Summary
Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.
Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to α-methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.
A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.
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References
Christenson, J.G., Dairman, W., Udenfriend, S.: Preparation and properties of a homogeneous aromatic L-amino acid decarboxylase from hog kidney. Arch. Biochem. Biophys. 141, 356–367 (1970)
Drum, D.E., Harrison, H.H., Li, T.-K., Bethune, J.L., Vallee, B.L.: Structural and functional zinc in horse liver alcohol dehydrogenase. Proc. nat. Acad. Sci. (Wash.) 57, 1434–1440 (1967)
Edsall, J.T.: The size and shape of protein molecules. In: The proteins (Neurath, H. and Bailey, K., eds.) Vol. 1B, pp. 549–726. New York: Academic Press 1953
Fellman, B.K.: Purification and properties of adrenal L-dopa decarboxylase. Enzymologia 20, 366–376 (1959)
Fragoulis, E.G., Sekeris, C.E.: Induction of dopa (3,4-dihydroxyphenyl-alanine) decarboxylase in the blowfly integument by ecdysone. Biochem. J. 146, 121–126 (1975a)
Fragoulis, E.G., Sekeris, C.E.: Translation of mRNA for 3,4-dihydroxyphenylalanine decarboxylase isolated from epidermis tissue of Calliphora vicina R.-D. in an heterologous system. Eur. J. Biochem. 51, 305–316 (1975b)
Fragousli, E.G., Sekeris, C.E.: Purification and characteristics of DOPA-decarboxylase from the integument of Calliphora vicina larvae. Arch. Biochem. Biophys. 168, 15–25 (1975c)
Hayes, J.E., Velick, S.F.: Yeast alcohol dehydrogenase: molecular weight, co-enzyme binding, and reaction equilibria. J. biol. Chem. 207, 225–244 (1954)
Hedrick, J.L., Smith, A.J.: Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155–164 (1968)
Hodgetts, R.B.: The response of dopa decarboxylase to variations in gene dosage in Drosophila: a possible location of the structural gene. Genetics 79, 45–54 (1975)
Hodgetts, R.B., Sage, B., O'Connor, J.D.: Ecdysone titers during postembryonic development of Drosophila melanogaster. Develop. Biol. 60, 310–317 (1977)
Karlson, P., Sekeris, C.E.: Ecdysone, an insect steroid hormone and its mode of action. Recent Progr. Hormone Res. 22, 473–502 (1966)
Kikuchi, Y., King, J.: Genetic control of bacteriophage T4 baseplate morphogenesis. I. Sequential assembly of the major precursor, in vivo and in vitro. J. molec. Biol. 99, 645–672 (1975)
Laurell, C.-B.: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15, 45–52 (1966)
Lindsley, D.L., Grell, E.H.: Genetic variations of Drosophila melanogaster. Carnegie Inst. Washington Publ. 627 (1968)
Loeb, G.I., Scheraga, H.A.: Hydrodynamic and thermodynamic properties of bovine serum albumin at low pH. J. phys. Chem. 60, 1633–1644 (1956)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–269 (1951)
Lunan, K.D., Mitchell, H.K.: The metabolism of tyrosine-O-phosphate in Drosophila. Arch. Biochem. Biophys. 132, 450–456 (1969)
Maranda, B., Hodgetts, R.B.: A characterization of dopamine acetyltransferase in Drosophila melanogaster. Insect Biochem. 7, 33–43 (1977)
Marmaras, V.J., Sekeris, C.E., Karlson, P.: Activity of 5-hydroxytryptophan decarboxylase during development of the blow-fly Calliphora erythrocephala, in relation to the ecdysone titer. Acta. Biochim. Polon. 13, 305–309 (1966)
Martin, R.G., Ames, B.N.: A method for determining the sedimentation behaviour of enzymes: application to protein mixtures. J. biol. Chem. 236, 1372–1379 (1960)
McCaman, M.W., McCaman, R.E., Lees, G.: Liquid cation exchange — a basis for sensitive radiometric assays for aromatic amino acid decarboxylases. Analyt. Biochem. 45, 242–252 (1972)
Müller-Hill, B.L., Crapo, L., Gilbert, W.: Mutants that make more lac repressor. Proc. nat. Acad. Sci. (Wash.) 59, 1259–1264 (1968)
Peacock, W.J., Brutlag, D., Goldring, E., Appels, R., Hinton, C.W., Lindsley, D.L.: The organization of highly repeated DNA sequnces in Drosophila melanogaster chromosomes. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 38, 405–416 (1974)
Sekeris, C.E., Karlson, P.: Biosynthesis of catecholamines in insects. Pharmacol. Rev. 18, 89–94 (1966)
Shaaya, E., Karlson, P.: Der Ecdysontiter während der Insektenentwicklung. II. Die postembryonale Entwicklung der Schmeissfliege Calliphora erythrocephala. J. Insect Physiol. 11, 65–69 (1965)
Sparrow, J.C., Wright, T.R.F.: The selection for mutants in Drosophila melanogaster hypersensitive to α-methyl dopa, a dopa decarboxylase inhibitor. Molec. gen. Genet. 130, 127–141 (1974)
Suzuki, Y., Gage, P.L., Brown, D.D.: The genes for silk fibroin in Bombyx mori. J. molec. Biol. 70, 637–649 (1972)
Vallee, B.L., Hoch, F.L.: Zinc, a component of yeast alcohol dehydrogenase. Proc. nat. Acad. Sci. (Wash.) 41, 327–338 (1955)
Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406–4412 (1969)
Wrigley, C.W.: Gel Electrofocusing. In: Methods in enzymology (Jakoby, W.B. ed.) Vol. 22, pp. 559–564. New York: Academic Press 1971
Wright, T.R.F., Hodgetts, R.B., Sherald, A.F.: The genetics of dopa decarboxylase in Drosophila melanogaster. I. Isolation and characterization of deficiencies that delete the dopa-decarboxylase-dosage-sensitive region and the α-methyl dopa hypersensitive locus. Genetics 84, 267–285 (1976a)
Wright, T.R.F., Bewley, G.C., Sherald, A.F.: The genetics of dopa decarboxylase in Drosophila melanogaster. II. Isolation and characterization of dopa decarboxylase deficient mutants and their relationship to the α-methyl dopa hypersensitive mutants. Genetics 84, 287–310 (1976b)
Yoshida, A.: Glucose-6-phosphate dehydrogenase of human erythrocytes. J. biol. Chem. 241, 4966–4976 (1966)
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Clark, W.C., Pass, P.S., Venkataraman, B. et al. Dopa decarboxylase from Drosophila melanogaster . Molec. Gen. Genet. 162, 287–297 (1978). https://doi.org/10.1007/BF00268854
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DOI: https://doi.org/10.1007/BF00268854