Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 245 to 246. Peroamon Press. Printed in Great Britain
DISTRIBUTION OF INORGANIC PYROPHOSPHATASE IN SOME MARINE INVERTEBRATES OF THE JAPAN SEA V. N. KASHO, A. A. BAYKOV AND S. M. AVAEVA Laboratory of Bioorganic Chemistry, Moscow State University, Moscow 117234, USSR
(Received 20 June 1974) Abstract--1. Fifteen marine invertebrates were investigated with respect to the content of inorganic pyrophosphatase by measurements of activity and by disk electrophoresis of various tissue extracts. 2. Isoelectric points of the enzyme from four sources were obtained using the isoelectrofocusing technique. INTRODUCTION
MATERIALS AND METHODS
INORGANIC pyrophosphatase (E.C. 3.6.1.1), an enzyme hydrolyzing pyrophosphate in the presence of some bivalent cations, has been isolated in the homogeneous crystalline form from several organisms (Kunitz, 1952; Josse, 1966; Niculescu et al., 1972). Studies of pyrophosphatases from different sources have shown that, though the enzymes are very similar in many respects, some differences in the structure and the mechanism of their action exist (Josse & Wong, 1970; Butler, 1970; Pynes & Younathan, 1967). A possible approach for elucidating the general principles of the mechanism of the inorganic pyrophosphatase reaction is to study the enzyme from the organisms which are at different evolutional levels. The present paper deals with the distribution of inorganic pyrophosphatase in some marine invertebrates; the aim of the work was to find good sources of the enzyme.
Enzyme sources All marine invertebrates were collected in the summer of 1973 in the Posjet Bay of the Japan Sea. Reagents The reagents for disk electrophoresis were obtained from Reanal (Hungary). Ampholines were purchased,from LKB (Sweden). Preparation of extracts Small lots of tissue were ground with a small amount of acid washed sand in a chilled mortar and pestle. The homogenates were extracted 3 times with 0"05 M Tris-HCl buffer (pH 7.5). The final volume of each extract was 5 ml/g of tissue. The extracts were centrifuged for 10 mill at 8000 g and kept at 4°C. Assay of protein concentration and activity Protein concentration was determined according to the method proposed by Lowry et al. (1951). Activity was mea-
Table 1. Distribution and some properties of marine invertebrate pyrophosphatases
No.
Source
Tissue
Protein mg/ml
Specific activity U/mg
Relative mobility
Isoelectric point
0"50; 0"56; 0"85
3"7; 4"0; 4'8
Coelenterata Anthozoa Metridium senile fibriatum Annelida
body only
1.6
ff14
2.
Chaetopterus variopedatus
3.
Neoamphitrite orayi
body only digestive way whole organism
2'1 2.7 4"6
0.11 0-21 0-10
midgut
4-1
0'47
0'75; 0"85
0-31 0.11 0"20 0.18 0-27 0"22 0.17 0.40
0'55
0" 13
1.
Polychaeta
Arthropoda Crustacea 4.
Hemigrapsussan#uineus Mollusca
Gastropoda 5.
Acmaea pallida
hepatopancreas
Mactra sulcataria Pecten (Patinopecten) yessoensis
hepatopancreas hepatopancreas
Swiftopecten swifti Modiolus difficiles
hepatopancreas hepatopancreas
Crassostrea yigas Tellina lutea Echinodermata
hepatopanereas hepatopancreas
5.5 4.6 3"8 6"6 4.7 2"3 3"7 3.8
Distolasterias nippon
digestive way
2- I
Bivalvia 6. 7. 8. 9. 10. I 1.
5"2
0"70
Asteroidea 12.
Eehinoidea 13.
Echinocardiumcordatum Tunicata
Ascidiae 14. 15.
Halocynthia auranthium Halocynthia roretzi
digestive way
1.7
0.13
body only hepatopancreas hepatopancreas
2"5 4-5 4.3
0.22 0.41 0.49
245
0"70 0"550"61
4'2 4"0; 4.4; 5"2
246
V.N. KASHO,A. A. BAYKOVAND S. M. AVAEVA
sured by following the liberation of Pi due to hydrolysis of PPi. A manual variant of the previously reported sensitive automatic method (Baykov & Avaeva, 1973) was used to measure phosphate. 0.04 to 1 ml aliquots of the reaction mixture were mixed with 1 ml of 1-75% solution of (NH4)6 Mo~O24 × 4 H 2 0 in 11% H2SO4 and then with aqueous solution of methyl green dye and Triton X305 to a total volume of 2.25 ml. The final concentration of the dye and detergent were 40 and 20 mg/1, respectively. The optical density at 650 nm was measured immediately on a "Spekol" spectrocolorimeter (Karl Zeiss, Jena). Disk eleetrophoresis Disk electrophoresis was performed according to Devis (1964). Staining pyrophosphatase by activity was done as described elsewhere (Tono & Kornberg, 1967). lsoeleetrofocusing in polyacrylamide (4el. Isoelectrofocusing experiments were carried out according to Beeley (1969) in 10-cm gels with the pH gradient from 3-10.
RESULTS AND DISCUSSION The data on pyrophosphatases from various sources are summarized in Table 1. Pyrophosphatase activity was detected in all the organisms studied. As follows from the experiments with various tissues of the same organism, the enzyme is mainly located in the organs corresponding to liver of mammals. The pyrophosphatase content of these organs varies within an order of magnitude for different organisms. In several cases no moving pyrophosphatase zone was found when the extracts were subjected to disk electrophoresis. The activity did not enter the gel, which indicated that the pyrophosphatase had a high mol. wt. This observation is consistent with the view that this enzyme may be strongly bound to membrane fragments (Schick & Butler, 1969; Gould & Winget, 1973). Extracts from several organisms contained more than one migrating active zone. This fact may be rationalized in terms of existence of isoenzymes. Evidence for this comes from the recent studies of other pyrophosphatases (Buruiana & Hadarag, 1968; Bennett et al., 1973). Our results show that this property is not as uncommon for this enzyme as has been believed. It should be noted that the number of pyrophosphatase zones in the isoelectrofocusing patterns was the same as after eleetrophoresis. Isoelectric points of the enzymes from four species were determined. It was found that they all lay in the acidic region.
Acknowledgement~The authors wish to thank Dr. N. V. Molotzov, Dr. M. G. Vafina and Miss E. V. Sundukova of the Pacific 'Ocean Institute of Bioorganic Chemistry for their help in this work.
REFERENCES
BAYKOVA. A. & AVAEVAS. M. (1973) Regulation of yeast inorganic pyrophosphatase activity by divalent cations. Eur. J. Biochem. 32, 136-142. BEELEYJ. A. (1969) Separation of human salivary proteins by isoelectric focusing in polyacrylamide gels. Arch. oral. Biol. 14, 559-561. BENNETTV. L., RISTROPHED. L., HAMMINGJ. J. • BUTLER L. G. (1973) Maize Leaf Inorganic pyrophosphatase: isoenzymes, specifity for substrates, inhibitors, and divalent metal ions, and pH optima. Biochim. biophys. Acta, 293, 232-241. BURUIANAL. M. & HADARAG E. (1968) Isozymes of the pyrophosphatase from the brain. Experientia, 24, 664-665. BUTLER L. G. (1971) Yeast and other inorganic pyrophosphatases. In The Enzymes, 3rd Edn, Vol. 4, pp. 529541. Academic Press, New York. DAVISB. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. N.Y. Acad, Sci. 121, 404-427. GOULD J. M. & WINGETJ. D. (1973) A Membrane--bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts. Arch. Biochem. Biophys. 154, 606-613. JOSSE J. (1966) Constitutive inorganic pyrophosphatase of Escherichia coll. J. Biol. Chem. 241, 1938-1947. JOSSEJ. & WONG S. C. K. (1971) Inorganic Pyrophosphatase of Escherichia coll. In The Enzymes 3rd Edn, Vol. 4, pp. 49%527. Academic Press, New York. KUNITZ M. (1952) Crystalline inorganic pyrophosphatase isolated from baker's yeast. J. 9en. Physiol. 35, 423-454. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275. NICULESCU S., DUMITRU I. F., & IORDACHESCUD. (1972) Purification and crystallisation and some physico-chemical properties of acid polyphosphate phosphohydrolase of vegetable origin (Lathirus saticus seedsl. Aeta vitaminol. enzymol. 26, 13-22. PVNES J. D. & YOUNATHANE. S. (1967) Purification and some properties of inorganic pyrophosphatase from human erythrocytes. J. Biol. Chem. 242, 2119 2123. SCHICK L. & BUTLERL. G. (1969) Inorganic Pyrophosphatase of Rat Liver Mitochondria. J. Cell Biol. 42, 235-240. Key Word lndex--Electrophoresis; isoelectric focusing; marine invertebrates; pyrophosphatase.
DISTRIBUTION OF INORGANIC PYROPHOSPHATASE IN SOME MARINE INVERTEBRATES OF THE JAPAN SEA V. N. KASHO, A. A. BAYKOV AND S. M. AVAEVA Laboratory of Bioorganic Chemistry, Moscow State University, Moscow 117234, USSR
(Received 20 June 1974) Abstract--1. Fifteen marine invertebrates were investigated with respect to the content of inorganic pyrophosphatase by measurements of activity and by disk electrophoresis of various tissue extracts. 2. Isoelectric points of the enzyme from four sources were obtained using the isoelectrofocusing technique. INTRODUCTION
MATERIALS AND METHODS
INORGANIC pyrophosphatase (E.C. 3.6.1.1), an enzyme hydrolyzing pyrophosphate in the presence of some bivalent cations, has been isolated in the homogeneous crystalline form from several organisms (Kunitz, 1952; Josse, 1966; Niculescu et al., 1972). Studies of pyrophosphatases from different sources have shown that, though the enzymes are very similar in many respects, some differences in the structure and the mechanism of their action exist (Josse & Wong, 1970; Butler, 1970; Pynes & Younathan, 1967). A possible approach for elucidating the general principles of the mechanism of the inorganic pyrophosphatase reaction is to study the enzyme from the organisms which are at different evolutional levels. The present paper deals with the distribution of inorganic pyrophosphatase in some marine invertebrates; the aim of the work was to find good sources of the enzyme.
Enzyme sources All marine invertebrates were collected in the summer of 1973 in the Posjet Bay of the Japan Sea. Reagents The reagents for disk electrophoresis were obtained from Reanal (Hungary). Ampholines were purchased,from LKB (Sweden). Preparation of extracts Small lots of tissue were ground with a small amount of acid washed sand in a chilled mortar and pestle. The homogenates were extracted 3 times with 0"05 M Tris-HCl buffer (pH 7.5). The final volume of each extract was 5 ml/g of tissue. The extracts were centrifuged for 10 mill at 8000 g and kept at 4°C. Assay of protein concentration and activity Protein concentration was determined according to the method proposed by Lowry et al. (1951). Activity was mea-
Table 1. Distribution and some properties of marine invertebrate pyrophosphatases
No.
Source
Tissue
Protein mg/ml
Specific activity U/mg
Relative mobility
Isoelectric point
0"50; 0"56; 0"85
3"7; 4"0; 4'8
Coelenterata Anthozoa Metridium senile fibriatum Annelida
body only
1.6
ff14
2.
Chaetopterus variopedatus
3.
Neoamphitrite orayi
body only digestive way whole organism
2'1 2.7 4"6
0.11 0-21 0-10
midgut
4-1
0'47
0'75; 0"85
0-31 0.11 0"20 0.18 0-27 0"22 0.17 0.40
0'55
0" 13
1.
Polychaeta
Arthropoda Crustacea 4.
Hemigrapsussan#uineus Mollusca
Gastropoda 5.
Acmaea pallida
hepatopancreas
Mactra sulcataria Pecten (Patinopecten) yessoensis
hepatopancreas hepatopancreas
Swiftopecten swifti Modiolus difficiles
hepatopancreas hepatopancreas
Crassostrea yigas Tellina lutea Echinodermata
hepatopanereas hepatopancreas
5.5 4.6 3"8 6"6 4.7 2"3 3"7 3.8
Distolasterias nippon
digestive way
2- I
Bivalvia 6. 7. 8. 9. 10. I 1.
5"2
0"70
Asteroidea 12.
Eehinoidea 13.
Echinocardiumcordatum Tunicata
Ascidiae 14. 15.
Halocynthia auranthium Halocynthia roretzi
digestive way
1.7
0.13
body only hepatopancreas hepatopancreas
2"5 4-5 4.3
0.22 0.41 0.49
245
0"70 0"550"61
4'2 4"0; 4.4; 5"2
246
V.N. KASHO,A. A. BAYKOVAND S. M. AVAEVA
sured by following the liberation of Pi due to hydrolysis of PPi. A manual variant of the previously reported sensitive automatic method (Baykov & Avaeva, 1973) was used to measure phosphate. 0.04 to 1 ml aliquots of the reaction mixture were mixed with 1 ml of 1-75% solution of (NH4)6 Mo~O24 × 4 H 2 0 in 11% H2SO4 and then with aqueous solution of methyl green dye and Triton X305 to a total volume of 2.25 ml. The final concentration of the dye and detergent were 40 and 20 mg/1, respectively. The optical density at 650 nm was measured immediately on a "Spekol" spectrocolorimeter (Karl Zeiss, Jena). Disk eleetrophoresis Disk electrophoresis was performed according to Devis (1964). Staining pyrophosphatase by activity was done as described elsewhere (Tono & Kornberg, 1967). lsoeleetrofocusing in polyacrylamide (4el. Isoelectrofocusing experiments were carried out according to Beeley (1969) in 10-cm gels with the pH gradient from 3-10.
RESULTS AND DISCUSSION The data on pyrophosphatases from various sources are summarized in Table 1. Pyrophosphatase activity was detected in all the organisms studied. As follows from the experiments with various tissues of the same organism, the enzyme is mainly located in the organs corresponding to liver of mammals. The pyrophosphatase content of these organs varies within an order of magnitude for different organisms. In several cases no moving pyrophosphatase zone was found when the extracts were subjected to disk electrophoresis. The activity did not enter the gel, which indicated that the pyrophosphatase had a high mol. wt. This observation is consistent with the view that this enzyme may be strongly bound to membrane fragments (Schick & Butler, 1969; Gould & Winget, 1973). Extracts from several organisms contained more than one migrating active zone. This fact may be rationalized in terms of existence of isoenzymes. Evidence for this comes from the recent studies of other pyrophosphatases (Buruiana & Hadarag, 1968; Bennett et al., 1973). Our results show that this property is not as uncommon for this enzyme as has been believed. It should be noted that the number of pyrophosphatase zones in the isoelectrofocusing patterns was the same as after eleetrophoresis. Isoelectric points of the enzymes from four species were determined. It was found that they all lay in the acidic region.
Acknowledgement~The authors wish to thank Dr. N. V. Molotzov, Dr. M. G. Vafina and Miss E. V. Sundukova of the Pacific 'Ocean Institute of Bioorganic Chemistry for their help in this work.
REFERENCES
BAYKOVA. A. & AVAEVAS. M. (1973) Regulation of yeast inorganic pyrophosphatase activity by divalent cations. Eur. J. Biochem. 32, 136-142. BEELEYJ. A. (1969) Separation of human salivary proteins by isoelectric focusing in polyacrylamide gels. Arch. oral. Biol. 14, 559-561. BENNETTV. L., RISTROPHED. L., HAMMINGJ. J. • BUTLER L. G. (1973) Maize Leaf Inorganic pyrophosphatase: isoenzymes, specifity for substrates, inhibitors, and divalent metal ions, and pH optima. Biochim. biophys. Acta, 293, 232-241. BURUIANAL. M. & HADARAG E. (1968) Isozymes of the pyrophosphatase from the brain. Experientia, 24, 664-665. BUTLER L. G. (1971) Yeast and other inorganic pyrophosphatases. In The Enzymes, 3rd Edn, Vol. 4, pp. 529541. Academic Press, New York. DAVISB. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. N.Y. Acad, Sci. 121, 404-427. GOULD J. M. & WINGETJ. D. (1973) A Membrane--bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts. Arch. Biochem. Biophys. 154, 606-613. JOSSE J. (1966) Constitutive inorganic pyrophosphatase of Escherichia coll. J. Biol. Chem. 241, 1938-1947. JOSSEJ. & WONG S. C. K. (1971) Inorganic Pyrophosphatase of Escherichia coll. In The Enzymes 3rd Edn, Vol. 4, pp. 49%527. Academic Press, New York. KUNITZ M. (1952) Crystalline inorganic pyrophosphatase isolated from baker's yeast. J. 9en. Physiol. 35, 423-454. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275. NICULESCU S., DUMITRU I. F., & IORDACHESCUD. (1972) Purification and crystallisation and some physico-chemical properties of acid polyphosphate phosphohydrolase of vegetable origin (Lathirus saticus seedsl. Aeta vitaminol. enzymol. 26, 13-22. PVNES J. D. & YOUNATHANE. S. (1967) Purification and some properties of inorganic pyrophosphatase from human erythrocytes. J. Biol. Chem. 242, 2119 2123. SCHICK L. & BUTLERL. G. (1969) Inorganic Pyrophosphatase of Rat Liver Mitochondria. J. Cell Biol. 42, 235-240. Key Word lndex--Electrophoresis; isoelectric focusing; marine invertebrates; pyrophosphatase.
