Gae E. Kovalick

Photoaffinity labels for insect juvenile hormone binding proteins: Synthesis and evaluation in vitro

Abstract The interactions of insect juvenile hormones (JH) with proteins are critically important to titer regulation, transport, and hormone action at a molecular level. We have synthesized several JH analogs bearing photolabile diazocarbonyl groups as potential photoaffinity labels for JH binding proteins (JHBP). The most promising compound, 10, 11-epoxyfarnesyl diazoacetate ( 2 ) (EFDA) competes with JH III for The JH binding site of JHBP from Leucophaea hemolymph and ovaries and from cultured Drosophila cells . Moreover, irradiation of protein solutions containing micromolar amounts of EFDA gave irreversible loss of [ 3 H]-JH III binding capacity with no change in binding affinity of the unlabelled protein. The protein could be protected against photoinactivation by the presence of equimolar JH III during irradiation. High specific activity [10- 3 H]-EFDA was prepared and used to demonstrate specific, finite binding of EFDA to the JH III receptor site of the binding protein. Further applications of photoaffinity labelling technique to characterization and cellular localization of the JHBP are discussed.

Assay and identification of juvenile hormone binding proteins in Leucophaea maderae.

We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.

Calmodulin transcription is limited to the nervous system during Drosophila embryogenesis

We have examined the RNA expression pattern for the Drosophila calmodulin gene during embryogenesis by in situ hybridization to transcripts in whole embryos. Our results indicate that maternally derived calmodulin mRNA is homogeneously distributed throughout the early embryo, but that these maternal transcripts are lost by maximal germ band extension. Zygotic transcription of the gene in mid- to late-stage embryos is restricted to neural cell precursors and their progeny in both the central and peripheral nervous systems. Thus, activation of calmodulin transcription during embryonic development appears to mark a commitment to a neural fate. Northern blot analysis revealed that the two transcripts from the calmodulin gene are differentially expressed during embryogenesis. Comparison of Northern blot and in situ hybridization data indicates that the longer calmodulin mRNA is a nervous tissue-specific transcript. This suggests that neural-specific regulation of polyadenylation site usage occurs. We have also examined calmodulin expression in embryos homozygous for mutations in four loci which are known to affect nervous system development: numb, the achaete-scute complex, daughterless, and mastermind. The calmodulin transcription pattern is altered in embryos mutant for each of these loci, suggesting that regulation by these genes, either directly or indirectly, is taking place.

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaea maderae

Abstract The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [3H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaea maderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.

Photoaffinity Labelling of Juvenile Hormone-Binding Proteins in Leucophaea maderae

Juvenile hormone (JH) binding proteins have been identified in tissues from several insect species [1–3]. In the adult female cockroach Leucophaea maderae, JH-binding proteins have been found in the fat body [4], ovaries [5], and hemolymph [4,5]. Although some properties of these binding proteins have been described, further characterization has been hampered by the extremely rapid dissociation of JH from the binding protein under nonequilibrium conditions. In an attempt to circumvent this problem, we have initiated studies on the possible use of a photoactivatable JH III analog (Fig. 1), 10,11-epoxyfarnesyl diazoacetate (EFDA), to form an irreversible covalent attachment at the JH binding site on the hormone binding protein. Our preliminary investigations suggested that EFDA covalently attached to JH-binding proteins in the hemolymph and ovaries [6]. Recent studies have focused on the specificity of [3H]-EFDA binding to both the hemolymph and the ovarian binding proteins.