Abstract
Techniques for performing numerous enzyme kinetic assays with minimum time and effort would be valuable to studies of the evolutionary genetics of metabolic control and the quantitative genetics of determinants of kinetic parameters. Microtiter plate readers have been used for a variety of repetitious analytical techniques, and instruments are available that can take repetitive readings with sufficient speed to perform kinetic assays. The ability of these instruments to assay rapidly the kinetic properties of small samples makes them potentially useful for a number of problems in population genetics. While the ability to handle large numbers of samples is very attractive, the small sample volumes and optical imprecision of microtiter plates result in some sacrifice in accuracy. This paper presents methods for performing kinetic assays on individual field-caughtDrosophila, quantifies the precision of these methods, and characterizes differences amongDrosophila melanogaster andD. simulans from samples caught in California and Pennsylvania. Comparisons between field-caught and laboratory rearedD. melanogaster show that most of the characters are very similar, with the exception of αGPDH, which has a threefold higher mean activity among field-caught flies. The phenotypic correlations are presented with a brief discussion of their relevance to assessing the evolution of metabolic control of these enzymes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barnes, P. T., and Laurie-Ahlberg, C. C. (1986). Genetic variability of flight metabolism inDrosophila melanogaster. III. Effects of GPDH allozymes and environmental temperature on power output.Genetics 112267.
Bucolo, G., and David, H. (1973). Quantitative determination of serum triglycerides by the use of enzymes.Clin. Chem. 19476.
Burton, R. S., and La Spada, A. (1986). Trehalase polymorphism inDrosophila melanogaster.Biochem. Genet. 24715.
Canova-Davis, E., Redemann, C. T., Vollmer, Y. P., and Kung, V. T. (1986). Use of a reversed-phase evaporation vesicle formulation for a homogeneous liposome immunoassay.Clin. Chem. 321687.
Clark, A. G. (1989a). Causes and consequences of variation in energy storage inDrosophila melanogaster, Genetics, in press.
Clark, A. G. (1989b). Genetic components of variation in energy storage inDrosophila melanogaster, Evolution, in press.
Clark, A. G., and Gellman, W. (1985). A rapid spectrophotometric assay of triglycerides inDrosophila.Dros. Info. Serv. 61190.
Clark, A. G., and Keith, L. E. (1988). Variation among extracted lines ofDrosophila melanogaster in triacylglycerol and carbohydrate storage.Genetics 119595.
Doane, W. W., and Treat-Clemons, L. (1982). Biochemical map of the “fruit fly”Drosophila melanogaster. In O'Brien, S. J. (ed.),Genetic Maps Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Vol. 2.
Geer, B. W., Bowman, J. T., and Simmons, J. R. (1974). The pentose shunt in wild type and glucose-6-phosphate dehydrogenase deficientDrosophila melanogaster.J. Exp. Zool. 18777.
Geer, B. W., Langevin, M. L., and McKechnie, S. W. (1985). Dietary ethanol and lipid synthesis inDrosophila melanogaster.Biochem. Genet. 23607.
Laurie-Ahlberg, C. C., Wilton, A. N., Curtsinger, J. W., and Emigh, T. H. (1982). Naturally occurring enzyme activity variation inDrosophila melanogaster. I. Sources of variation for 23 enzymes.Genetics 102191.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193265.
Lucchesi, J. C., and Rawls, J. M. (1973). A comparison of X-linked enzyme activities in relation to gene dosage in diploids and triploids ofDrosophila melanogaster.Biochem. Genet. 941.
Michal, G., Möllering, H., and Siedel, J. (1983). Chemical design of indicator reactions for the visible range. In Bergmeyer, H. U. (ed.),Methods of Enzymatic Analysis 3rd ed., Verlag Chemie, Weinheim, pp. 197–209.
Miller, R. G., Jr. (1981).Simultaneous Statistical Inference McGraw-Hill, New York.
Murphy, T. A., and Wyatt, G. R. (1965). The enzymes of glycogen and trehalose synthesis in silkmoth fat body.J. Biol. Chem. 2401500.
Nepokroeff, C. M., Lakshmanan, M. R., and Porter, J. W. (1972). Fatty acid synthase from rat liver. InMethods in Enzymology, Vol. 23 Academic Press, New York, pp. 37–44.
O'Brien, S. J., and MacIntyre, R. J. (1978). Genetics and biochemistry of enzymes and specific proteins ofDrosophila. In Ashburner, M., and Wright, T. (eds.),The Genetics and Biology of Drosophila Academic Press, New York, Vol. 2a, pp. 396–552.
Raabo, E., and Terkildsen, T. C. (1960). On the enzymatic determination of blood glucose.Scand. J. Clinic. Lab. Invest. 12402.
Sacktor, B. (1975). Biochemistry of insect flight. In Candy, D. J., and Kilby, B. A. (eds.),Insect Biochemistry and Function John Wiley and Sons, New York, pp. 3–88.
Stam, L. F., and Laurie-Ahlberg, C. C. (1982). A semi-automated procedure for the assay of 23 enzymes fromDrosophila melanogaster.Insect Biochem. 12537.
Steele, J. E. (1982). Glycogen phosphorylase in insects.Insect Biochem. 12131.
Wilton, A. N., Laurie-Ahlberg, C. C., Emigh, T. H., and Curtsinger, J. W. (1982). Naturally occurring enzyme activity variation inDrosophila melanogaster. II. Relationship among enzymes.Genetics 102207–221.
Ziegler, R., Ashida, M., Fallon, A. M., Winner, L. T., Wyatt, S. S., and Wyatt, G. R. (1979). Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae.J. Comp. Physiol. 131321.
Author information
Authors and Affiliations
Additional information
This work was supported by Grant BSR 8717495 from the National Science Foundation and by Grants HD 18379 and HD 00743 from the U.S. Public Health Service.
Rights and permissions
About this article
Cite this article
Clark, A.G., Keith, L.E. Rapid enzyme kinetic assays of individualDrosophila and comparisons of field-caughtD. melanogaster andD. simulans . Biochem Genet 27, 263–277 (1989). https://doi.org/10.1007/BF00554162
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00554162