Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK_a values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO⁻CH₂NH₂⁺CH₂PO₃²⁻, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K_i = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K_i′ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate].     

The nucleoside sequence of tyrosine tRNA from Bacillus stearothermophilus.

The nucleotide sequence of tRNATyr from B. stearothermophilus has been determined: pG-G-A-G-G-G-G-s4U-A-G-C-G-A-A-G-U-Gm-G-C-U-A-A-m1A-C-G-C-G-G-C-G-G-A-C-U-Q-U-A-ms2i6A-A-psi-C-C-G-C-U-C-C-C-U-U-U-G-G-G-U-U-C-G-G-C-G-G-T-psi-C-G-A-A-U-C-C-G-U-C-C-C-C-C-U-C-C-A-C-C-AOH. A combination of classical fingerprinting methods, partial nuclease P1 digestion and two-dimensional homochromatography and a rapid "read off" sequencing gel technique were used to establish the complete nucleotide sequence.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Depletion of Mg²⁺ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by ³H-thymidine incorporation, and concomitant decreases in cellular K⁺ and Mg²⁺ and increases in Na⁺ and Ca²⁺. In cells grown in media containing 0.2 mM Ca²⁺, graded reduction of Mg²⁺ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg²⁺. Concomitantly, cell cations showed graded changes, Na⁺ increasing to 227%, K⁺ decreasing to 52.5%, Mg²⁺ decreasing to 57.5% and Ca²⁺ increasing to 153.5% of control. The effects of Mg²⁺ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca²⁺ concentration, the effects being larger at low Ca²⁺ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg²⁺ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg²⁺ depletion. The effects of Mg²⁺ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg²⁺ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg²⁺ in the coordinate control of growth and metabolism in animal cells.     

Antagonistic effects of insulin and cortisol on coordinate control of metabolism and growth in cultured fibroblasts

A variety of metabolic and biosynthetic pathways in chick embryo fibroblasts are stimulated coordinately by many unrelated exogenous agents. Three of the best characterized components of this coordinate response are the uptake of 2-deoxy-D-glucose (2-dGlc) and of uridine and the incorporation of thymidine into DNA. Insulin stimulates and cortisol inhibits the coordinate response. In cortisol-treated cultures, as little as 10^?3 units/ml of insulin may stimulate thymidine incorporation 4-fold and 10^?1 units/ml may stimulate as much as 40-fold. The higher concentrations of insulin completely override the inhibitory effect of cortisol. They also cause about a 5-fold stimulation of the uptake of 2-dGlc and of uridine and a 2-fold stimulation of proline incorporation into protein. The uptake rates of 2-dGlc and uridine double within 30 minutes after addition of insulin to cortisol-inhibited cultures, but the incorporation of thymidine only begins to increase markedly after a 4-hour delay. When cortisol is added to cultures in the absence of insulin, the rates of uptake of 2-dGlc and uridine begin to decrease within two hours, but the incorporation of thymidine remains constant for two hours before beginning to decrease. Deprivation of Mg²⁺ inhibits the accelerated coordinate response maintained by insulin, but does not further the inhibition induced by cortisol. Results with metabolic inhibitors indicate that the stimulation of 2-dGlc and uridine uptake by insulin do not require RNA synthesis, and also suggest that they do not require protein synthesis. These and other findings can be explained by a model for coordinate control in which insulin increases and cortisol decreases the availability of Mg²⁺ for a wide spectrum of regulatory reactions in different metabolic pathways. In this model both hormones affect only the rates of ongoing reactions and do not instruct the cell to carry out specific new reactions unless the cell was predetermined to do so.     

Control of urea transport across toad urinary bladder by vasopressin: Effect of periodate oxidation of the mucosal cell surface

The Journal of Membrane Biology - Oxidation of toad urinary bladder epithelial cell membranes by periodate in the bathing medium altered vasopressin-stimulated transport of urea or of water and...     

Distribution and regulation of cyclic nucleotide levels in cerebellum, in vivo.

Abstract— In mouse cerebellum, in vivo. cyclic GMP levels are 7 pmol/mg protein in the vermis and 40% lower in the hemispheres, whereas cyclic AMP levels are 7 9 pmol/mg protein in both regions. In the vermis. most of the cyclic GMP is contained in the molecular layer; cyclic AMP levels are highest in the granular layer. Amphetamine, harmaline. pentylenetetrazol and physical shaking elevate, and diazepam and reserpine depress levels of cyclic GMP in both vermis and hemispheres. Oxotremorine and atropine, respectively, increase and decrease cyclic GMP levels only in vermis. Regardless of the agent used, most of the change (67 89%) in cyclic GMP levels occurs in the molecular layer of the vermis; the remainder occurs in the granular layer. Of the drugs tested, only pentylenetetrazol affects cyclic AMP levels, and this drug increases cyclic AMP levels in both vermis and hemispheres and causes equal elevations in the molecular and granular layers of the vermis. In incubated slices of mouse cerebellum, none of the drugs produces changes in cyclic nucleotide levels which are similar to those in vivo. These data indicate that many drugs and conditions that alter cyclic GMP levels in cerebellum act via a common, but indirect, process. We suggest that cyclic GMP levels in cerebellum are regulated by the activity of both the climbing fiber and mossy fiber cerebellar afferent systems. Increased activity in these afferent pathways causes elevation of cyclic GMP levels in Purkinje cells and perhaps in other cells; decreased activity leads to depressed cyclic GMP levels.     

THE PERMISSIVE ROLE OF HYPERTHERMIA IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA OR d-AMPHETAMINE1

Abstract— l -DOPA or d -amphetamine administration disaggregates brain polyribosomes in animals maintained in an environment warm enough (26°C) so that the drugs concurrently elevate their body temperatures to above 39°C. The production of equivalent hyperthermia (by keeping control rats at ambient temperatures of 40–44° C) does not cause similar disaggregation of brain polysomes. Hence, the role of hyperthermia in the drug-induced disaggregation is permissive.     

Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts.

In the course of adapting O'Farrell's (1975, J. Biol. Chem.250, 4007–4021) two-dimensional separation technique for proteins to eukaryotic material, we have made some modifications. During sample preparation, sodium dodecyl sulfate (SDS) can be included, with a resulting enhancement in reproducibility of gel patterns. However, heating in the presence of SDS leads to artifactual spots in the gels, probably as a result of protein charge modifications. Ultracentrifugation reduces the clogging at the top of the isoelectric focussing gel. For electrophoresis, some modifications of apparatus and technique are suggested. For the analysis of gels, a simple high-efficiency method for the counting of radioactivity in spots from dried gel slabs is described. In addition, an inexpensive microdensitometer option is described for the analysis of the autoradiographs. Patterns of proteins obtained from superior cervical sympathetic ganglia of rats and from other eukaryotic tissues are illustrated. Finally, a few of the proteins commonly found in mammalian tissue are identified on the gels.