Reorganization of the endoplasmic reticulum in epidermal cells of onion bulb scales after cold stress: Involvement of cytoskeletal elements

In the epidermal cells of onion (Allium cepa L.) bulb scales the endoplasmic reticulum (ER) can be subdivided into three domains: a peripheral tubular network, cisternae, and long tubular strands. The latter are the form in which the ER is moved in onion cells. During cold treatment the arrangement of the three domains changes drastically. The cisternae and long tubular strands disintegrate into short ER tubules which show rapid agitational motion. Long-distance movement is inhibited. The peripheral tubular ER network is presumably retained during cold treatment. Rewarming of previously chilled bulb scales initiates the reorganization of the ER into the three domains. The ER is partly relocated during recovery from cold treatment. Redistribution and reorganization of the ER is not affected by the microtubule-destabilizing herbicides oryzalin and trifluralin (5 M). Cytochalasin D (2M), however, inhibits not only the relocation of ER material, as is evident by the absence of long tubular ER strands, but also the movement of other cell organelles. The latter cluster on top of the cisternae in a manner which is characteristic of treatment with the actin-filament inhibitor. The array of actin filaments is similar in unstressed, cold-treated cells, and cells which recover from low temperatures in the presence of oryzalin or tap water alone. In the presence of cytochalasin D the actin filaments are severely fragmented. The results indicate that low temperatures most likely influence either the interaction of the force-generating system, probably myosin, with actin filaments, or the force-generating mechanism of the actomyosin-driven intracellular movement, but do not affect actin-filament integrity.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum     

Methylglyoxal — ein toxisches Fermentationsprodukt

Methylglyoxal (CH₃COCHO) is a toxic intermediate of the carbon metabolism. Its accumulation in the growth medium of microorganisms may be an important danger for many fermentation processes. In this paper data will be presented which show the physiological effect of this compound on the living cell, the enzymes responsible for the synthesis and the turnover of methylglyoxal including the environmental conditions necessary for the accumulation of lethal concentrations in the medium.     

The reaction of diethyl pyrocarbonate with pyruvate kinase (Short Communication)

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.     

Reaction of yeast phosphofructokinase with succinic and maleic anhydride.

Modification of yeast phosphofructokinase by succinic and maleic anhydride influences the catalytic activity and the allosteric behaviour of the enzyme. Depending on the degree of succinylation and maleinylation a decrease of maximum activity, an increase of the apparent affinity for fructose-6-phosphate, a decrease of the Hill-coefficient and a diminution of ATP-inhibition are observed. Up to about 40% of the lysyl residues could be succinylated without dissociation of the hexameric protein, however with a decrease of the enzyme activity. More extensive succinylation or maleinylation causes a dissociation into subunits. The sedimentation coefficient is lowered from 20 S to about 3 S. The molecular weight of the smallest dissociation product was determined to 50 000 (+/- 10 000) by the sedimentation equilibrium method. The number of bound succinyl groups, as determined from radioactivity incorporation, exceeds the content of lysyl groups of the enzyme, indicating that the modifying reagent is also reacting with other amino acid residues.     

Affinity elution of pyruvate kinase from phosphocellulose.

Pyruvate kinase from ascites tumour cells can be eluted from phosphocellulose by very low concentrations of phosphoenolpyruvate, fructose 1,6-bisphosphate, adenosine 5'-diphosphate and pyrophosphate, respectively. The appropriate limiting conditions for "facilitated desorption" of the enzyme from phosphocellulose by these ligands have been elaborated for achieving maximum selectivity and recovery in the process of its purification. This method has been designated as "affinity elution chromatography" owing to the specific interactions between a ligand as a constituent of the eluting medium with the adsorbed enzyme, which causes its selective desorption from the ion-exchanger. Affinity elution with phosphoenolpyruvate has been found to be very effective for preparation of the M-types of pyruvate kinase. A specific activity of 420 for an almost homogeneous preparation of pyruvate kinase from ascites tumour cells has maximally been obtained.     

Differentiation of the drug-binding sites of calmodulin

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5-10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 microM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 microM) and four low affinity sites (apparent Kd 44 microM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 microM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.