Bisulfite compounds as metabolic inhibitors: nonspecific effects on membranes

Bisulfite compounds are shown to be nonspecific inhibitors ofphotosynthetic processes and of ion transport in green tissues.CO₂ fixation and light-dependent transient changes in externalpH are inhibited about 50% by 5x10^–4 M glyoxal-Na-bisulfite.Chloride uptake in the light and in the dark is inhibited tothe same extent at this concentration. At 5x10^–3 M theinhibitor reduces ATP levels in the light and in the dark, andeffects on glycolate oxidase and PEP carboxylase are observed.The extent of inhibition is dependent on time of treatment withglyoxal-Na-bisulfite and freshly prepared NaHSO₃ is equallyas effective as the addition compound. Possible explanations of the bisulfite effects and the relationshipsto SO₂ effects on photosynthesis are discussed. (Received September 1, 1971; )     

On the Mechanism of Reinitiation of Endogenous Crassulacean Acid Metabolism Rhythm by Temperature Changes

Under continuous light the endogenous Crassulacean acid metabolism (CAM) rhythm of Kalanchoe daigremontiana Hamet et Perrier de la Bathie disappears at high (>29.0[deg]C) or low (<8.0[deg]C) temperatures. We investigated the reinitiation of rhythmicity when temperature was reduced from above the upper and increased from below the lower threshold level via measurements of (a) short-term changes in carbon-isotope discrimination to illustrate shifts between C3 and C4 carboxylation in vivo, and (b) the malate sensitivity of phosphoenolpyruvate carboxylase (PEPC) in vitro. When the net CO2-exchange rhythm disappears at both temperatures, the instantaneous discrimination indicates low PEPC activity. Leaf malate concentration and osmolarity attain high and low values at low and high temperatures, respectively. After small temperature increases or reductions from the low and high temperatures, respectively, the rhythm is reinitiated, with phases shifted by 180[deg] relative to each other. This can be related to the contrasting low and high leaf malate concentrations due to direct inhibition of PEPC and possibly also of the phosphorylation of PEPC by malate. The experimental results were satisfactorily simulated by a mathematical CAM-cycle model, with temperature acting only on the passive efflux of malate from the vacuole. We stress the important role of the tonoplast in malate compartmentation and of malate itself for the reinitiation and generation of endogenous CAM rhythmicity.     

Influence of Estrogen and Progesterone on Glutamic Acid Decarboxylase Activity in Discrete Regions of Rat Brain

Abstract: Female Charles River rats were ovariectomized and treated for three days with 17/8-estradiol benzoate (E) (1.0 /μg/day), progesterone (P) (500 μg/day), vehicle, or a combined treatment (2 days E, one day P). Animals were killed on day 3 and the brains were dissected by the micropunch technique. Glutamic acid decarboxylase (GAD) activity was measured by collection of ¹⁴CO². Estradiol benzoate and progesterone were potent inhibitors of GAD activity in regions such as the arcuate nucleus, ventromedial hypothalamus and corticomedial amygdala. Estrogen reduced the V_max of GAD for glutamate as a substrate without changing the K_m. Estrogen also failed to change the K_m for pyridoxal phosphate. Combined treatment with estrogen and progesterone did not reduce GAD from ovariectomized levels except in the septum, indicating an interaction of the two hormones at the level of GAD. The suggestion is made that under conditions that inhibit LH secretion GAD activity is low, but when LH secretion is stimulated GAD activity may be comparatively high.     

ATP-dependent stabilization against microsomal factor-induced loss of unoccupied glucocorticoid receptors

ATP stabilizes the unoccupied glucocorticoid receptor from brain at 12 degrees C, but only in the presence of a destabilizing microsomal factor. This stabilization is optimal at an ATP concentration of about 1 mM, higher concentrations resulting in an increase in the rate of heat inactivation. Other nucleotides, including CTP, GTP, UTP, ADP, cAMP and cGMP were ineffective in stabilizing receptors, although additions of some of these nucleotides actually resulted in further destabilization of the unoccupied glucocorticoid receptor.     

Inhibition of testosterone 5alpha-reductase activity and growth of accessory sex tissues in castrated male mice

The steroid 4-androsten-3-one-17beta-carboxylic acid (17betaC) reduced the growth-promoting actions of testosterone, but not those of DHT in accessory sex tissues of castrated mice. The 5alpha-reduction of testosterone to DHT in these tissues was also reduced by 17betaC treatment, suggesting that DHT formation is a required step in the mechanism of action of testosterone.     

Activation of Glucocorticoid-Type II Receptor Complexes in Brain Cytosol Leads to an Increase in Surface Hydrophobicity as Determined by Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography has been used to demonstrate an increase in the surface hydrophobicity of [3H]triamcinolone acetonide ([3H]TA)-labeled type II receptors in mouse brain cytosol following transformation of these receptor complexes to the activated DNA-binding form. After removing unbound [3H]TA and molybdate (which prevents activation) by gel filtration, [3H]TA-type II receptors were activated by incubation at 22 degrees C for 20 min. Gel filtration was then used to remove newly dissociated steroid and to readjust the molybdate and/or KCl concentration. Unactivated and activated receptors were then added to propyl, butyl, pentyl, hexyl, octyl, decyl, and dodecyl alkyl agarose, phenyl agarose, or unmodified agarose columns equilibrated and eluted with buffers of various molybdate and KCl concentrations and/or other additions, including glycerol, ethylene glycol, and urea. Under high-salt conditions, activated receptors were retained longer than unactivated receptors run on butyl, pentyl, hexyl, and phenyl agaroses. With the longer alkyl chain columns, essentially none of the [3H]TA was eluted in association with receptor macromolecules. Removal of the remaining steroid required receptor denaturation with urea. Under low-salt conditions, both receptor forms were retained more avidly on all alkyl agarose columns; however, on phenyl agarose only activated receptors displayed this increased retention. Further studies revealed that optimal separation and subsequent recovery of unactivated and activated [3H]TA-type II receptor complexes were achieved on pentyl agarose columns equilibrated and eluted with buffers containing 50 mM molybdate and 600-1,200 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed.     

Intracerebral prostaglandin E2: Effects upon sexual behavior, open field activity and body temperature in ovariectomized female rats

A single 27 gauge implant of PGE₂ into the periventricular region of the hypothalamus resulted in a significant increase in sexual receptivity in estrogen primed, ovariectomized female rats. Open field activity levels were only slightly decreased while rectal body temperature increased significantly over control values. It is postulated that the effects upon sexual receptivity might be mediated by PGE₂ stimulated LRF release.     

Effects of Polyhydric and Monohydric Compounds on the Stability of Type I Receptors for Adrenal Steroids in Brain Cytosol

We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aldosterone ([3H]ALDO) in a time- and temperature-dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0 degree C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22 degrees C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 degrees C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0 degree C on the subsequent binding of [3H]ALDO yielded an inverse U-shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective Inhibition of Absorption and Long Distance Transport in Relation to the Dual Mechanisms of Ion Absorption in Maize Seedlings

The influence of several uncouplers of oxidative phosphorylation and inhibitors of terminal electron transport was studied on absorption and long distance transport of both K and C1 at concentrations within each range of the dual isotherm typical of ion uptake by maize roots. At low concentrations in the range of system 1, the system considered to implement ion movement through the plasma membrane, root absorption and long distance transport are equally inhibited by a given inhibitor. In the high range of system 2, the system considered to mediate ion passage through the tonoplast, long distance transport is markedly less sensitive to inhibitors than is absorption. The observations are in accord with the hypothesis that only system 1 is involved in the uptake of ions from the external solution into the symplast, and hence into the xylem. At high concentrations, entrance into the symplasm is deemed to be largely by diffusion and therefore less inhibitor sensitive.With respect to absorption by the roots, the plasma membrane system is more inhibitor sensitive than is the tonoplast system. It is suggested that the difference in sensitivity is real, and not the consequence of an inequality of inhibitor concentration in the vicinity of the plasma membrane and tonoplast respectively.