Abdulaziz A. Al-Jafari

Kinetics of human acetylcholinesterase inhibition by the novel experimental alzheimer therapeutic agent, tolserine

Characterization of the kinetic parameters of tolserine, a novel acetylcholinesterase (AChE) inhibitor of potential in the therapy of Alzheimers disease, to inhibit purified human erythrocyte AChE was undertaken for the first time. An IC(50) value was estimated by three methods. Its mean value was found to be 8.13 nM, whereas the IC(100) was observed to be 25.5 nM as calculated by single graphical method. The Michaelis-Menten constant (K(m)) for the hydrolysis of the substrate acetylthiocholine iodide was found to be 0.08 mM. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the partial non-competitive type. The value of K(i) was estimated as 4.69 nM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. Four new kinetic constants were also investigated by polynomial regression analysis of the relationship between the apparent K(i) (K(Iapp)) and substrate concentration, which may open new avenues for the kinetic study of the inhibition of several enzymes by a wide variety of inhibitors in vitro. Tolserine proved to be a highly potent inhibitor of human AChE compared to its structural analogues physostigmine and phenserine.

Determination of l-carnitine, acylcarnitine and total carnitine levels in plasma and tissues of camel (Camelus dromedarius)

The total, free and acyl carnitine levels were measured in plasma and tissues of the Arabian camel (Camelus dromedarius). Significant variation in carnitine concentrations were observed in plasma and tissues of the camel when compared with other animal species. A higher proportion of acyl carnitine was found in plasma and skeletal muscle of the camel than other animal species. Among the camel tissues, skeletal muscle possessed the highest amount of carnitine while the lowest amount was found in kidney. The higher carnitine content and a higher proportion of acyl carnitine in plasma and tissues of the Arabian camel suggest an adaptive mechanism that could be common to desert animal species.

Kinetic analysis of the toxicological effect of tacrine (Cognex) on human retinal acetylcholinesterase activity.

For the first time, kinetic parameters of the effect of tacrine, an anti-cholinesterase inhibitor of therapeutic potential in Alzheimers disease has been studied on human retinal acetyl-cholinesterase (AChE). Tacrine inhibited the AChE activity in a concentration dependent manner, the IC(50) being about 45 nM. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.120 mM and this value was increased by 4-52.8% in the presence of tacrine. V(max) was observed to be 2.23 micromol/h per mg protein for the control system, while it was decreased by 14.73-56.25% in the tacrine treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the mixed type, i. e. a combination of competitive and noncompetitive inhibition. The values of K(i) and K(I) were estimated to be as 37.76 and 64.36 nM, respectively.

Inhibition of human acetylcholinesterase by cyclophosphamide

The inhibitory effect of cyclophosphamide (CP) on human erythrocyte membrane bound acetylcholinesterase (AChE) was investigated in the present study. It was found that CP inhibits the AChE reversibly with an IC50 of 511 microM. The Michaelis-Menten constant (Km) was 132 microM for AChE in the control system; a value increased by 78% in the CP treated system. The Vmax was 73.8 mumol/h/mg protein for the control system. The Lineweaver-Burk plot and Dixon plot indicated that the nature of this inhibition is of the linear mixed type, i.e., partially competitive and purely noncompetitive. The values of Ki and KI were estimated as 378 and 582 microM, respectively. The KI was greater than Ki indicating that noncompetitive inhibition was predominant over competitive.

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the arabian camel (Camelus dromedarius)

This study was conducted to determine the concentration of total, free, peptide‐bound, protein‐bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide‐bound, protein‐bound, soluble and insoluble collagen Hyp in various tissues (P < 0·01). Camel kidney showed a significantly high concentration of total, free, peptide‐bound and protein‐bound Hyp and collagen content as compared to other tissues examined (P < 0·01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0·01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0·01). These variations may result from differences in the collagen structure and/or composition in this species. Copyright

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: Kinetic characterization

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (AChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration‐dependent manner; IC50 was found to be 8.07 nM. The Michaelis‐Menten constant (Ka) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54–67% in the tacrine‐treated systems. The Vmax was 0.701 μ mole/min per milligram protein for the control system, but it was decreased by 26–69% in the tacrine‐treated systems. The Lineweaver–Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of Ki and Kt were estimated to be as 4.475 and 8.517 nM, respectively.

The toxicological effect of cyclophosphamide on acetylcholinesterase activity.

Cyclophosphamide (CP; 2.5-10.0 mM) reversibly inhibits acetylcholinesterase (AChE) activity of chicken brain (58-84%) in a concentration-dependent manner, the IC50 (the concentration causing 50% inhibition) being about 2.0 mM. Lineweaver-Burk plots indicated that the nature of this inhibition is mixed; intercept and slope replots are straight lines, showing that the inhibition is complete rather than partial. The value of Km for AChE is found to be 108 microM, while values of KI and Ki are found to be 1.5 and 5.1 mM, respectively, Ki is greater than KI indicating that uncompetitive inhibition is predominant over noncompetitive inhibition.

Acetylcholinesterase from desert Cobra (Walterinnesia aegyptia) venom: Optimization and kinetics study

Acetylcholinesterase (AChE) was investigated inWalterinnesia aegyptia venom and characterized with respect to its kinetic properties. It was found that 4.0 ug of crude venom protein and an incubation time of 4.0 min were suitable conditions for linearity of AChE activity at 25°C. The optimum strength of the sodium phosphate buffer was 0.05 M, and the optimum pH was 7.75. The optimum temperature was 30°C. The activation energy and the heat of activation were observed to be 6510 and 5922 cal/mole. The AChE was specific for acetylthiocholine but it did not hydrolyse butyrylthiocholine. The optimum substrate concentration was 3.0 mM but at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (Km, Vmax, kcat, and ksp) were established at each order of the reaction.

The Effect of Tetrabutylammonium Ion on Chicken Brain Acetylcholinesterase Activity in Vitro

The effect of tetrabutylammonium ion on chicken brain acetylcholinesterase activity was investigated. Tetrabutylammonium ion (1-4mM) reversibly inhibited acetylcholinesterase activity of chicken brain (34-73%) in a concentration dependent manner, the IC50 being about 1.1mM. A Lineweaver-Burk plot, as well as a Dixon plot, indicated that the nature of inhibition is noncompetitive with a Km value of 0.134mM and a Ki value of 1.58mM at 37 degrees C. An Arrhenius plot showed that the transition temperature is significantly reduced in the presence of tetrabutylammonium ion.

PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINESTERASE FROM DESERT COBRA (WALTERINNESIA AEGYPTIA) VENOM

Acetylcholinesterase (AChE) has been identified and purified from the venom of desert cobra (W aegyptia) to apparent homogeneity using a TSK G 3000 SW gel filtration column and a Mono Q anion-exchange column. AChE was purified to homogeneity as established by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The specific activity of AChE was 357 IU/mg with acetylthiocholine iodide as substrate. The denatured W aegyptia venom AChE displayed a molecular mass of 67000 +/- 3000 Da suggesting it was a single polypeptide. Isoelectric focusing of AChE revealed that the enzyme exists in different isoforms, with isoelectric points ranging between pH 7.4-7.9. The kinetic parameters (Km and Vmax) and IC50 of AChE inhibition by procaine, tetracaine and physostigmine were investigated in the present study.