively active over a wide range of pH (Schmidt, 1928 ; Conway & Cooke, 1939 ; Brady, 1942; Kalckar, 1947c). The enzyme in the blowfly larva has an optimum at pH 8.3, according to Lennox (1941). The activity of the enzyme from Drosophila larva was determined at pH 6.5 (Wagner & Mitchell, 1948). The adenosine aminohydrolase in the ammonium sulphate fraction of the lobster hepatopancreas was studied at pH 7-0 (Roush & Betz, 1956). The maximum activity of the enzyme in Bacillus cereus spore is shown at pH 8"7 (Powell & Hunter, 1956), and the non-specific adenosine aminohydrolase from taka-diastase has an optimum at pH 6.8 (Kaplan et al., 1952). Previously the author reported that the homogenate of the mid-gut gland of the clam showed the aminohydrolase activity over a wide range of pH 3-8 (Aikawa, 1959). That the purified enzyme has a much more restricted range of activity is interesting from the physiological viewpoint. A similar tendency is observed in the same enzyme in the clam gill (Umemori, unpublished). On the other hand, the activity of the adenosine aminohydrolase in the adductor and foot muscles of the clam is sharpely intensified at pH 7-5-8.0 (Aikawa, 1959). Therefore, these adenosine aminohydrolases in various tissues of the clam, should be studied further in purified form. Under the present experimental conditions, a rectilinear proportionality is found between the initial velocity and the enzyme concentration, and the reaction producing inosine and ammonia from adenosine must be of the first order, and it was found that one mole of inosine is produced from one mole of adenosine. The production of inosine was confirmed by the increase of optical density change at 240 m~. The highly specific clam enzyme is in contrast with the adenosine aminohydrolase from taka-diastase which is non-specific (Kaplan et al., 1952). The amino groups of free adenine and other purine and pyrimidine compounds are resistant to the clam enzyme. Deoxyadenosine is the only substrate which can be hydrolysed
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281
equally as adenosine, and it is probable that both nucleosides are attacked by the same enzyme, as already suggested by Brady (1942) and Schaedel et al. (1947). Roush & Betz (1956) found that an ammonium sulphate fraction of the lobster hepatopancreas has enzymatic activities sufficient to decompose adenosine and deoxyadenosine. They consider that two different enzymes are contained in this fraction, since they obtained two different values of specific activity, 0.66 for adenosine and 0.28 for deoxyadenosine. According to Brady (1942), the blood extracts of the rabbit, fowl, rat, and man, and also some tissue extracts of the rabbit hydrolyse deoxyadenosine at half the rate of adenosine, while the enzyme from the calf mucosa hydrolyses both substrates at an equal rate. He concludes from the experiment, in which both substrates are mixed, that these activities do not belong to separate enzymes. Considering Brady's result, the activities of the lobster hepatopancreas for adenosine and deoxyadenosine might be exerted by a single enzyme. Schaedel et al. (1947) suggested that the high specificity of the intestinal aminohydrolase is not only due to a perfect fit of the purine ring to the enzyme but to many other structural restrictions of the substrate. According to Byrne (1954) the highly purified adenosine aminohydrolase from beef spleen can hydrolize amino groups of several nucleotides with the optimum at pH 5.2. As shown in Table 2, fluoride ion is strongly inhibitory to the clam enzyme. This inhibition is proportional to fluoride concentration and is noncompetitive, though a mechanism in more detail is unknown. The activity is extensively inhibited by Hg 2+, while most other metal ions have little or no effect. Versene and pyrophosphate inhibit more than 10 per cent of the activity. These results suggest that the metals may not be required for the enzyme. Among the sulphhydryl binding agents only PCMB, the mercuric compound, is obviously inhibitory. The enzyme may be very sensitive to Hg in the PCMB molecule, for its chemical behavior toward this substance is like that toward the metallic Hg. The present results are rather similar to those of the calf enzyme which in general is scarcely affected by common inhibitors (Brady, 1942). Wagner & Mitchell (1948) observed a proportionality between the growth rate and the adenosine aminohydrolase activity in the larva of Drosophila melanogaster. In the chick embryo, the enzyme was formed by an adaptive induction (Gordon & Roder, 1953). Stern et al. (1952), investigated vertebrate tissues and found adenosine aminohydrolase and guanine aminohydrolase (guanase) in cytoplasm but not in nuclei. Thus they consider that these enzymes are not essential for the universal pattern of nuclei metabolism. Furthermore, they found that the adenosine aminohydrolase activity was forty-fold higher in the cytoplasm of the adult tissue than in that of the fetal calf intestinal mucosa. In view of these reports, it seems likely that the adenosine aminohydrolase in general is very changeable in its activity (or amount), either in the clam tissues or in other animal tissues. In addition, constancy in nature and amount of contaminated proteins may not be expected in enzyme solutions from the clam mid-gut gland, and it may be considered that the enzyme purity at different purification steps will be affected by many factors which vary with seasons, age and others. Further studies are needed on seasonal changes of the
282
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enzymatic activity in relation to changes in other metabolic enzymes and also on amino acid constituents of relevant proteins. Adenosine has a strong vasodepressor effect, according to Drury & SzentGySrgyi (1929), Ewing et al. (1949), and Clarke et al. (1952). They discuss that the effect of adenosine could be temporary owing to the decomposing activity of the enzyme, while some other analogs retain the vasodepressor effect longer since they are resistant to the enzyme. Assuming from the work of Roush & Betz (1956) on crustaceans, the adenosine aminohydrolase may also play a part in pharmacological protection in the clam. It is also suggested that the enzyme participates in offering amino groups for the synthesis of amino compounds and in changing the permeability of cells (Conway & Cooke, 1939). Despite the existence of adenosine aminohydrolase, adenosine is effectively incorporated into adenosine monophosphate in the biosynthesis in the vertebrate, according to many authors (Caputto, 1951; Kornberg & Pricer, 1951 ; Lowy et al., 1952), and the enzyme action in metabolism is not elucidated yet. Recently, Umemori found in the homogenate of the clam mid-gut gland a high phosphatase activity toward adenosine-5'-monophosphate, inosine-5'-monophosphate, and a guanylic acid (unpublished). This phosphatase has two optimal peaks at pH 4.0-4.2 and 8-5-9.0, and always shows a depression near the neutrality. The activity is considerably higher on the alkaline side than on the acid. Further studies on these nucleotidases in more purified forms are required to make clear the role of the adenosine aminohydrolase in the pathway of adenylate metabolism, in the clam mid-gut gland and other tissues. SUMMARY 1. Adenosine aminohydrolase was extracted from the mid-gut gland of the clam, Meretrix meretrix lusoria, and purified by several fractionation methods. Preparation with high specific activity was obtained. 2. The purified enzyme is not resistant to heat, but can be stored unchanged at 0°C if dissolved in deionized and distilled water. Preserved in buffer solution of pH 7.2 at 0°C, it remained almost unchanged for 6 days. 3. The apparent activation energy of the enzyme activity was approximately 14,600 cal. Under usual conditions the reaction of the enzymatic decomposition of adenosine was of the first order, and the velocity constant, k, was of 13.18 x 10 -2 min -1. Change of reaction velocity was proportional to that of the enzyme concentration. 4. Optimal pH of the enzyme in citrate-phosphate buffer was 5-0 which was a sharply elevated peak. 5. At pH 5.0 and 26°C, the Michaelis-Menten constant, Kin, was 6-9 x 10 -a M. 6. The enzyme in purified form is highly specific. While it decomposes both adenosine and deoxyadenosine equally, it is not effective on most purines and pyrimidines, their nucleosides and nucleotides.
ADENOSINE AMINOHYDROLASE FROM THE CLAM
283
7. Effects of anions, metals, s u l p h h y d r y l binders and some o t h e r agents were examined. S t r o n g inhibitory effects were s h o w n b y fluoride and b y metallic or organic m e r c u r y . 8. T h e properties and metabolic significance of the e n z y m e were discussed in c o m p a r i s o n with that f r o m other sources. Acknowledgements--The author expresses his cordial thanks to Professor Shuzo Ishida and Professor Kiyoshi Aoki for their encouragement and criticisms. He is also grateful to Miss Yoko Umemori of the Chiba University for her help in many ways. This work was supported in part by the Grant-in-Aid for Fundamental Scientific Research of the Ministry of Education of Japan. REFERENCES AIKAWA T. (1959) Deamination of adenosine and adenylic acids in the clam, Meretrix meretrix lusoria (Gmelin). Sci. Rep. T6hoku Univ. (Biol.) 25, 73-80. BRADYT. (1942) Adenosine deaminase. Biochem..7. 36, 478-484. BYRNE W. L. (1954) The purification of an adenylic acid deaminase. Abstr. 126th National Meeting Am. Chem. Soc., N.Y., p. 73c (1955) In The Nucleic Acids (Edited by CHARGAFF E. & DAVmSON J. N.), Vol. 1, p. 597. Academic Press, N.Y. CAPUTTO R. (1951) The enzymic synthesis of adenylic acid; adenosinekinase. 07. Biol. Chem. 189, 801-814. CHARGAFFE. 86 DAVIDSONJ. N. (1955) The Nucleic Acids, Vol. 1, p. 595, and Vol. 2, p. 423. Academic Press, N.Y. CLARKED. A., DAVOLLJ., PHILIPS F. S. & BROWNG. B. (1952) Enzymatic deamination and vasodepressor effects of adenosine analogs..7. Pharm. Exp. Therap. 106, 291-302. CONWAY E. J. & COOKE R. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem. 07. 33, 479-492. DIXON M. & WEBS E. C. (1964) Enzymes 2nd Ed. p. 756. Longmans, London. DRURY A. N. & SZENT-GYSROYIA. (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. 07. Physiol. 68, 213-237. EWING P. L., SCHLENKF. & EMERSONG. A. (1949) Comparison of smooth muscle effects of crotonoside (isoguanosine) and adenosine. 07. Pharm. Exp. Therap. 97, 379-383. FLORKIN M. (1945) L'evolution du m6tabolisme des substances azot6es chez les animaux. Actualitds biochimiques, No. 3. Paris et Lifige. FLORKIN M. & MASON H. S. (1960) Comparative Biochemistry, Vol. 2, p. 178. Academic Press, N.Y. GORDON M. W. & RODER M. (1953) Adaptive enzyme formation in the chick embryo. 07. Biol. Chem. 200, 859-866. GR~ENBERQ D. M. (1961) Metabolic Pathways, Vol. 2, p. 419. Academic Press, N.Y. GY{SRGYP. & R{STHLERH. (1927) l~ber Bedingungen der autolytischen Ammoniakbildung in Geweben. III. Mitteilung. Die Beziehungen des Gewebsammoniaks zum Purinstoffwechsel. Biochem. Z. 187, 194--219. ISHIDA S. (1955) Metabolic patterns in bivalves. VII. Some aspects of the purine metabolism in the clam, Meretrix meretrix lusoria (Gmelin). ~. Coll. Arts Sci. Chiba Univ. 1, 270-272. ISmDA S. (1956) Metabolic patterns in bivalves. V I I I . Distribution of the guaninedeaminating activity in the soft parts of some marine bivalves. Bull. Mar. Biol. Sta. Asamushi 8, 9-18. KALCK~a~H. M. (1947a) Differential spectrophotometry of purine compounds by means of specific enzymes. I. Determination of hydroxypurine compounds, ft. Biol. Chem. 167, 429-443.
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KALCKARH. M. (1947b) Differential spectrophotometry of purine compounds by means of specific enzymes. I I. Determination of adenine compounds. J. Biol. Chem. 167, 4 4 5 4 5 9 . KALCKAR H. M. (1947c) Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167, 461-475. KAPLAN N. O., COLOWICKS. P. • CIOTTI M. M. (1952) Enzymatic deamination of adenosine derivatives. J. Biol. Chem. 194, 579-591. KEILIN D. & HARTREE E. F. (1938) On the mechanism of the decomposition of hydrogen peroxide by catalase. Proc. R. Soc. London (Ser. B) 124, 397-405. KORNBERG A. & PRICER W. E. JR. (1951) Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J. Biol. Chem. 193, 481-495. LANG C. A. (1958) Simple microdetermination of Kjeldahl nitrogen in biological materials. Anal. Chem. 30, 1692-1694. LENNOX F. G. (1941) The physiology and toxicology of blowflies. IX. T h e enzymes responsible for ammonia production by larvae of Lucillia cuprina. Aust. Council Sci. Industr. Res. Pamphlet No. 109, 37-64. Cited from Chem. Abstr. 36, 3559 (1942). LINEWEAVER H. & BURK D. (1934) T h e determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56, 658-666. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. T. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. LowY B. A., DAVOLL J. & BROWN G. B. (1952) The utilization of purine nucleosides for nucleic acid synthesis in the rat. J. Biol. Chem. 197, 591-600. POWELL J. F. & HUNTER J. R. (1956) Adenosine deaminase and ribosidase in spores of Bacillus cereus. Biochem. J. 62, 381-387. RousH A. H. & BETZ R. B. (1956) The adenosine deaminase of crustaceans. Biochim. biophys. Acta 19, 579-580. ROUSH A. & NORRIS E. R. (1950) Deamination of 8-azaguanine by guanase. Arch. Biochem. 29, 124-129. SCrIAEDEL M. L., WALDVOGEL M. J. & SCHLENK F. (1947) T h e specificity of adenosine deaminase and purine nucleosidase. J. Biol. Chem. 171, 135-141. SCHMIDT G. (1928) fiber fermentative Desaminierung im Muskel. Z. Physiol. Chem. 179, 243-282. STERN H., ALLFREYV., MIRSKYA. E. & SAETRENH. (1952) Some enzymes of isolated nuclei. -7. Gen. Physiol. 35, 559-578. WAGNER R. P. & MITCHELL H. K. (1948) An enzymatic assay for studying the nutrition of Drosophila melanogaster. Arch. Biochem. 17, 87-96. WANG T. V., SABLE H. Z. & LAMPEN O. (1950) Enzymatic deamination of cytosine nucleosides..7. Biol. Chem. 184, 17-28. WILLSTATTER R. & KRAUT H. (1923) Ober ein Tonerde-Gel yon der Formel AI(OH)3. II. Mitteilung tiber Hydrate und Hydrogele. Ber. Dtsch. Chem. Ges. 56, 1117-1121.