Changes in peroxidase and polyphenol oxidase activity and transcript levels of related genes in two egyptian bread wheat cultivars (Triticum aestivum L.) affected by gamma irradiation and salinity stress

The effect of gamma irradiation (100, 200, and 300 Gy) and NaCl (60 and 120 mM) on peroxidase (POD) and polyphenol oxidase (PPO) activities in Sids-1 and Sakha-93 wheat cultivars were investigated. POD and PPO activities increased significantly with increasing NaCl concentrations, and the maximal activity increased at 120 mM NaCl, while PPO activity was higher than POD at the same concentration of NaCl in both the two cultivars. POD and PPO isozymes profile revealed a total of six bands, whereas all of them are variable and exposed the effect of gamma ray and sodium chloride with different genetic response of the two wheat cultivars. The expression level of POD gene in Sakha-93 cultivar was more than in Sids-1. While it was more pronounced at 120 mM NaCl combined with gamma irradiation dose 300 Gy. Gamma ray can be used as a useful tool for gene expression in plants and salt tolerance could be attributed to the constitutive induced antioxidant gene, leading to more efficient enzyme stimulation and protection in wheat.

N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease.

Alanine Reverses the Inhibitory Effect of Phenylalanine on Acetylcholinesterase Activity

The aim of this work was to evaluate, in vitro, the effect of ʟ-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by ʟ-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mm of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 μᴍ substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, KM was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.

A polylysine-induced aggregation of substrate accompanies the stimulation of casein kinase II by polylysine

Casein kinase II (CK-II) activation by polylysine parallels an aggregation of substrates promoted by the polycation. CK-II is known to be stimulated by basic polypeptides and polyamines. The mechanism by which this stimulation takes place, however, is not yet fully understood. Here we show that, in the usual CK-II assay, polylysine induces the aggregation of casein. This aggregation has been monitored by turbidimetry, electron microscopy and gel filtration. The polylysine-concentration-dependence of the casein aggregation parallels the polylysine-concentration-dependence of the enzyme stimulation. In the presence of polylysine the enzyme is incorporated into the casein aggregates promoted by the polycation, thus supporting the view that this substrate aggregation is directly related to the mechanism of CK-II stimulation. Preliminary results show that a similar parallelism occurs with other natural substrates of the enzyme. The physiological meaning of this substrate aggregation, and its possible relation to other polylysine-stimulated enzymes and polylysine-aggregated proteins, are discussed.

Control of the Activity of Brain Synaptosome-Associated Acetylcholinesterase by Acidic Phospholipids

Incubation of synaptosomal plasma membranes (SPM) with liposomes of phosphatidylserine (PS), phosphatidylinositol (PIN) or phosphatidylglycerol (PGL), led to an increase of acetylcholinesterase (AchE) activity at concentrations of 0.1-1 μmol phospholip ids per mg SPM protein. The use of higher concentrations (1-7 μmol/mg protein), however, led to a progressive inhibition of the activity with respect to the maximal percentage of enzyme stimulation. To explain the enzyme stimulation by the acidic phospholipids, AchE was solubilized with the detergent Lubrol-PX and showed no change in the enzyme activity at any PS, PIN or PGL concentration used, indicating that these compounds do not act on the protein molecule directly. Arrhenius plots of AchE activities in untreated SPM (control), exhibited a break point at 23 °C , which was decreased to 16-17 °C in PS-treated SPM. Moreover, the Arrhenius activation energy (Ea) value in PS-treated SPM was increased related to the Ea below the break point in the control. These results indicate that acidic phospholipids do not act on AchE directly, but indirectly, affecting the membrane fluidity probably. Such modifications of interactions between lipid and AchE may control physiological processes in the central nervous system .