Synthetic organic food colouring agents and their degraded products: effects on human and rat cholinesterases.

Most synthetic coloured additives are carcinogenic; teratogenic and cause allergic reactions. In this study, the effects of synthetic azo dyes (sunset yellow FCF and carmoisine), as well as their degraded products (sulphanilic acid and naphthionic acid), on both true and pseudo-cholinesterases (ChEs) are studied. The results indicate that the synthetic azo dyes and their degraded products inhibit both human true and pseudo-ChE activities in vitro. The concentration of coloured additive that cause 50% inhibition (IC50) and enzyme inhibitor dissociation constant (Ki) show that sunset yellow FCF produces greater inhibition of both true and pseudo-ChEs than does carmoisine and sulphanilic acid, while naphthionic acid produces greater inhibition of pseudo-ChE only. Ki indicates that the affinity of sulphanilic acid for both true and pseudo-ChEs is higher than the other three inhibitors. Inhibition of both true and pseudo-ChEs by sunset yellow FCF is of mixed (competitive and non-competitive) type, but carmoisine and sulphanilic acid are non-competitive. Naphthionic acid produces a competitive inhibition kinetic with plasma ChE only. This inhibition is abolished by dialysis, indicating that their effects are reversible. The effects of sunset yellow FCF, carmoisine, sulphanilic acid and naphthionic acid on rat true and pseudo-ChEs are investigated. The data clearly show that there is a significant decrease in enzyme activity. Sulphanilic acid and sunset yellow FCF are the most potent in vivo inhibitors of true ChE and pseudo-ChE, respectively.

Effect of the carbamate "aldicarb" on acetyl cholinesterase extracted from whole and different parts of rat brain (in vitro and in vivo studies).

Aldicarb, a synthetic carbamate compound used as an insecticide and herbicide, is also employed as a pharmacological cholinergic agent in therapeutic forms. Inhibition of the enzyme AChE, which results in accumulation of ACh, is the basis for its use as an insecticide and it resulted in toxicity in a variety of animals including man. In this work, experiments were carried out in vitro and in vivo to study the effects of aldicarb on the activity of ChE enzyme extracted from whole and five different parts of rat brain, namely: basal ganglia, frontal cortex, medulla oblongata, pons and cerebellum. Kinetic measurements of Michaelis constants (Km), enzyme inhibitor dissociation constants (Ki) as well as the rate of carbamylation (k2c) and binding constants (Kl) for the inhibition of the enzyme obtained from the different parts were done. The carbamylation rate (degree of inhibition of AChE by carbamate) and binding constant for inhibition of whole and different parts of brain in vitro, indicated that the carbamylation rate constants of pons, medulla oblongata and cerebellum were higher than those of other parts. Inhibition of AChE in the different parts of the brain in vitro by aldicarb was of the competitive type, with different enzyme inhibitor dissociation constants (Ki). In vivo studies showed that the inhibition was marked in the enzyme obtained from pons, medulla oblongata and cerebellum; the parts responsible for vital centers and balance which coinsize with the in vitro results.

Purification and properties of three cellobiases from Aspergillus niger A20.

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gel electrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55-60 degrees C. The pattern of their amino acid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K(m) values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2+ and Mg2+ ions stimulated cellobiase A. The purified enzymes hydrolyzed cellobiose and aryl-beta-D-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylase reaction, and the major product formed from cellobiose was tetramer of glucose.

Effect of physostigmine on cholinesterase activity in different parts of rat brain.

Inhibition of acetylcholinesterase (AChE) in the whole brain, cerebellum, pons, frontal cortex, basal ganglia and medulla oblongata of rat brain by physostigmine (CAS 57-47-6) in vitro was studied. Constants characterizing this inhibition namely: binding constant (KI) and bimolecular rate constant (K'2) were determined. The highest values of K'2 were obtained in the cases of pons and medulla oblongata, the parts responsible for the respiratory and vital centers. The difference in the inhibition of AChE in the different parts of the brain could be due to the difference in the grey and white matters contents in these parts.

Effect of dietary trans fatty acids on cholinesterase and monoamine oxidase activities in different organs of rats.

The effect of trans fatty acids and essential fatty acid deficiency upon the activities of cholinesterase and monoamine oxidase in livers, hearts, brains and lungs of rats was studied. This study was performed using three groups of male albino rats. Two out of these three groups were fed essential fatty acid low diets containing 10% hydrogenated coconut oil (HCNO) or margarine stock (MS, partially hydrogenated soybean oil) while the third group was fed an adequate supply of essential fatty acids through a diet containing 10% corn oil (CO). In the group of rats fed HCNO the activities of cholinesterase and monoamine oxidase in their livers, hearts, brains and lungs were not significantly different from those of the control group fed CO. In the group of rats fed MS, the activity of cholinesterase was significantly decreased in the livers, hearts and brains, but not affected in the lungs, while the activity of monoamine oxidase was significantly decreased in the livers and hearts but not affected in the brains and lungs as compared to the control group fed CO. The levels of serum total lipids, total cholesterol and triglycerides were elevated in both groups of rats fed HCNO or MS than in the group fed CO, but this elevation was more highly significant in the group fed MS than in the group fed HCNO.

Inhibition of erythrocyte and plasma cholinesterase by 5-hydroxytryptamine.

The inhibition of human erythrocytes and plasma cholinesterase (ChE) by 5-hydroxytryptamine in vitro was studied. Constants characterising this inhibition, namely the inhibition rate for red blood cells RBCs (2.5 x 10(4) (mol/l)-1 min-1) and plasma ChE (1.08 x 10(4) (mol/l)-1 min-1) as well as the rate constant for spontaneous reactivation for RBCs (0.3 min-1) and plasma ChE (0.26 min-1) were calculated. The inhibition of RBCs and plasma ChE by 5-hydroxy-tryptamine was found to be of the competitive type.

Separation of dog brain renin-like activity from acid protease activity.

A renin-like enzyme and acid protease (cathepsin) from whole and saline-pefused dog brains were separated by CM-cellulose chromatography with a linear NaCl gradient. Plasma renin and cathepsin were also separated using the same system. During the separation steps (in all the above cases) the specific activity of the brain renin-like enzyme was increased, while the specific activity of the brain cathepsin was decreased. Approximately a 70-fold increase in the specific activity of brain renin-like enzyme and a sixfold decrease in brain cathepsin specific activity was obtained from saline-perfused brain. The separation made it possible to study the pH optimum of the brain renin-like enzyme and acid protease. The brain renin-like enzyme showed optimal activity in the range of pH 6-7. Immunologically, the renin-like enzyme was distinctly different from dog kidney renin.

Inhibition of cholinesterase by epinephrine and norepinephrine.

Acetylcholinesterase and monoamine oxidase were found to be inhibited by each other's substrate. The inhibition of acetylcholinesterase by low concentration of epinephrine or norepinephrine was found to follow first-order reaction kinetics. The constants characterising this inhibition, the bimolecular rate ka (51.8 M-1 min-1 for epinephrine and 15.9 M-1 min-1 for norepinephrine) and the enzyme inhibitor dissociation constant (8.52 mM for epinephrine and 12.2 mM norepinephrine) were determined. The inhibition of acetylcholinesterase by epinephrine was found to be of the mixed type while its inhibition by norepinephrine was of the competitive type.

Inhibition of cholinesterase by 5-hydroxytryptamine.

On the basis of structural relationships between 5-hydroxytryptamine (5-HT, serotonine) and eserine, the effect of 5-HT on partially purified brain cholinesterase (ChE) was studied. The addition of 5-HT to brain ChE in vitro resulted in its inhibition, and the constants characterizing this inhibition, namely, the inhibition rate ki (5.44 X 10(2) mol-1/l - min-1), the equilibrium constant K (1.86 X 10(-3), and the rate constant for spontaneous reactivation kr (1.01 min-1) were determined. The inhibition of AChE by 5-HT in vitro was found to be of the competitive type.