[en] A short synthesis of [4,5-³H₂] (E)-9-oxo-2-decenoic acid (ODA), a high-specific-activity tritium-containing isotopomer of the queen bee pheromone, is described. Catalytic tritiation of the ketal of ethyl 9-oxo-4-decenoate introduces tritium into two positions, one of which is completely unactivated. Subsequent transformation by selenation, oxidation, and hydrolysis affords the labeled 9-ODA at >60 Ci/mmol. The material is suitable for biochemical studies of binding and catabolism in ovarian, antennal, and other target tissues

[en] Silver-staining of polyacrylamide gel electrophoresis (PAGE)-separated proteins allows sensitive detection of proteins but severely reduces the ability to detect weak beta-emitters present in the protein band. A simple procedure is described in which silver can be removed from a silver-stained PAGE gel (deargentation) using photographic fixer, and the silver-free gel can be enhanced and used for fluorography. A quantitative study of sensitivity is reported for ³H-labeled bovine serum albumin with a one-dimensional sodium dodecyl sulfate-PAGE slab gel

[en] A series of mono, di-, and trihalogenated acetate analogs of Z11-16:Ac were prepared and examined for electrophysiological activity in antennae of males of the diamondback moth, Plutella xylostella. In addition, two potential affinity labels, a diazoacetate (Dza) and a trifluoromethyl ketone (Tfp), were evaluated for EAG activity. The Z11-16:Ac showed the highest activity in EAG assays, followed by the fluorinated acetates, but other haloacetates were essentially inactive. The effects of these analogs on the hydrolysis of [³H]Z11-16:Ac to [³H]Z11-16:OH by antennal esterases was also examined. The three fluorinated acetates showed the greatest activity as inhibitors in competition assays, with rank order F₂Ac > F₃Ac > FAc > AC > Cl₂Ac > ClAc > Dza > Br₂Ac > BrAc > Tfp > I > Cl₃Ac > Br₃Ac > OH. The relative polarities of the haloacetates, as determined by TLC mobility, are in the order mono- > di- > trihalo, but F, Cl, Br, and I all confer similar polarities within a substitution group. Thus, the steric size appears to be the predominant parameter affecting the interactions of the haloacetate analogs with both receptor and catabolic proteins in P. xylostella males

[en] A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites

[en] Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, [3H](Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes

[en] Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography

[en] A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-[³H]hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specific fashion with a biologically relevant odorant

[en] The synthesis of 1-(1,2-O-diundecyl-sn-glycerylphosphoryl) 4,5-D-myo-inosityl biphosphate and its tritiated analog are described. The convergent synthesis employed optically-pure inositol and glycerol derivatives. In the final step, hydrogenation of an alkyl chain gave the saturated diether PIP₂ and tritiation gave the high-specific activity, tritium-labelled analog. (author)

[en] A new photoaffinity analog of inositol hexakisphosphate (InsP₆, or phytic acid) was prepared for investigation of InsP₆-binding proteins. The racemic P-1-(O-6-aminohexyl) derivative of InsP₆ was synthesized in six steps from inositol, and a tritium-labeled benzophenone-containing photophore was attached in the final step to give P-1 [³H]BZDC-InsP₆ (sp. act. 35 Ci/mmol). (author)

[en] Both natural D- and L-enantiomers of myo-Ins(1,4,5)P₃ were synthesized with specific activities 14-16 Ci/mmol. A suitable inositol derivative was resolved as the diastereomeric camphanate esters, and the chiral inositol derivatives were oxidized to the protected inosose. Reduction of each chiral ketone with sodium borotritide and manipulation of protecting groups gave the enantiomeric [1-³H]-2,3,6-tri-O-benzyl-myo-inositols in 55% radiochemical yield. Phosphorylation with tetrabenzylpyrophosphate and complete hydrogenolytic debenzylation provided the separate D-myo and L-myo-[1-³H]-Ins(1,4,5)P₃ enantiomers in 30% radiochemical yield. (author)