Protein Synthesis in Dormant and Non-Dormant Cocklebur Seed Segments

Using the axial and cotyledonary segments of lower cocklebur (Xanthium pensylvanicum Wallr.) seeds, protein synthesis as shown by incorporation of radioactive leucine was examined in relation to their dormant status. During the first 9 h of water imbibition, the protein synthesis was higher in the dormant axes than in the non-dormant, after- ripened ones. When imbibed for more than 12 h non-dormant axes had a higher activity than dormant ones. This was also the case with the cotyledonary segments. Cyctoheximide, an inhibitor of protein synthesis, blocked protein synthesis in the axial tissue regardless of its dormant status, and thereby inhibited germination of the non-dormant seeds. In the dormant seeds, however, cycloheximide at 3 mM slightly stimulated germination without stimulating the C₂H₄ production. Based on these results, it is suggested that in cocklebur seeds there may be some proteinaceous system which is involved in the maintenance of dormancy.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Protein synthesis

Summary Primary fetal hepatocytes derived from Zucker rats with expectedfa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75fa gene cultures is significantly less than in 0.0fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0fa gene cultures but has no effect on 0.75fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH₄Cl inhibits protein synthesis in 0.0 but not in 0.75fa gene cultures. These data suggest that fetal hepatocytes bearing thefa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without thefa gene. These diminished capacities may be expressions of a genetic error in lysosomal function. A portion of this work was presented in preliminary form at the 1980 meeting of the Tissue Culture Association. This work was supported in part by National Institutes of Health Grants AM19382 and AM06197.     

Identification of newly synthesized wing imaginal disc proteins induced by 20-hydroxyecdysone inGalleria mellonella

Summary Wing imaginal discs from 7th instarGalleria mellonella L. larvae evaginate and exhibit tracheolar elongation when exposed to 20-hydroxyecdysone in vitro. This response was elicited within 24 h of treatment as was a greater than fourfold stimulation of the incorporation of [³H]leucine into disc proteins. Autoradiographic analyses of [³⁵S]methionine labeled polypeptides separated on two-dimensional gels, however, revealed no differences in protein profiles between control and treated discs until 48 h following exposure to molting hormone. At this time, wing imaginal discs exposed to 1 μg/ml 20-hydroxyecdysone synthesized four unique polypeptides not detected either in controls or in discs treated for 24 h. These four new proteins were also found to be synthesized by imaginal discs that had evaginated in vivo. These results suggest that these proteins are normally synthesized subsequent to evagination and do not play a role in the morphological events necessary for evagination. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA. S. G. M. is employed through a cooperative agreement between the Insect Attractants, Behavior and Basic Biology Laboratory and the Department of Entomology, University of Florida.     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Ultracentrifugation in polyethylene tubing: An efficient method to concentrate protein solutions on the microliter scale

An ultracentrifugation procedure is described to concentrate protein solutions on the microliter scale. Protein solutions were centrifuged in U-shaped lengths of polyethylene tubing at 160 000 × g for 15 h and thereafter fractionated by cutting the tubing. The method can be performed in a conventional ultracentrifuge and needs no special equipment.     

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, and cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 ± 1.5 pmole cGMP per mg dry weight, flash-illuminated ones –45 ± 2 pmole/mg. 3-s bright illumination (ca. 10⁷ rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP within the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.