Ali S. Duhaiman

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.

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.

Glycation of human lens proteins from diabetic and (nondiabetic) senile cataract patients.

Glycation (nonenzymatic glycosylation) in the human lens (cortex and nucleus) in senile (nondiabetic) and diabetic cataracts was studied by measuring the extent of early and late glycation products, the content of free ε-amino groups and the formation of disulfide bonds in the soluble lens proteins. There was a significant (p<0.001) increase in early and late glycation in the lens nucleus compared to the cortex in both the senile and diabetic groups. Overall these changes were much larger in the diabetic group. The concentration of free ε-amino groups was decreased in the senile nucleus as well as in the diabetic nucleus when compared with the senile and diabetic cortex (p<0.001). Disulfide bond content was in the order of diabetic nucleus > diabetic cortex > senile nucleus > senile cortex. Glycation of the lens proteins is a generalized feature which is enhanced in the diabetic lens compared to senile lens proteins and is associated with a decrease in free ε-amino groups and an increase in disulfide bonds formation in the lens proteins.

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.

CAMEL LENS CRYSTALLINS GLYCOSYLATION AND HIGH MOLECULAR WEIGHT AGGREGATE FORMATION IN THE PRESENCE OF FERROUS IONS AND GLUCOSE

The incubation of camel lens cortex homogenate with 100 microM ferrous ions and 5.5 mM glucose under sterile conditions caused rapid protein aggregation, but little or no reaction was seen with either 100 microM ferrous ions or 5.5 mM glucose alone. The formation of glycosylated high molecular weight (HMW) protein aggregates was confirmed by light scattering studies, a decreased level of free -SH groups, incorporation of [14C]-glucose and elution of HMW protein aggregate just after the void volume of a Sephacryl S-1000 column. The bonding involved in the formation of these aggregates was found to be mainly disulfide in nature. Isoelectric focusing (IEF) in the presence and absence of reducing conditions indicated that gamma-crystallins may be involved in the formation of HMW protein aggregates. The modifications observed were found to mimic those seen in cataractous lenses.

Purification and some properties of camel lens crystallins.

Five major crystallin fractions were found in camel lens fractionated by Sepharose CL-6B column chromatography. These crystallin fractions were named alpha high (alpha H), alpha low (alpha L), beta high (beta H), beta low (beta L) and gamma (gamma) by comparison with the elution profiles and molecular weights of rabbit crystallins. The amino acid composition and isoelectric focusing bands for crystallins of camel and rabbit were remarkably similar, but individual differences were found. By means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two alpha H crystallin subunits, of 23K and 20K molecular weight, were found in camel and rabbit. Likewise, a single 21K molecular weight band was found in the gamma crystallin of camel and rabbit. In camel, the beta high crystallin consisted of five major subunits, while rabbit beta high crystallin consisted of only three subunits. On SDS-PAGE, camel and rabbit beta low crystallins both showed two major subunits of 27K and 23K molecular weight but camel beta low crystallin showed an additional 35K molecular weight subunit. Characterization of camel lens crystallins may contribute to understanding the effect of aging on aggregation of camel crystallins.

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.

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.

Kinetic properties of purified carnitine acetyltransferase from the skeletal muscle of Arabian camel (Camelus dromedarius)

The kinetic properties of carnitine acetyltransferase from the skeletal muscle of the Arabian camel (Camelus dromedarius) were studied. The enzyme showed an optimum pH between 7.2 and 8.2. Reciprocal plots of data obtained by varying one substrate concentration while keeping the other constant revealed lines that converged on the abscissa, indicating that the enzyme possible follows a random mechanism of catalysis. The Kms for L-carnitine and acetyl-coenzyme A were 244 and 44 microM respectively, while those for acetyl-DL-carnitine and coenzyme A (Co A) were 307 and 39 microM respectively. The Km for one substrate was found to be independent of the concentration of the second substrate used. Corresponding Vmax values for L-CA, acetyl-Co A, acetyl-DL-carnitine and Co A are 98, 98, 102 and 100 mumol min-1 mg-1 protein respectively. The low Km obtained for acetyl-DL-carnitine suggests an adaptive mechanism in this desert species for enduring prolonged dry spells without food and water.

High-affinity binding of NADPH to camel lens ζ-crystallin

Fluorescence spectrum of camel lens ζ-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including α-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with ζ-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and ζ-crystallin with an apparent Km<0.45 μM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of ζ-crystallin, also was able to quench the fluorescence of ζ-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that ζ-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in ζ-crystallin.