Hostname: page-component-586b7cd67f-rcrh6 Total loading time: 0 Render date: 2024-11-24T13:35:22.006Z Has data issue: false hasContentIssue false
  • English
  • Français

Purification and properties of phosphoenolpyruvate carboxylase from Molinema dessetae (Nematoda: Filarioidea)

Published online by Cambridge University Press:  06 April 2009

P. M. Loiseau ,
P. Gayral  and
F. Petek
P. M. Loiseau*
Affiliation:
Biologie et Contrôle des Organismes Parasites, Université de Paris-Sud, 92296- Châtenay-Malabry Cédex
P. Gayral
Affiliation:
Biologie et Contrôle des Organismes Parasites, Université de Paris-Sud, 92296- Châtenay-Malabry Cédex
F. Petek
Affiliation:
Régulation de l’Expression des Gènes, U.P.R. 37 C.N.R.S., Institut de Recherches Scientifiques sur le Cancer, 94802-Veillejuif Cédex
*
*Reprint requests to Dr P. M. Loiseau
Get access Rights & Permissions [Opens in a new window]

Summary

The presence of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), an enzyme at the branchpoint of glycolysis and the Krebs cycle was detected in the Filaria Molinema dessetae. This enzyme has not previously been identified in Helminths, which have so far been found to only possess a phosphoenolpyruvate carboxykinase (EC 4.1.1.32). This enzyme had a level of activity comparable to that of pyruvate kinase, and was relatively less active than enzymes such as malate dehydrogenase or lactate dehydrogenase. We propose here a method of purification of M. dessetae PEP-carboxylase. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 64 kDa. Kinetic studies indicated that the carboxylation reaction had an optimal pH of 5·8. The enzyme was inhibited by cations such as Fe2+, Zn2+, Cd2+, Cu2+ but required the presence of Mg2+ or Mn2+. The enzyme was thermostable. The apparent Km value of 2·38 mmol for phosphoenolpyruvate for the carboxylation reaction was higher than previously reported values. The Km value for KHCO3 was found to be 1·6 mmol. PEP-carboxylase did not catalyse the reverse reaction.

Keywords

phosphoenolpyruvate carboxylaseMolinema dessetaeFilarioideaFilaria.
Type
Research Article
Information
Parasitology , Volume 106 , Issue 1 , January 1993 , pp. 55 - 61
Copyright
Copyright © Cambridge University Press 1993

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Anwar, N., Ansari, A. A., Ghatak, S. & Krishna Murti, C. R. (1977). Setaria cervi: enzymes of glycolysis and PEP-succinate pathway. Zeitschrift für Parasitenkunde 51, 275–83.CrossRefGoogle ScholarPubMed
Barrett, J. (1981). Catabolism and energy production. In Biochemistry of Parasitic Helminths, pp. 92–94. Macmillan, London.CrossRefGoogle Scholar
Barrett, J. (1983). Biochemistry of filarial worms. Helminthological Abstracts, series S 52, 1–16.Google Scholar
Behm, C. A. & Bryant, C. (1975). Studies of regulatory metabolism in Moniezia expansa: the role of phosphoenolpyruvate carboxykinase. International Journal for Parasitology 5, 347–54.CrossRefGoogle ScholarPubMed
Behm, C. A. & Bryant, C. (1982). Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology 12, 271–8.CrossRefGoogle ScholarPubMed
Brazier, J. B. & Jaffe, J. J. (1973). Two types of pyruvate kinase in schistosomes and filariae. Comparative Biochemistry and Physiology 44B, 145–55.Google ScholarPubMed
Cornish, R. A., Wilkes, J. & Mettrick, D. F. (1981). A study of phosphoenolpyruvate carboxykinase from Moniliformis dubius (Acanthocephala). Molecular and Biochemical Parasitology 2, 151–66.CrossRefGoogle ScholarPubMed
Dean, R. B. & Dixon, W. J. (1951). Simplified statistics for small numbers of observations. Analytical Chemistry 23, 636–8.CrossRefGoogle Scholar
Gayral, P., Bories, C., Loiseau, P. & Gueyouche, C. (1987). Molinema (ex Dipetalonema) dessetae for in vitro and in vivo evaluations of filaricidal activities. Tropical Medicine and Parasitology 38, 65.Google Scholar
Ginger, C. D. (1991). Filarial worms: targets for drugs. Parasitology 7, 262–4.Google ScholarPubMed
Görg, A., Postel, W., Weser, J., Schiwara, H. W. & Boesken, W. H. (1985). Horizontal SDS electrophoresis in ultrathin pore-gradient gels for the analysis of urinary proteins. Science Tools 32, 1–8.Google Scholar
Goto, Y., Shimizu, J., Okazaki, T. & Shukuta, R. (1979). Purification and characterization of phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbiana) liver. Journal of Biochemistry 86, 77–8.Google Scholar
Hammond, K. D. & Balinski, D. (1978). Kinetic studies on phosphoenolpyruvate carboxykinase purified from the mitochondrial and cytosol fractions of monkey liver. International Journal of Biochemistry 9, 199–211.CrossRefGoogle ScholarPubMed
Köhler, P. (1991). The pathways of energy generation of filarial parasites. Parasitology Today 7, 21–5.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T 14. Nature, London 227, 680–5.CrossRefGoogle Scholar
Lineweaver, H. & Burk, D. (1934). The determination of enzyme dissociation constants. Journal of the American Chemical Society 56, 658–66.CrossRefGoogle Scholar
Lloyd, G. M. & Barrett, J. (1983). Fasciola hepatica: Inhibition of phosphoenolpyruvate carboxykinase, and end-product formation by quinolinic acid and 3-mercaptopicolic acid. Experimental Parasitology 56, 259–65.CrossRefGoogle ScholarPubMed
Moon, T. W., Mustafa, T., Hulbert, W. C., Podesta, R. B. & Mettrick, D. F. (1977). The phosphoenolpyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenolpyruvate carboxykinase. Journal of Experimental Zoology 200, 325–36.CrossRefGoogle Scholar
Prichard, R. K. (1976). Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda). International Journal for Parasitology 6, 227–33.CrossRefGoogle ScholarPubMed
Prichard, R. K. & Schofield, P. J. (1968). Phosphoenolpyruvate carboxykinase in the adult liver fluke Fasciola hepatica. Comparative Biochemistry and Physiology 24, 773–85.CrossRefGoogle ScholarPubMed
Saz, H. J. (1981). Biochemical aspects of filarial parasites. Trends in Biochemical Sciences 6, 117–19.CrossRefGoogle Scholar
Sedmak, J. J. & Grossberg, S. E. (1977). A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G 250. Analytical Biochemistry 79, 544–52.CrossRefGoogle Scholar
Srivastava, V. M. L., Chatterjee, R. K., Sen, A. B., Ghatak, S. & Krishna Murti, C. R. (1970). Glycolysis in Litomosoides carinii, the filarial parasite of the cotton rat. Experimental Parasitology 28, 176–85.CrossRefGoogle ScholarPubMed
Srivastava, V. M. L. & Ghatak, S. (1971). Glycolytic and carbon dioxide metabolizing enzymes in Chandlerella hawkingi. Indian Journal of Biochemistry and Biophysics 8, 108–11.Google Scholar
Utter, M. F. & Kolenbrander, H. M. (1972). Formation of oxaloacetate by CO2 fixation on phosphoenolpyruvate. In The Enzymes, vol. VI. Carboxylation and Decarboxylation (non-oxidative), (ed., Boyer, P.), pp. 117–168. New York: Academic Press.Google Scholar
Van Den Bossche, H. (1969). Phosphoenolpyruvate carboxykinase activity in Ascaris suum muscle. Comparative Biochemistry and Physiology 31, 789–97.CrossRefGoogle ScholarPubMed
Walter, R. D. & Albiez, E. J. (1986). Phosphoenolpyruvate carboxykinase from Onchocerca volvulus and Onchocerca gibsoni. Tropical Medicine and Parasitology 37, 356–8.Google Scholar
Wilkes, J., Cornish, R. A. & Mettrick, D. F. (1981). Purification and properties of phosphoenolpyruvate carboxykinase from Hymenolepis diminuta (Cestoda). Journal of Parasitology 67, 832–40.CrossRefGoogle ScholarPubMed