Dithioerythritol Research Articles

Studies on the Chemistry and Fine Structure of Elastic Fibers from Normal Adult Skin

Collagenase-prepared, purified elastic fibers from adult human skin consist of at least two morphologically distinct components, the unstained amorphous component, and internally-located, deeply staining microfibrils. Separation and isolation of the amorphous component was achieved by alkali preparation of the elastic fibers. Electron micrographs of alkali prepared fibers showed essentially amorphous component as most of the microfibrils had been solubilized. The amino acid composition of the amorphous component was that of classical elastin. The microfibrillar component embedded deep within the interstices of the fiber was separated by, first, removing the amorphous component with elastase, and subsequently solubilizing, with dithioerythritol (DTE), the cystine-containing microfibrils contained in the elastase-produced residue. The amino acid composition of this DTE-extracted microfibrillar material was similar to that of the peripheral microfibrillar component enzymatically removed from bovine fetal elastic fibers. Electron microscopic monitoring showed that only stained microfibrils remained after elastase digestion. A protein portion of the microfibrillar component was not solubilized by the DTE and was presumed to contain a low concentration of disulfide bonds. Studies on purified elastin (amorphous component) revealed that alanine is concentrated around desmosine crosslinks and that the pyrollidines are uniformly distributed along the elastin molecule thereby precluding a α-helix conformation. Dark field electron microscopy suggested that the desmosine crosslink region is dumbell-shaped and that the desmosine crosslinks are not equidistant from each other on the peptide chains.

Inhibition of adenyl cyclase preparations from rat kidney by calcium ions and various diuretics

Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 3′∶5′-monophosphate (Ado-3′∶5′-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-3′∶5′-P formed from 1 mM 14C-ATP in the presence of 10 mM Mg++, an ATP regenerating system, and 5 mM unlabeled Ado-3′∶5′-P to reduce the enzymatic degradation of the labeled Ado-3′∶5′-P. Calcium ions reduced the rate of Ado-3′∶5′-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg++ or Mn++. With 10 mM Mg++, 1 mM Ca++ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca++. The inhibition was independent of the ATP concentration, but was influenced by the Mg++ content of the incubation medium. Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-3′∶5′-P in the regulation of sodium transport is as yet unclear. Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration. The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 3′∶5′-monophosphate phosphodiesterase, reduced the rate of Ado-3′∶5′-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues. Instead of phosphodiesterase inhibitors, unlabeled Ado-3′∶5′-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-3′∶5′-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-3′∶5′-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-3′∶5′-P by 35%.

Inactivation of the pseudorabies virus by dithiothreitol

The virucidal nature of reduced dithiothreitol (DTT) for the pseudorabies (PR) virus (PRV) is presented. The rate of decay of PRV in DTT increased exponentially as the pH rose from 6.5 to 8. Effective virucidal concentrations of DTT decreased in concentration as the pH was elevated. The reaction rate was temperature dependent under mild alkaline conditions, being essentially nil at 0° and at 20°, but progressively more rapid from 30° to 41°. Electron micrographs indicated substantial disruption of the architecture of the DTT inactivated virions. Preincubation of DTT with catalytic amounts of Cu 2+ or with high concentrations of Mg 2+ decreased the virucidal activity; preincubation with catalytic levels of Fe 3+ lowered activity slightly. Thirteen additional compounds, mostly thiols, were tested for virucidal activities. Reduced dithioerythritol (DTE), an asymmetric isomer of DTT, was considerably less active than DTT. British anti-lewisite (BAL), (2,3-dimercapto-1-propanol), was of the same order of activity as DTE, but 1,3-dimercapto-2-propanol, an isomer of BAL, was essentially nonvirucidal. Thimerosal and iodoacetamide at 0.01 M were virucidal, but 2-mercaptoethanol at 0.01 M was inactive. Trithiodiglycol at 0.01 M, pH 8.0, was moderately virucidal. Oxidized dithiothreitol was nonvirucidal.

Toxicity of Radioprotective Agents to Yeast

These studies consider the toxic effects to baker's yeast of four sulfhydryl compounds frequently used as radioprotectants: cystamine, cysteamine, cysteine, and dithioerythritol (DTE). All but cysteine markedly reduce colony formation at concentrations above 10-4 M. The rate of action on colony formation varied. Cystamine required at least an hour of exposure to produce an effect, and then it developed slowly, while the others manifested their action rapidly. All the agents inhibit exogenous respiration with dextrose to some degree, while their action on endogenous respiration was either small and variable or a stimulation. All cause a small but significant increase in the number of cells staining with methylene blue dye, but this is quite insufficient to account for the inhibition of colony formation by death of the cells. None of the effects measured bear a consistent relation to the radioprotective ability of these compounds in single cells or in higher organisms. Therefore, it is unlikely that identic...