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Ribonucleotide phosphohydrolases in the clam, Meretrix meretrix lusoria (Gmelin)
Comp. Biochem. Physiol., 1967, Vol. 20, pp. 635 to 639. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION RIBONUCLEOTIDE PHOSPHOHYDROLASES IN THE CLAM, M E R E T R I X M E R E T R I X L U S O R I A (GMELIN) YOKO UMEMORI Biological Institute, Chiba University, Chiba, Japan (Received 30 August 1966)
A b s t r a c t - - 1 . Phosphohydrolytic activity was studied in the homogenized mid-gut gland tissue, using nucleotides (5"-AMP, 3"-AMP, 5'-IMP and GMP) and fl-giycerophosphate as substrates. 2. For nucleotides there were two optima of pH (8"5-9"7, 4"0-4"2). For fl-glycerophosphate the activity was restricted to the alkaline region (pH 9"5). INTRODUCTION MOST studies on 5'-ribonucleotide phosphoydrolases have been concerned with m a m m a l i a n tissues (Heppel & Hilmoe, 1951; Segal & Brenner, 1960). Regarding such enzymes in lower animals, information is rather poor. Participating in the work to elucidate pathways of nucleotide metabolism in the clam, M e r e t r i x raeretrix lusoria (Gmelin), the present author investigated the phosphohydrolytic decomposition of nucleotides in the homogenized mid-gut gland tissue of this species. MATERIALS AND M E T H O D S Fresh material was made free from sperm and other tissues, washed several times with chilled water and homogenized with 9 vol. of chilled water in a Waring Blendor. After standing for 30 min at 5°C, the homogenate was used as the enzyme solution. Substrates were 5"-adenosine monophosphate (5'-AMP), 3"-adenosine monophosphate (3"-AMP), 5'-inosine monophosphate (5"-IMP), guanosine monophosphate (GMP) and fl-glycerophosphate (5 x 10 -s M solutions). Buffers were acetate-hydrochloric acid, Tris-maleate and glycine-sodium hydroxide (1 M solutions) which covered the ranges of pH 2"5-5"0, 6"0-8"0 and 9"0-12"0 respectively. Chemicals were the products of Zellstoff-fabrik Waldhof, Nutritional Biochemical Corporation, Sigma Chemical Company, Wako Pure Chemical Industries and Kanto Chemical Co. Deionized water was always used. Instruments were Toyo Rika glass electrode pH meter (GA-S type), Marusan Centrifuge and Ito Electric Photometer (KL type). The reaction mixture consisted of 1-5 nd of buffers, 0"5 nd of substrate and 1 ml of enzyme, contained in a conical flask, and incubated for 20 rain at 30°C. The enzyme was replaced by water in the control mixture which was similarly incubated. The reaction was stopped by the addition of 0"5 ml of 35~o trichloroacetic acid after the desired period. The precipitate was centrifuged off 15 rain later. Phosphate-phosphorus in the supernatant 635
636
YOKO UMEMORI
was determined by the method of Fiske & Subbarow (1925). The net amount of the enzymatically liberated phosphorus was represented by the difference in P (/zg) between the final contents of the experimental and control flasks. One unit of the enzyme was defined as the activity to transform 1/zM of substrate in 1 rain, and the specific activity was defined as units per mg of protein nitrogen, which was determined by a modification of Lang's method (1958). Apparent velocity was represented by the enzymic liberation of phosphorus in p.g/min, averaged during a 20-min period of incubation.
To show the enzymic activities for different substrates at different pH's the apparent velocities are plotted in Fig. 1 (a-e). Each of the curves for the nucleotides used has two conspicuous peaks both in the acid and alkaline regions, and a depression at neutrality. The optimal pH's and the values of specific activity for different substrates are summarized in Table 1. In the alkaline region the present enzyme behaved in nearly a uniform manner against all the substrates used, though the optimum varied from pH 8.5 to 9.7 and the specific activity also fluctuated somewhat. Results were less uniform in the acid region; the specific activity toward GMP in this region was exceptionally higher than that in the alkaline region, whereas fl-glycerophosphate in the acid
1.0 -
a
-, 0,8 •~
0.6 0,4
0.2 0-0
2
I 4
I 6
I 8
10
pH
1.0 ~
b
0-8
0.6 0-4
<
0.2 0.0 2
4
6
OH
8
10
637
RIBONUCLEOTIDE PHOSPHOHYDROLABES I N THE CLAM
1.o
(:
_>' 0.8 -~ 0
®
0.6
c:
0"4-
0
0.20-0
I 4
2
I 6
I 8
I 10
pH
1,2 -
d
1.0 0-8 > 0"6 c
0.4
o.
,~ 0.2 t 4
0.0 2
I 6
I 8
I 10
pH
1.0
-
Q
->' 0-8 "~
0.6 0.4 0.2 0.0
I
2
4
6
8
lO
12
pH FIG. I. Effect of pH on rate of hydrolysis of several substrates by the homogenate of the mld-gut gland from the clam: (a) 5'-AMP, (b) 3"-AMP, (c) 5'-IMP, (d) GMP and (e) fl-glycerophosphate as substrate.
638
Y o x o UMEMORI
region was little affected by this enzyme. At any rate, the value of optimal pH in the acid region varied less (pH 4.0-4.2). Evidently the clam mid-gut gland contains an enzyme which should be called 5'-AMP phosphohydrolase. TABLE 1--THE
OPTIMA
OF
pH
AND
THE
VALUES
OF
SPECIFIC
ACTIVITY
FO R
DIFFERENT
SUBSTRATES IN T H E CLAM M I D - G U T GLAND
Substrate (5 × 10 -8 M )
Optimal p H
Specific activity ( u n i t s / p r o t e i n N rag)
5'-AMP
4"2 8.8
32-1 × 10 -3 54-8 × 10 3
3'-AMP
4"2 9"7
41"6 × 10 3 42"1 × 10 3
5'-IMP
4"1 8"5
46-0 × 10 -3 58-6 × 10 -3
GMP
4-0 8"8
70-0 × 10 -3 51"6 x 10 -~
}3-Glycerophosphate
9"5
58"6 × 10 -3
Bull semen contains a 5'-AMP phosphohydrolase with an optimum at pH 8-5 (Heppel & Hilmoe, 1951), whereas rat liver microsomes have a similar enzyme which is most active at neutrality (Segal & Brenner, 1960). Towards/3-glycerophosphate the branchial tissue homogenate of the clam resembles the present material in respect of optimal pH (Usuki, 1959). But the oyster has in its gills an enzyme that attacks the same substrate exclusively in the acid region (Usuki, 1959). The present results suggest that the position of the amino group or of the phosphate group in the nucleotide molecules might affect the behaviour of the enzyme, especially in the acid region, or they suggest the coexistence of two or more different phosphohydrolases in the homogenate. Also in the homogenized tissue of the clam enzymic activities of deaminating 5'-AMP and adenosine have been demonstrated around pH 5 and over a wide range of pH 3.0-8.0 respectively (Aikawa, 1959). A further study on a more purified form of the clam adenosine aminohydrolase shows that it has a narrow and sharply peaked optimum at pH 5-0 (Aikawa, 1966). In respect of these and the present results the 5'-AMP in the clam tissue can probably follow at least two pathways of decomposition towards inosine; in one pathway this nucleotide may first be deaminated and then be dephosphorylated, and in the other it may first be acted upon by the phosphohydrolase and thereafter by the aminohydrolase.
Acknowledgements--This w o r k was s u p p o r t e d b y the G r a n t - i n - A i d for F u n d a m e n t a l Scientific R e s e a r c h of t h e M i n i s t r y of E d u c a t i o n of Japan. Also t h a n k e d are Professors S h u z o Ishida a n d T o y o o Aikawa for t h e i r criticisms a n d suggestions.
R I B O N U C I ~ O T I D E PHOSPHOHYDROLASES I N THE CLAM
639
REFERENCES AIKAWA T. (1959) Deamination of adenosine and adenylic acids in the dam, Meretrix meretrix lusoria (Gmelin). Sci. Rep. TOhoku Univ. 25, 73-80. AXKAWAT. (1966) Adenosine aminohydrolase from the clam, Meretrix meretrix lusoria (Gmelin). Comp. Biochem. Physiol. 17, 271-284. FISKE C. H. & SUBBAROWY. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. HgPPEL L. A. & HILMOER. J. (1951) Purification and properties of 5-nucleotidase. J. biol. Chem. 188, 665-676. L^NO C. A. (1958) Simple microdetermination of Kjeldahl nitrogen in biological materials. Analyt. Chem. 30, 1692-1694. SEOALH. L. & BRSNNERB. M. (1960) 5'-Nudeotidase of rat liver microsomes..7, biol. Chem. 235, 471-474. USUKX I. (1959) Alkaline and acid phosphatase activities in the ciliated tissues of certain bivalves and frogs. Sci. Rep. T~hoku Univ. 25, 53-64.
SHORT COMMUNICATION RIBONUCLEOTIDE PHOSPHOHYDROLASES IN THE CLAM, M E R E T R I X M E R E T R I X L U S O R I A (GMELIN) YOKO UMEMORI Biological Institute, Chiba University, Chiba, Japan (Received 30 August 1966)
A b s t r a c t - - 1 . Phosphohydrolytic activity was studied in the homogenized mid-gut gland tissue, using nucleotides (5"-AMP, 3"-AMP, 5'-IMP and GMP) and fl-giycerophosphate as substrates. 2. For nucleotides there were two optima of pH (8"5-9"7, 4"0-4"2). For fl-glycerophosphate the activity was restricted to the alkaline region (pH 9"5). INTRODUCTION MOST studies on 5'-ribonucleotide phosphoydrolases have been concerned with m a m m a l i a n tissues (Heppel & Hilmoe, 1951; Segal & Brenner, 1960). Regarding such enzymes in lower animals, information is rather poor. Participating in the work to elucidate pathways of nucleotide metabolism in the clam, M e r e t r i x raeretrix lusoria (Gmelin), the present author investigated the phosphohydrolytic decomposition of nucleotides in the homogenized mid-gut gland tissue of this species. MATERIALS AND M E T H O D S Fresh material was made free from sperm and other tissues, washed several times with chilled water and homogenized with 9 vol. of chilled water in a Waring Blendor. After standing for 30 min at 5°C, the homogenate was used as the enzyme solution. Substrates were 5"-adenosine monophosphate (5'-AMP), 3"-adenosine monophosphate (3"-AMP), 5'-inosine monophosphate (5"-IMP), guanosine monophosphate (GMP) and fl-glycerophosphate (5 x 10 -s M solutions). Buffers were acetate-hydrochloric acid, Tris-maleate and glycine-sodium hydroxide (1 M solutions) which covered the ranges of pH 2"5-5"0, 6"0-8"0 and 9"0-12"0 respectively. Chemicals were the products of Zellstoff-fabrik Waldhof, Nutritional Biochemical Corporation, Sigma Chemical Company, Wako Pure Chemical Industries and Kanto Chemical Co. Deionized water was always used. Instruments were Toyo Rika glass electrode pH meter (GA-S type), Marusan Centrifuge and Ito Electric Photometer (KL type). The reaction mixture consisted of 1-5 nd of buffers, 0"5 nd of substrate and 1 ml of enzyme, contained in a conical flask, and incubated for 20 rain at 30°C. The enzyme was replaced by water in the control mixture which was similarly incubated. The reaction was stopped by the addition of 0"5 ml of 35~o trichloroacetic acid after the desired period. The precipitate was centrifuged off 15 rain later. Phosphate-phosphorus in the supernatant 635
636
YOKO UMEMORI
was determined by the method of Fiske & Subbarow (1925). The net amount of the enzymatically liberated phosphorus was represented by the difference in P (/zg) between the final contents of the experimental and control flasks. One unit of the enzyme was defined as the activity to transform 1/zM of substrate in 1 rain, and the specific activity was defined as units per mg of protein nitrogen, which was determined by a modification of Lang's method (1958). Apparent velocity was represented by the enzymic liberation of phosphorus in p.g/min, averaged during a 20-min period of incubation.
To show the enzymic activities for different substrates at different pH's the apparent velocities are plotted in Fig. 1 (a-e). Each of the curves for the nucleotides used has two conspicuous peaks both in the acid and alkaline regions, and a depression at neutrality. The optimal pH's and the values of specific activity for different substrates are summarized in Table 1. In the alkaline region the present enzyme behaved in nearly a uniform manner against all the substrates used, though the optimum varied from pH 8.5 to 9.7 and the specific activity also fluctuated somewhat. Results were less uniform in the acid region; the specific activity toward GMP in this region was exceptionally higher than that in the alkaline region, whereas fl-glycerophosphate in the acid
1.0 -
a
-, 0,8 •~
0.6 0,4
0.2 0-0
2
I 4
I 6
I 8
10
pH
1.0 ~
b
0-8
0.6 0-4
<
0.2 0.0 2
4
6
OH
8
10
637
RIBONUCLEOTIDE PHOSPHOHYDROLABES I N THE CLAM
1.o
(:
_>' 0.8 -~ 0
®
0.6
c:
0"4-
0
0.20-0
I 4
2
I 6
I 8
I 10
pH
1,2 -
d
1.0 0-8 > 0"6 c
0.4
o.
,~ 0.2 t 4
0.0 2
I 6
I 8
I 10
pH
1.0
-
Q
->' 0-8 "~
0.6 0.4 0.2 0.0
I
2
4
6
8
lO
12
pH FIG. I. Effect of pH on rate of hydrolysis of several substrates by the homogenate of the mld-gut gland from the clam: (a) 5'-AMP, (b) 3"-AMP, (c) 5'-IMP, (d) GMP and (e) fl-glycerophosphate as substrate.
638
Y o x o UMEMORI
region was little affected by this enzyme. At any rate, the value of optimal pH in the acid region varied less (pH 4.0-4.2). Evidently the clam mid-gut gland contains an enzyme which should be called 5'-AMP phosphohydrolase. TABLE 1--THE
OPTIMA
OF
pH
AND
THE
VALUES
OF
SPECIFIC
ACTIVITY
FO R
DIFFERENT
SUBSTRATES IN T H E CLAM M I D - G U T GLAND
Substrate (5 × 10 -8 M )
Optimal p H
Specific activity ( u n i t s / p r o t e i n N rag)
5'-AMP
4"2 8.8
32-1 × 10 -3 54-8 × 10 3
3'-AMP
4"2 9"7
41"6 × 10 3 42"1 × 10 3
5'-IMP
4"1 8"5
46-0 × 10 -3 58-6 × 10 -3
GMP
4-0 8"8
70-0 × 10 -3 51"6 x 10 -~
}3-Glycerophosphate
9"5
58"6 × 10 -3
Bull semen contains a 5'-AMP phosphohydrolase with an optimum at pH 8-5 (Heppel & Hilmoe, 1951), whereas rat liver microsomes have a similar enzyme which is most active at neutrality (Segal & Brenner, 1960). Towards/3-glycerophosphate the branchial tissue homogenate of the clam resembles the present material in respect of optimal pH (Usuki, 1959). But the oyster has in its gills an enzyme that attacks the same substrate exclusively in the acid region (Usuki, 1959). The present results suggest that the position of the amino group or of the phosphate group in the nucleotide molecules might affect the behaviour of the enzyme, especially in the acid region, or they suggest the coexistence of two or more different phosphohydrolases in the homogenate. Also in the homogenized tissue of the clam enzymic activities of deaminating 5'-AMP and adenosine have been demonstrated around pH 5 and over a wide range of pH 3.0-8.0 respectively (Aikawa, 1959). A further study on a more purified form of the clam adenosine aminohydrolase shows that it has a narrow and sharply peaked optimum at pH 5-0 (Aikawa, 1966). In respect of these and the present results the 5'-AMP in the clam tissue can probably follow at least two pathways of decomposition towards inosine; in one pathway this nucleotide may first be deaminated and then be dephosphorylated, and in the other it may first be acted upon by the phosphohydrolase and thereafter by the aminohydrolase.
Acknowledgements--This w o r k was s u p p o r t e d b y the G r a n t - i n - A i d for F u n d a m e n t a l Scientific R e s e a r c h of t h e M i n i s t r y of E d u c a t i o n of Japan. Also t h a n k e d are Professors S h u z o Ishida a n d T o y o o Aikawa for t h e i r criticisms a n d suggestions.
R I B O N U C I ~ O T I D E PHOSPHOHYDROLASES I N THE CLAM
639
REFERENCES AIKAWA T. (1959) Deamination of adenosine and adenylic acids in the dam, Meretrix meretrix lusoria (Gmelin). Sci. Rep. TOhoku Univ. 25, 73-80. AXKAWAT. (1966) Adenosine aminohydrolase from the clam, Meretrix meretrix lusoria (Gmelin). Comp. Biochem. Physiol. 17, 271-284. FISKE C. H. & SUBBAROWY. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. HgPPEL L. A. & HILMOER. J. (1951) Purification and properties of 5-nucleotidase. J. biol. Chem. 188, 665-676. L^NO C. A. (1958) Simple microdetermination of Kjeldahl nitrogen in biological materials. Analyt. Chem. 30, 1692-1694. SEOALH. L. & BRSNNERB. M. (1960) 5'-Nudeotidase of rat liver microsomes..7, biol. Chem. 235, 471-474. USUKX I. (1959) Alkaline and acid phosphatase activities in the ciliated tissues of certain bivalves and frogs. Sci. Rep. T~hoku Univ. 25, 53-64.